Disclosed herein are methods and apparatuses for sequencing a nucleic acid. These methods permit a very large number of independent sequencing reactions to be arrayed in parallel, permitting simultaneous sequencing of a very large number (>10,000) of different oligonucleotides.
DescriptionRELATED APPLICATIONS
This application claims the benefit of priority to U.S. Ser. No. 10/104,280 filed Mar. 21, 2002; which is a CIP of 09/814,338 filed Mar. 21, 2001; which is a CIP of U.S. Ser. No. 09/664,197 filed Sep. 18, 2000; which is a CIP of U.S. Ser. No. 09/398,833 filed Sep. 16, 1999, now U.S. Pat. No. 6,274,320. Each of the above referenced patent and patent applications are incorporated herein by reference in their entireties.
The invention relates to apparatus and methods for determining the sequence of a nucleic acid.
Many diseases are associated with particular DNA sequences. The DNA sequences are often referred to as DNA sequence polymorphisms to indicate that the DNA sequence associated with a diseased state differs from the corresponding DNA sequence in non-afflicted individuals. DNA sequence polymorphisms can include, e.g., insertions, deletions, or substitutions of nucleotides in one sequence relative to a second sequence. An example of a particular DNA sequence polymorphism is 5â²-ATCG-3, relative to the sequence 5â²-ATGG-3â² at a particular location in the human genome. The first nucleotide âGâ in the latter sequence has been replaced by the nucleotide âCâ in the former sequence. The former sequence is associated with a particular disease state, whereas the latter sequence is found in individuals not suffering from the disease. Thus, the presence of the nucleotide sequence â5-ATCG-3â indicates the individual has the particular disease. This particular type of sequence polymorphism is known as a single-nucleotide polymorphism, or SNP, because the sequence difference is due to a change in one nucleotide.
Techniques which enable the rapid detection of as little as a single DNA base change are therefore important methodologies for use in genetic analysis. Because the size of the human genome is large, on the order of 3 billion base pairs, techniques for identifying polymorphisms must be sensitive enough to specifically identify the sequence containing the polymorphism in a potentially large population of nucleic acids.
Typically a DNA sequence polymorphism analysis is performed by isolating DNA from an individual, manipulating the isolated DNA, e.g., by digesting the DNA with restriction enzymes and/or amplifying a subset of sequences in the isolated DNA. The manipulated DNA is then examined further to determine if a particular sequence is present.
Commonly used procedures for analyzing the DNA include electrophoresis. Common applications of electrophoresis include agarose or polyacrylamide gel electrophoresis. DNA sequences are inserted, or loaded, on the gels and subjected to an electric field. Because DNA carries a uniform negative charge, DNA will migrate through the gel based on properties including sequence length, three-dimensional conformation and interactions with the gel matrix upon application of the electrical field. In most applications, smaller DNA molecules will migrate more rapidly through the gel than larger fragments. After electrophoresis has been continued for a sufficient length of time, the DNA molecules in the initial population of DNA sequences will have been separated according to their relative sizes.
Particular DNA molecules can then be detected using a variety of detection methodologies. For some applications, particular DNA sequences are identified by the presence of detectable tags, such as radioactive labels, attached to specific DNA molecules.
Electrophoretic-based separation analyses can be less desirable for applications in which it is desirable to rapidly, economically, and accurately analyze a large number of nucleic acid samples for particular sequence polymorphisms. For example, electrophoretic-based analysis can require a large amount of input DNA. In addition, processing the large number of samples required for electrophoretic-based nucleic acid based analyses can be labor intensive. Furthermore, these techniques can require samples of identical DNA molecules, which must be created prior to electrophoresis at costs that can be considerable.
Recently, automated electrophoresis systems have become available. However, electrophoresis can be ill suited for applications such as clinical sequencing, where relatively cost-effective units with high throughput are needed. Thus, the need for non-electrophoretic methods for sequencing is great. For many applications, electrophoresis is used in conjunction with DNA sequence analysis.
Several alternatives to electrophoretic-based sequencing have been described. These include scanning tunnel electron microscopy, sequencing by hybridization, and single molecule detection methods.
Another alternative to electrophoretic-based separation analysis is solid substrate-based nucleic acid analyses. These methods typically rely upon the use of large numbers of nucleic acid probes affixed to different locations on a solid support. These solid supports can include, e.g., glass surfaces, plastic microtiter plates, plastic sheets, thin polymers, or semi-conductors. The probes can be, e.g., adsorbed or covalently attached to the support, or can be microencapsulated or otherwise entrapped within a substrate matrix, membrane, or film.
Substrate-based nucleic acid analyses can include applying a sample nucleic acid known or suspected of containing a particular sequence polymorphism to an array of probes attached to the solid substrate. The nucleic acids in the population are allowed to hybridize to complementary sequences attached to the substrate, if present. Hybridizing nucleic acid sequences are then detected in a detection step.
Solid support matrix-based hybridization and sequencing methodologies can require a high sample-DNA concentration and can be hampered by the relatively slow hybridization kinetics of nucleic acid samples with immobilized oligonucleotide probes. Often, only a small amount of template DNA is available, and it can be desirable to have high concentrations of the target nucleic acid sequence. Thus, substrate based detection analyses often include a step in which copies of the target nucleic acid, or a subset of sequences in the target nucleic acid, is amplified. Methods based on the Polymerase Chain Reaction (PCR), e.g., can increase a small number of probe targets by several orders of magnitude in solution. However, PCR can be difficult to incorporate into a solid-phase approach because the amplified DNA is not immobilized onto the surface of the solid support matrix.
Solid-phase based detection of sequence polymorphisms has been described. An example is a âmini-sequencingâ protocol based upon a solid phase principle described by Hultman, et al., 1988. Nucl. Acid. Res. 17: 4937-4946; Syvanen, et al., 1990. Genomics 8: 684-692. In this study, the incorporation of a radiolabeled nucleotide was measured and used for analysis of a three-allelic polymorphism of the human apolipoprotein E gene. However, such radioactive methods are not well suited for routine clinical applications, and hence the development of a simple, highly sensitive non-radioactive method for rapid DNA sequence analysis has also been of great interest.
The invention is based in part on the use of arrays for determining the sequences of nucleic acids.
Accordingly, in one aspect, the invention involves an array including a planar surface with a plurality of reaction chambers disposed thereon, wherein the reaction chambers have a center to center spacing of between 5 to 200 μm and each chamber has a width in at least one dimension of between 0.3 μm and 100 μm. In some embodiments, the array is a planar surface with a plurality of cavities thereon, where each cavity forms an analyte reaction chamber. In a preferred embodiment, the array is fashioned from a sliced fiber optic bundle (i.e., a bundle of fused fiber optic cables) and the reaction chambers are formed by etching one surface of the fiber optic reactor array (âFORAâ). The cavities can also be formed in the substrate via etching, molding or micromachining.
Specifically, each reaction chamber in the array typically has a width in at least one dimension of between 0.3 μm and 100 μm, preferably between 0.3 μm and 20 μm, mst preferably between 0.3 μm and 10 μm. In a separate embodiment, we contemplate larger reaction chambers, preferably having a width in at least one dimension of between 20 μm and 70 μm.
The array typically contains more than 1,000 reaction chambers, preferably more than 400,000, more preferably between 400,000 and 20,000,000, and most preferably between 1,000,000 and 16,000,000 cavities or reaction chambers. The shape of each cavity is frequently substantially hexagonal, but the cavities can also be cylindrical. In some embodiments, each cavity has a smooth wall surface, however, we contemplate that each cavity may also have at least one irregular wall surface. The bottom of each of the cavities can be planar or concave.
The array is typically constructed to have cavities or reaction chambers with a center-to-center spacing between 10 to 150 μm, preferably between 50 to 100 μm.
Each cavity or reaction chamber typically has a depth of between 10 μm and 100 μm; alternatively, the depth is between 0.25 and 5 times the size of the width of the cavity, preferably between 0.3 and 1 times the size of the width of the cavity.
In one embodiment, the arrays described herein typically include a planar top surface and a planar bottom surface, which is optically conductive such that optical signals from the reaction chambers can be detected through the bottom planar surface. In these arrays, typically the distance between the top surface and the bottom surface is no greater than 10 cm, preferably no greater than 3 cm, most preferably no greater than 2 cm, and usually between 0.5 mm to 5 mm.
In one embodiment, each cavity of the array contains reagents for analyzing a nucleic acid or protein. The array can also include a second surface spaced apart from the planar array and in opposing contact therewith such that a flow chamber is formed over the array.
In another aspect, the invention involves an array means for carrying out separate parallel common reactions in an aqueous environment, wherein the array means includes a substrate having at least 1,000 discrete reaction chambers. These chambers contain a starting material that is capable of reacting with a reagent. Each of the reaction chambers are dimensioned such that when one or more fluids containing at least one reagent is delivered into each reaction chamber, the diffusion time for the reagent to diffuse out of the well exceeds the time required for the starting material to react with the reagent to form a product. The reaction chambers can be formed by generating a plurality of cavities on the substrate, or by generating discrete patches on a planar surface, the patches having a different surface chemistry than the surrounding planar surface.
In one embodiment, each cavity or reaction chamber of the array contains reagents for analyzing a nucleic acid or protein. Typically those reaction chambers that contain a nucleic acid (not all reaction chambers in the array are required to) contain only a single species of nucleic acid (i.e., a single sequence that is of interest). There may be a single copy of this species of nucleic acid in any particular reaction chamber, or they may be multiple copies. It is generally preferred that a reaction chamber contain at least 100 copies of a nucleic acid sequence, preferably at least 100,000 copies, and most preferably between 100,000 to 1,000,000 copies of the nucleic acid. In one embodiment the nucleic acid species is amplified to provide the desired number of copies using PCR, RCA, ligase chain reaction, other isothermal amplification, or other conventional means of nucleic acid amplification. In one embodimant, the nucleic acid is single stranded. In other embodiments the single stranded DNA is a concatamer with each copy covalently linked end to end.
The nucleic acid may be immobilized in the reaction chamber, either by attachment to the chamber itself or by attachment to a mobile solid support that is delivered to the chamber. A bioactive agent could be delivered to the array, by dispersing over the array a plurality of mobile solid supports, each mobile solid support having at least one reagent immobilized thereon, wherein the reagent is suitable for use in a nucleic acid sequencing reaction.
The array can also include a population of mobile solid supports disposed in the reaction chambers, each mobile solid support having one or more bioactive agents (such as a nucleic acid or a sequencing enzyme) attached thereto. The diameter of each mobile solid support can vary, we prefer the diameter of tie mobile solid support to be between 0.01 to 0.1 times the width of each cavity. Not every reaction chamber need contain one or more mobile solid supports. There are three contemplated embodiments; one where at least 5% to 20% of of the reaction chambers can have a mobile solid support having at least one reagent immobilized thereon; a second embodiment where 20% to 60% of the reaction chambers can have a mobile solid support having at least one reagent immobilized thereon; and a third embodiment where 50% to 100% of the reaction chambers can have a mobile solid support having at least one reagent immobilized thereon.
The mobile solid support typically has at least one reagent immobilized thereon. For the embodiments relating to pyrosequencing reactions or more generally to ATP detection, the reagent may be a polypeptide with sulfurylase or luciferase activity, or both. The mobile solid supports can be used in methods for dispersing over the array a plurality of mobile solid supports having one or more nucleic sequences or proteins or enzymes immobilized thereon.
In another aspect, the invention involves an apparatus for simultaneously monitoring the array of reaction chambers for light generation, indicating that a reaction is taking place at a particular site. In this embodiment, the reaction chambers are sensors, adapted to contain analytes and an enzymatic or fluorescent means for generating light in the reaction chambers. In this embodiment of the invention, the sensor is suitable for use in a biochemical or cell-based assay. The apparatus also includes an optically sensitive device arranged so that in use the light from a particular reaction chamber would impinge upon a particular predetermined region of the optically sensitive device, as well as means for determining the light level impinging upon each of the predetermined regions and means to record the variation of the light level with time for each of the reaction chamber.
In one specific embodiment, the instrument includes a light detection means having a light capture means and a second fiber optic bundle for transmitting light to the light detecting means. We contemplate one light capture means to be a CCD camera. The second fiber optic bundle is typically in optical contact with the array, such that light generated in an individual reaction chamber is captured by a separate fiber or groups of separate fibers of the second fiber optic bundle for transmission to the light capture means.
The above arrays may be used for carrying out separate parallel common reactions in an aqueous environment. The method includes delivering a fluid containing at least one reagent to the described arrays, wherein certain reaction chambers (not necessarily all) on the array contain a starting material that is capable of reacting with the reagent. Each of the reaction chambers is dimensioned such that when the fluid is delivered into each reaction chamber, the diffusion time for the reagent to diffuse out of the well exceeds the time required for the starting material to react with the reagent to form a product. The method also includes washing the fluid from the array in the time period after the starting material has reacted with the reagent to form a product in each reaction chamber but before the reagent delivered to any one reaction chamber has diffused out of that reaction chamber into any other reaction chamber. In one embodiment, the product formed in any one reaction chamber is independent of the product formed in any other reaction chamber, but is generated using one or more common reagents. The starting material can be a nucleic acid sequence and at least one reagent in the fluid is a nucleotide or nucleotide analog. The fluid can additionally have a polymerase capable of reacting the nucleic acid sequence and the nucleotide or nucleotide analog. The steps of the method can be repeated sequentially.
The apparatus includes a novel reagent delivery cuvette adapted for use with the arrays described herein, to provide fluid reagents to the array, and a reagent delivery means in communication with the reagent delivery cuvette.
The disclosures of one or more embodiments of the invention are set forth in the accompanying description below. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described. Other features, objects, and advantages of the invention will be apparent from the description and from the claims. In the specification and the appended claims, the singular forms include plural referents unless the context clearly dictates otherwise. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Unless expressly stated otherwise, the techniques employed or contemplated herein are standard methodologies well known to one of ordinary skill in the art. The examples of embodiments are for illustration purposes only. All patents and publications cited in this specification are incorporated by reference.
FIGS. 1A-D are schematic illustrations of rolling circle-based amplification using an anchor primer.
FIG. 2 is a drawing of a sequencing apparatus according to the present invention.
FIG. 3 is a drawing of a perfusion chamber according to the present invention.
FIG. 4 is a drawing of a cavitated fiber optic terminus of the present invention.
FIG. 5 is a tracing of a sequence output of a concatemeric template generated using rolling circle amplification.
FIG. 6 is a micrograph of a Fiber Optic Reactor Array (FORA).
FIG. 7 is a schematic illustration for the the preparation of a carpeted FORA.
FIG. 8 is a micrograph for single well DNA delivery.
FIG. 9 is a schematic illustration of the Flow Chamber and FORA.
FIG. 10 is a diagram of the analytical instrument of the present invention.
FIG. 11 is a schematic illustration of microscopic parallel sequencing reactions within a FORA.
FIG. 12 is a micrograph of single well reactions.
The methods and apparatuses described herein allow for the determination of nucleic acid sequence information without the need for first cloning a nucleic acid. In addition, the method is highly sensitive and can be used to determine the nucleotide sequence of a template nucleic acid, which is present in only a few copies in a starting population of nucleic acids. Further, the method can be used to determine simultaneously the sequences of a large number of nucleic acids.
The methods and apparatuses described are generally useful for any application in which the identification of any particular nucleic acid sequence is desired. For example, the methods allow for identification of single nucleotide polymorphisms (SNPs), haplotypes involving multiple SNPs or other polymorphisms on a single chromosome, and transcript profiling. Other uses include sequencing of artificial DNA constructs to confirm or elicit their primary sequence, or to identify specific mutant clones from random mutagenesis screens, as well as to obtain the sequence of cDNA from single cells, whole tissues or organisms from any developmental stage or environmental circumstance in order to determine the gene expression profile from that specimen. In addition, the methods allow for the sequencing of PCR products and/or cloned DNA fragments of any size isolated from any source.
The methods described herein include a sample preparation process that results in a solid or a mobile solid substrate array containing a plurality of anchor primers covalently linked to a nucleic acid containing one or more copies complementary to a target nucleic acid. Formation of the covalently linked anchor primer and one or more copies of the target nucleic acid preferably occurs by annealing the anchor primer to a complementary region of a circular nucleic acid, and then extending the annealed anchor primer with a polymerase to result in formation of a nucleic acid containing one or more copies of a sequence complementary to the circular nucleic acid.
Attachment of the anchor primer to a solid or mobile solid substrate can occur before, during, or subsequent to extension of the annealed anchor primer. Thus, in one embodiment, one or more anchor primers are linked to the solid or a mobile solid substrate, after which the anchor primer is annealed to a target nucleic acid and extended in the presence of a polymerase. Alternatively, in a second embodiment, an anchor primer is first annealed to a target nucleic acid, and a 3â²OH terminus of the annealed anchor primer is extended with a polymerase. The extended anchor primer is then linked to the solid or mobile solid substrate. By varying the sequence of anchor primers, it is possible to specifically amplify distinct target nucleic acids present in a population of nucleic acids.
Sequences in the target nucleic acid can be identified in a number of ways. Preferably, a sequencing primer is annealed to the amplified nucleic acid and used to generate a sequencing product. The nucleotide sequence of the sequence product is then determined, thereby allowing for the determination of the nucleic acid. Similarly, in one embodiment, the template nucleic acid is amplified prior to its attachment to the bead or other mobile solid support. In other embodiments, the template nucleic acid is attached to the bead prior to its amplification.
The methods of the present invention can be also used for the sequencing of DNA fragments generated by analytical techniques that probe higher order DNA structure by their differential sensitivity to enzymes, radiation or chemical treatment (e.g., partial DNase treatment of chromatin), or for the determination of the methylation status of DNA by comparing sequence generated from a given tissue with or without prior treatment with chemicals that convert methyl-cytosine to thymidine (or other nucleotide) as the effective base recognized by the polymerase. Further, the methods of the present invention can be used to assay cellular physiology changes occurring during development or senescence at the level of primary sequence.
The invention also provides methods of preparing nucleic acid sequences for subsequent analysis, e.g., sequencing.
I. Apparatus for Sequencing Nucleic Acids
This invention provides an apparatus for sequencing nucleic acids, which generally comprises one or more reaction chambers for conducting a sequencing reaction, means for delivering reactants to and from the reaction chamber(s), and means for detecting a sequencing reaction event. In another embodiment, the apparatus includes a reagent delivery cuvette containing a plurality of cavities on a planar surface. In a preferred embodiment, the apparatus is connected to at least one computer for controlling the individual components of the apparatus and for storing and/or analyzing the information obtained from detection of the sequence reaction event.
The invention also provides one or more reaction chambers are arranged in the form of an array on an inert substrate material, also referred to herein as a âsolid supportâ, that allows for combination of the reactants in a sequencing reaction in a defined space and for detection of the sequencing reaction event. Thus, as used herein, the terms âreaction chamberâ or âanalyte reaction chamberâ refer to a localized area on the substrate material that facilitates interaction of reactants, e.g., in a nucleic acid sequencing reaction. As discussed more fully below, the sequencing reactions contemplated by the invention preferably occur on numerous individual nucleic acid samples in tandem, in particular simultaneously sequencing numerous nucleic acid samples derived from genomic and chromosomal DNA. The apparatus of the invention therefore preferably comprises an array having a sufficient number of reaction chambers to carry out such numerous individual sequencing reactions. In one embodiment, the array comprises at least 1,000 reaction chambers. In another embodiment, the array comprises greater than 400,000 reaction chambers, preferably between 400,000 and 20,000,000 reaction chambers. In a more preferred embodiment, the array comprises between 1,000,000 and 16,000,000 reaction chambers.
The reaction chambers on the array typically take the form of a cavity or well in the substrate material, having a width and depth, into which reactants can be deposited. One or more of the reactants typically are bound to the substrate material in the reaction chamber and the remainder of the reactants are in a medium which facilitates the reaction and which flows through the reaction chamber. When formed as cavities or wells, the chambers are preferably of sufficient dimension and order to allow for (i) the introduction of the necessary reactants into the chambers, (ii) reactions to take place within the chamber and (iii) inhibition of mixing of reactants between chambers. The shape of the well or cavity is preferably circular or cylindrical, but can be multisided so as to approximate a circular or cylindrical shape. In another embodiment, the shape of the well or cavity is substantially hexagonal. The cavity can have a smooth wall surface. In an additional embodiment, the cavity can have at least one irregular wall surface. The cavities can have a planar bottom or a concave bottom. The reaction chambers can be spaced between 5 μm and 200 μm apart. Spacing is determined by measuring the center-to-center distance between two adjacent reaction chambers. Typically, the reaction chambers can be spaced between 10 μm and 150 μm apart, preferably between 50 μm and 100 μm apart. In one embodiment, the reaction chambers have a width in one dimension of between 0.3 μm and 100 μm. The reaction chambers can have a width in one dimension of between 0.3 μm and 20 μm, preferably between 0.3 μm and 10 μm, and most preferably about 6 μm. In another embodiment, the reaction chambers have a width of between 20 μm and 70 μm Ultimately the width of the chamber may be dependant on whether the nucleic acid samples require amplification. If no amplification is necessary, then smaller, e.g., 0.3 μm is preferred. If amplification is necessary, then larger, e.g., 6 μm is preferred. The depth of the reaction chambers are preferably between 10 μm and 100 μm. Alternatively, the reaction chambers may have a depth that is between 0.25 and 5 times the width in one dimension of the reaction chamber or, in another embodiment, between 0.3 and 1 times the width in one dimension of the reaction chamber.
In another aspect, the invention involves an apparatus for determining the nucleic acid sequence in a template nucleic acid polymer. The apparatus includes an array having a plurality of cavities on a planar surface. Each cavity forms an analyte reaction chamber, wherein the reaction chambers have a center-to-center spacing of between 5 to 200 μm. It also includes a nucleic acid delivery means for introducing a template nucleic acid polymers into the reaction chambers; and a nucleic acid delivery means to deliver reagents to the reaction chambers to create a polymerization environment in which the nucleic acid polymers will act as a template polymers for the synthesis of complementary nucleic acid polymers when nucleotides are added. The apparatus also includes a reagent delivery means for successively providing to the polymerization environment a series of feedstocks, each feedstock comprising a nucleotide selected from among the nucleotides from which the complementary nucleic acid polymer will be formed, such that if the nucleotide in the feedstock is complementary to the next nucleotide in the template polymer to be sequenced the nucleotide will be incorporated into the complementary polymer and inorganic pyrophosphate will be released. It also includes a detection means for detecting the formation of inorganic pyrophosphate enzymatically; and a data processing means to determine the identity of each nucleotide in the complementary polymers and thus the sequence of the template polymers.
In another aspect, the invention involves an apparatus for determining the base sequence of a plurality of nucleotides on an array. The apparatus includes a reagent cuvette containing a plurality of cavities on a planar surface. Each cavity forms an analyte reaction chamber, wherein the reaction chambers have a center-to-center spacing of between 5 to 200 μm. The apparatus also includes a reagent delivery means for adding an activated nucleotide 5â²-triphosphate precursor of one known nitrogenous base to a reaction mixture in each reaction chamber. Each reaction mixture has a template-directed nucleotide polymerase and a single-stranded polynucleotide template hybridized to a complementary oligonucleotide primer strand at least one nucleotide residue shorter than the templates to form at least one unpaired nucleotide residue in each template at the 3â²-end of the primer strand, under reaction conditions which allow incorporation of the activated nucleoside 5â²-triphosphate precursor onto the 3â²-end of the primer strands, provided the nitrogenous base of the activated nucleoside 5â²-triphosphate precursor is complementary to the nitrogenous base of the unpaired nucleotide residue of the templates. The apparatus also includes a detection means for detecting whether or not the nucleoside 5â²-triphosphate precursor was incorporated into the primer strands in which incorporation of the nucleoside 5â²-triphosphate precursor indicates that the unpaired nucleotide residue of the template has a nitrogenous base composition that is complementary to that of the incorporated nucleoside 5â²-triphosphate precursor. The apparatus also includes a means for sequentially repeating the second and third steps wherein each sequential repetition adds and, detects the incorporation of one type of activated nucleoside 5â²-triphosphate precursor of known nitrogenous base composition. The apparatus also includes a data processing means for determining the base sequence of the unpaired nucleotide residues of the template in each reaction chamber from the sequence of incorporation of the nucleoside precursors.
Solid Support Material
Any material can be used as the solid support material, as long as the surface allows for stable attachment of the primers and detection of nucleic acid sequences. The solid support material can be planar or can be cavitated, e.g., in a cavitated terminus of a fiber optic or in a microwell etched, molded, or otherwise micromachined into the planar surface, e.g. using techniques commonly used in the construction of microelectromechanical systems. See e.g., Rai-Choudhury HANDBOOK OF MICROLITHOGRAPHY, MICROMACHINING, AND MICROFABRICATION, VOLUME I: MICROLITHOGRAPHY, Volume PM 39, SPIE Press (1997); Madou, CRC Press (1997), Aoki, Biotech. Histochem. 67: 98-9 (1992); Kane et al., Biomaterials, 20: 2363-76 (1999); Deng et al., Anal. Chem. 72:3176-80 (2000); Zhu et al., Nat. Genet. 26:283-9 (2000). In some embodiments, the solid support is optically transparent, e.g., glass.
An array of attachment sites on an optically transparent solid support can be constructed using lithographic techniques commonly used in the construction of electronic integrated circuits as described in, e.g., techniques for attachment described in U.S. Pat. Nos. 5,143,854, 5,445,934, 5,744,305, and 5,800,992; Chee et al., Science 274: 610-614 (1996); Fodor et al., Nature 364: 555-556 (1993); Fodor et al., Science 251: 767-773 (1991); Gushin, et al., Anal. Biochem. 250: 203-211 (1997); Kinosita et al., Cell 93: 21-24 (1998); Kato-Yamada et al., J. Biol. Chem. 273: 19375-19377 (1998); and Yasuda et al., Cell 93: 1117-1124 (1998). Photolithography and electron beam lithography sensitize the solid support or substrate with a linking group that allows attachment of a modified biomolecule (e.g., proteins or nucleic acids). See e.g., Service, Science 283: 27-28 (1999); Rai-Choudhury, HANDBOOK OF MICROLITHOGRAPHY, MICROMACHINING, AND MICROFABRICATION, VOLUME I: MICROLITHOGRAPHY, Volume PM39, SPIE Press (1997). Alternatively, an array of sensitized sites can be generated using thin-film technology as described in Zasadzinski et al., Science 263: 1726-1733 (1994).
Fiber Optic Substrate Arrays
The substrate material is preferably made of a material that facilitates detection of the reaction event. For example, in a typical sequencing reaction, binding of a dNTP to a sample nucleic acid to be sequenced can be monitored by detection of photons generated by enzyme action on phosphate liberated in the sequencing reaction. Thus, having the substrate material made of a transparent or optically (i.e., light) conductive material facilitates detection of the photons.
In some embodiments, the solid support can be coupled to a bundle of optical fibers that are used to detect and transmit the light product. The total number of optical fibers within the bundle may be varied so as to match the number of individual reaction chambers in the array utilized in the sequencing reaction. The number of optical fibers incorporated into the bundle is designed to match the resolution of a detection device so as to allow 1:1 imaging. The overall sizes of the bundles are chosen so as to optimize the usable area of the detection device while maintaining desirable reagent (flow) characteristics in the reaction chamber. Thus, for a 4096Ã4096 pixel CCD (charge-coupled device) array with 15 μm pixels the fiber bundle is chosen to be approximately 60 mmÃ60 mm or to have a diameter of approximately 90 mm. The desired number of optical fibers are initially fused into a bundle or optical fiber array, the terminus of which can then be cut and polished so as to form a âwaferâ of the required thickness (e.g., 1.5 mm). The resulting optical fiber wafers possess similar handling properties to that of a plane of glass. The individual fibers can be any size diameter (e.g., 3 μm to 100 μm).
In some embodiments two fiber optic bundles are used: a first bundle is attached directly to the detection device (also referred to herein as the fiber bundle or connector) and a second bundle is used as the reaction chamber substrate (the wafer or substrate). In this case the two are placed in direct contact, optionally with the use of optical coupling fluid, in order to image the reaction centers onto the detection device. If a CCD is used as the detection device, the wafer could be slightly larger in order to maximize the use of the CCD area, or slightly smaller in order to match the format of a typical microscope slideâ25 mmÃ75 mm. The diameters of the individual fibers within the bundles are chosen so as to maximize the probability that a single reaction will be imaged onto a single pixel in the detection device, within the constraints of the state of the art. Exemplary diameters are 6-8 μm for the fiber bundle and 6-50 μm for the wafer, though any diameter in the range 3-100 μm can be used. Fiber bundles can be obtained commercially from CCD camera manufacturers. In these arrays, typically the distance between the top surface and the bottom surface is no greater than 10 cm, preferably no greater than 3 cm, most preferably no greater than 2 cm, and usually between 0.5 mm to 5 mm. For example, the wafer can be obtained from Incom, Inc. (Charlton, Mass.) and cut and polished from a large fusion of fiber optics, typically being 2 mm thick, though possibly being 0.5 to 5 mm thick. The wafer has handling properties similar to a pane of glass or a glass microscope slide.
Reaction chambers can be formed in the substrate made from fiber optic material. The surface of the optical fiber is cavitated by treating the termini of a bundle of fibers, e.g., with acid, to form an indentation in the fiber optic material. Thus, in one embodiment cavities are formed from a fiber optic bundle, preferably cavities can be formed by etching one end of the fiber optic bundle. Each cavitated surface can form a reaction chamber. Such arrays are referred to herein as fiber optic reactor arrays or FORA. The indentation ranges in depth from approximately one-half the diameter of an individual optical fiber up to two to three times the diameter of the fiber. Cavities can be introduced into the termini of the fibers by placing one side of the optical fiber wafer into an acid bath for a variable amount of time. The amount of time can vary depending upon the overall depth of the reaction cavity desired (see e.g., Walt, et al., 1996. Anal. Chem. 70: 1888). A wide channel cavity can have uniform flow velocity dimensions of approximately 14 mmÃ43 mm. Thus, with this approximate dimension and at approximately 4.82Ã10â4 cavities/um2 density, the apparatus can have approximately 290,000 fluidically accessible cavities. Several methods are known in the art for attaching molecules (and detecting the attached molecules) in the cavities etched in the ends of fiber optic bundles. See, e.g., Michael, et al., Anal. Chem. 70: 1242-1248 (1998); Ferguson, et al., Nature Biotechnology 14: 1681-1684 (1996); Healey and Walt, Anal. Chem. 69: 2213-2216 (1997). A pattern of reactive sites can also be created in the microwell, using photolithographic techniques similar to those used in the generation of a pattern of reaction pads on a planar support. See, Healey, et al., Science 269: 1078-1080 (1995); Munkholm and Walt, Anal. Chem. 58: 1427-1430 (1986), and Bronk, et al., Anal. Chem. 67: 2750-2757 (1995).
The opposing side of the optical fiber wafer (i.e., the non-etched side) is typically highly polished so as to allow optical-coupling (e.g., by immersion oil or other optical coupling fluids) to a second, optical fiber bundle. This second optical fiber bundle exactly matches the diameter of the optical wafer containing the reaction chambers, and serve to act as a conduit for the transmission of light product to the attached detection device, such as a CCD imaging system or camera.
In one preferred embodiment, the fiber optic wafer is thoroughly cleaned, e.g. by serial washes in 15% H2O2/15% NH4OH volume:volume in aqueous solution, then six deionized water rinses, then 0.5M EDTA, then six deionized water, then 15% H2O2/15% NH4OH, then six deionized water (one-half hour incubations in each wash).
The surface of the fiber optic wafer is preferably coated to facilitate its use in the sequencing reactions. A coated surface is preferably optically transparent, allows for easy attachment of proteins and nucleic acids, and does not negatively affect the activity of immobilized proteins. In addition, the surface preferably minimizes non-specific absorption of macromolecules and increases the stability of linked macromolecules (e.g., attached nucleic acids and proteins).
Suitable materials for coating the array include, e.g., plastic (e.g. polystyrene). The plastic can be preferably spin-coated or sputtered (0.1 μm thickness). Other materials for coating the array include gold layers, e.g. 24 karat gold, 0.1 μm thickness, with adsorbed self-assembling monolayers of long chain thiol alkanes. Biotin is then coupled covalently to the surface and saturated with a biotin-binding protein (e.g. streptavidin or avidin).
Coating materials can additionally include those systems used to attach an anchor primer to a substrate. Organosilane reagents, which allow for direct covalent coupling of proteins via amino, sulfhydryl or carboxyl groups, can also be used to coat the array. Additional coating substances include photoreactive linkers, e.g. photobiotin, (Amos et al., âBiomaterial Surface Modification Using Photochemical Coupling Technology,â in Encyclopedic Handbook of Biomaterials and Bioengineering, Part A: Materials, Wise et al. (eds.), New York, Marcel Dekker, pp. 895926, 1995).
Additional coating materials include hydrophilic polymer gels (polyacrylamide, polysaccharides), which preferably polymerize directly on the surface or polymer chains covalently attached post polymerization (Hjerten, J. Chromatogr. 347, 191 (1985); Novotny, Anal. Chem. 62, 2478 (1990), as well as pluronic polymers (triblock copolymers, e.g. PPO-PEO-PPO, also known as F-108), specifically adsorbed to either polystyrene or silanized glass surfaces (Ho et al., Langmuir 14:3889-94, 1998), as well as passively adsorbed layers of biotin-binding proteins. The surface can also be coated with an epoxide which allows the coupling of reagents via an amine linkage.
In addition, any of the above materials can be derivatized with one or more functional groups, commonly known in the art for the immobilization of enzymes and nucleotides, e.g. metal chelating groups (e.g. nitrilo triacetic acid, iminodiacetic acid, pentadentate chelator), which will bind 6ÃHis-tagged proteins and nucleic acids.
Surface coatings can be used that increase the number of available binding sites for subsequent treatments, e.g. attachment of enzymes (discussed later), beyond the theoretical binding capacity of a 2D surface.
In a preferred embodiment, the individual optical fibers utilized to generate the fused optical fiber bundle/wafer are larger in diameter (i.e., 6 μm to 12 μm) than those utilized in the optical imaging system (i.e., 3 μm). Thus, several of the optical imaging fibers can be utilized to image a single reaction site.
Summary of the Arrays of This Invention
In one aspect, the invention involves an array including a planar surface with a plurality of reaction chambers disposed thereon, wherein the reaction chambers have a center to center spacing of between 5 to 200 μm and each chamber has a width in at least one dimension of between 0.3 μm and 100 μm. In some embodiments, the array is a planar surface with a plurality of cavities thereon, where each cavity forms an analyte reaction chamber. In a preferred embodiment, the array is fashioned from a sliced fiber optic bundle (i.e., a bundle of fused fiber optic cables) and the reaction chambers are formed by etching one surface of the fiber optic reactor array (âFORAâ). The cavities can also be formed in the substrate via etching, molding or micromachining.
Specifically, each reaction chamber in the array typically has a width in at least one dimension of between 0.3 μm and 100 μm, preferably between 0.3 μm and 20 μm, mst preferably between 0.3 μm and 10 μm. In a separate embodiment, we contemplate larger reaction chambers, preferably having a width in at least one dimension of between 20 μm and 70 μm.
The array typically contains more than 1,000 reaction chambers, preferably more than 400,000, more preferably between 400,000 and 20,000,000, and most preferably between 1,000,000 and 16,000,000 cavities or reaction chambers. The shape of each cavity is frequently substantially hexagonal, but the cavities can also be cylindrical.. In some embodiments, each cavity has a smooth wall surface, however, we contemplate that each cavity may also have at least one irregular wall surface. The bottom of each of the cavities can be planar or concave.
The array is typically constructed to have cavities or reaction chambers with a center-to-center spacing between 10 to 150 μm, preferably between 50 to 100 μm.
Each cavity or reaction chamber typically has a depth of between 10 μm and 100 μm; alternatively, the depth is between 0.25 and 5 times the size of the width of the cavity, preferably between 0.3 and 1 times the size of the width of the cavity.
In one embodiment, the arrays described herein typically include a planar top surface and a planar bottom surface, which is optically conductive such that optical signals from the reaction chambers can be detected through the bottom planar surface. In these arrays, typically the distance between the top surface and the bottom surface is no greater than 10 cm, preferably no greater than 3 cm, most preferably no greater than 2 cm.
In one embodiment, each cavity of the array contains reagents for analyzing a nucleic acid or protein. The array can also include a second surface spaced apart from the planar array and in opposing contact therewith such that a flow chamber is formed over the array.
In another aspect, the invention involves an array means for carrying out separate parallel common reactions in an aqueous environment, wherein the array means includes a substrate having at least 1,000 discrete reaction chambers. These chambers contain a starting material that is capable of reacting with a reagent. Each of the reaction chambers are dimensioned such that when one or more fluids containing at least one reagent is delivered into each reaction chamber, the diffusion time for the reagent to diffuse out of the well exceeds the time required for the starting material to react with the reagent to form a product. The reaction chambers can be formed by generating a plurality of cavities on the substrate, or by generating discrete patches on a planar surface, the patches having a different surface chemistry than the surrounding planar surface.
In one embodiment, each cavity or reaction chamber of the array contains reagents for analyzing a nucleic acid or protein. Typically those reaction chambers that contain a nucleic acid (not all reaction chambers in the array are required to) contain only a single species of nucleic acid (i.e., a single sequence that is of interest). There may be a single copy of this species of nucleic acid in any particular reaction chamber, or they may be multiple copies. It is generally preferred that a reaction chamber contain at least 100 copies of a nucleic acid sequence, preferably at least 100,000 copies, and most preferably between 100,000 to 1,000,000 copies of the nucleic acid. The ordinarily skilled artisan will appreciate that changes in the number of copies of a nucleic acid species in any one reaction chamber will affect the number of photons generated in a pyrosequencing reaction, and can be routinely adjusted to provide more or less photon signal as is required.
In one embodiment the nucleic acid species is amplified to provide the desired number of copies using PCR, RCA, ligase chain reaction, other isothermal amplification, or other conventional means of nucleic acid amplification. In one embodimant, the nucleic acid is single stranded. In other embodiments the single stranded DNA is a concatamer with each copy covalently linked end to end.
Delivery Means
An example of the means for delivering reactants to the reaction chamber is the perfusion chamber of the present invention is illustrated in FIG. 3 . The perfusion chamber includes a sealed compartment with transparent upper and lower slide. It is designed to allow flow of solution over the surface of the substrate surface and to allow for fast exchange of reagents. Thus, it is suitable for carrying out, for example, the pyrophosphate sequencing reactions. The shape and dimensions of the chamber can be adjusted to optimize reagent exchange to include bulk flow exchange, diffusive exchange, or both in either a laminar flow or a turbulent flow regime.
The correct exchange of reactants to the reaction chamber is important for accurate measurements in the present invention. In the absence of convective flow of bulk fluid, transport of reaction participants (and cross-contamination or âcross-talkâ between adjacent reaction sites or microvessels) can take place only by diffusion. If the reaction site is considered to be a point source on a 2-D surface, the chemical species of interest (e.g., a reaction product) will diffuse radially from the site of its production, creating a substantially hemispherical concentration field above the surface.
The distance that a chemical entity can diffuse in any given time t may be estimated in a crude manner by considering the mathematics of diffusion (Crank, The Mathematics of Diffusion, 2nd ed. 1975). The rate of diffusive transport in any given direction x (cm) is given by Fick's law as j = - D ⢠â C â x Eq . â ⢠1
where j is the flux per unit area (g-mol/cm2-s) of a species with diffusion coefficient D (cm2/s), and âc/âx is the concentration gradient of that species. The mathematics of diffusion are such that a characteristic or âaverageâ distance an entity can travel by diffusion alone scales with the one-half power of both the diffusion coefficient and the time allowed for diffusion to occur. Indeed, to order of magnitude, this characteristic diffusion distance can be estimated as the square root of the product of the diffusion coefficient and timeâas adjusted by a numerical factor of order unity that takes into account the particulars of the system geometry and initial and/or boundary conditions imposed on the diffusion process.
It will be convenient to estimate this characteristic diffusion distance as the root-mean-square distance drms that a diffusing entity can travel in time t: d rms = 2 ⢠Dt Eq . â ⢠2
As stated above, the distance that a diffusing chemical typically travels varies with the square root of the time available for it to diffuseâand inversely, the time required for a diffusing chemical to travel a given distance scales with the square of the distance to be traversed by diffusion. Thus, for a simple, low-molecular-weight biomolecule characterized by a diffusion coefficient D of order 1·10â5 cm2/s, the root-mean-square diffusion distances drms that can be traversed in time intervals of 0.1 s, 1.0 s, 2.0 s, and 10 s are estimated by means of Equation 2 as 14 μm, 45 μm, 63 μm, and 141 μm, respectively.
The relative importance of convection and diffusion in a transport process that involves both mechanisms occurring simultaneously can be gauged with the aid of a dimensionless numberânamely, the Peclet number Pe. This Peclet number can be viewed as a ratio of two rates or velocitiesânamely, the rate of a convective flow divided by the rate of a diffusive âflowâ or flux. More particularly, the Peclet number is a ratio of a characteristic flow velocity V(in cm/s) divided by a characteristic diffusion velocity D/L (also expressed in units of cm/s)âboth taken in the same direction: Pe = VL D Eq . â ⢠3
In Equation 3, V is the average or characteristic speed of the convective flow, generally determined by dividing the volumetric flow rate Q (in cm3/s) by the cross-sectional area A (cm2) available for flow. The characteristic length L is a representative distance or system dimension measured in a direction parallel to the directions of flow and of diffusion (i.e., in the direction of the steepest concentration gradient) and selected to be representative of the typical or âaverageâ distance over which diffusion occurs in the process. And finally D (cm2/s) is the diffusion coefficient for the diffusing species in question. (An alternative but equivalent formulation of the Peclet number Pe views it as the ratio of two characteristic timesânamely, of representative times for diffusion and convection. Equation 3 for the Peclet number can equally well be obtained by dividing the characteristic diffusion time L2/D by the characteristic convection time L/V.)
The convective component of transport can be expected to dominate over the diffusive component in situations where the Peclet number Pe is large compared to unity. Conversely, the diffusive component of transport can be expected to dominate over the convective component in situations where the Peclet number Pe is small compared to unity. In extreme situations where the Peclet number is either very much larger or very much smaller than one, transport may be accurately presumed to occur either by convection or by diffusion alone, respectively. Finally, in situations where the estimated Peclet number is of order unity, then both convection and diffusion can be expected to play significant roles in the overall transport process.
The diffusion coefficient of a typical low-molecular-weight biomolecule will generally be of the order of 10â5 cm2/s (e.g., 0.52·10â5 Cm/s for sucrose, and 1.06·10â5 cm/s for glycine). Thus, for reaction centers, cavities, or wells separated by a distance of 100 μm (i.e.. 0.01 cm), the Peclet number Pe for low-molecular-weight solutes such as these will exceed unity for flow velocities greater than about 10 μm/sec (0.001 cm/s). For cavities separated by only 10 μm (i.e., 0.001 cm), the Peclet number Pe for low-molecular-weight solutes will exceed unity for flow velocities greater than about 100 μm/sec (0.01 cm/s). Convective transport is thus seen to dominate over diffusive transport for all but very slow flow rates and/or very short diffusion distances.
Where the molecular weight of a diffusible species is substantially largerâfor example as it is with large biomolecules like DNA/RNA, DNA fragments, oligonucleotides, proteins, and constructs of the formerâthen the species diffusivity will be corresponding smaller, and convection will play an even more important role relative to diffusion in a transport process involving both mechanisms. For instance, the aqueous-phase diffusion coefficients of proteins fall in about a 10-fold range (Tanford, Physical Chemistry of Macromolecules, 1961). Protein diffusivities are bracketed by values of 1.19Ã10â6 cm2/s for ribonuclease (a small protein with a molecular weight of 13,683 Daltons) and 1.16Ã10â7 cm2/s for myosin (a large protein with a molecular weight of 493,000 Daltons). Still larger entities (e.g., tobacco mosaic virus or TMV at 40.6 million Daltons) are characterized by still lower diffusivities (in particular, 4.6Ã10â8 cm2/s for TMV) (Lehninger, Biochemistry, 2nd ed. 1975). The fluid velocity at which convection and diffusion contribute roughly equally to transport (i.e., Pe of order unity) scales in direct proportion to species diffusivity.
With the aid of the Peclet number formalism it is possible to gauge the impact of convection on reactant supply toâand product removal fromâreaction chambers, cavities or wells. On the one hand, it is clear that even modest convective flows can appreciably increase the speed at which reactants are delivered to the interior of the cavities in an array or FORA. In particular, suppose for the sake of simplicity that the criteria for roughly equal convective and diffusive flows is considered to be Pe=1. One may then estimate that a convective flow velocity of the order of only 0.004 cm/s will suffice to carry reactant into a 25-μm-deep well at roughly the same rate as it could be supplied to the bottom of the well by diffusion alone, given an assumed value for reactant diffusivity of 1Ã10â5 cm2/s. The corresponding flow velocity required to match the rate of diffusion of such a species from the bottom to the top of a 2.5-μm-deep microwell is estimated to be of order 0.04 cm/s. Flow velocities through a FORA much higher than this are possible, thereby illustrating the degree to which a modest convective flow can augment the diffusive supply of reactants to FORA reaction centers, cavities or wells.
The perfusion chamber is preferably detached from the imaging system while it is being prepared and only placed on the imaging system when sequencing analysis is performed. In one embodiment, the solid support (i.e., a DNA chip or glass slide) is held in place by a metal or plastic housing, which may be assembled and disassembled to allow replacement of said solid support. The lower side of the solid support of the perfusion chamber carries the reaction chamber array and, with a traditional optical-based focal system, a high numerical aperture objective lens is used to focus the image of the reaction center array onto the CCD imaging system.
An alternative system for the analysis is to use an array format wherein samples are distributed over a surface, for example a microfabricated chip, and thereby an ordered set of samples may be immobilized in a 2-dimensional format. Many samples can thereby be analyzed in parallel. Using the method of the invention, many immobilized templates may be analyzed in this was by allowing the solution containing the enzymes and one nucleotide to flow over the surface and then detecting the signal produced for each sample. This procedure can then be repeated. Alternatively, several different oligonucleotides complementary to the template may be distributed over the surface followed by hybridization of the template. Incorporation of deoxynucleotides or dideoxynucleotides may be monitored for each oligonucleotide by the signal produced using the various oligonucleotides as primer. By combining the signals from different areas of the surface, sequence-based analyses may be performed by four cycles of polymerase reactions using the various dideoxynucleotides.
When the support is in the form of a cavitated array, e.g., in the termini of a FORA or other array of microwells, suitable delivery means for reagents include flowing and washing and also, e.g., flowing, spraying, electrospraying, ink jet delivery, stamping, ultrasonic atomization (Sonotek Corp., Milton, N.Y.) and rolling. Preferably, all reagent solutions contain 10-20% ethylene glycol to minimize evaporation. When spraying is used, reagents are delivered to the FORA surface in a homogeneous thin layer produced by industrial type spraying nozzles (Spraying Systems. Co., Wheaton, Ill.) or atomizers used in thin layer chromatography (TLC), such as CAMAG TLC Sprayer (Camag Scientific Inc., Wilmington, N.C.). These sprayers atomize reagents into aerosol spray particles in the size range of 0.3 to 10 μm.
Electrospray deposition (ESD) of protein and DNA solutions is currently used to generate ions for mass spectrometric analysis of these molecules. Deposition of charged electrospray products on certain areas of a FORA substrate under control of electrostatic forces is suggested. It was also demonstrated that the ES-deposited proteins and DNA retain their ability to specifically bind antibodies and matching DNA probes, respectively, enabling use of the ESD fabricated matrixes in Dot Immuno-Binding (DIB) and in DNA hybridization assays. (Morozov and Morozova Anal. Chem. 71(15):3110-7 (1999)).
Ink-jet delivery is applicable to protein solutions and other biomacromolecules, as documented in the literature (e.g. Roda et al., Biotechniques 28(3): 492-6 (2000)). It is also commercially available e.g. from MicroFab Technologies, Inc. (Plano, Tex.).
Reagent solutions can alternatively be delivered to the FORA surface by a method similar to lithography. Rollers (stamps; hydrophilic materials should be used) would be first covered with a reagent layer in reservoirs with dampening sponges and then rolled over (pressed against) the FORA surface.
Successive reagent delivery steps are preferably separated by wash steps using techniques commonly known in the art. These washes can be performed, e.g., using the above described methods, including high-flow sprayers or by a liquid flow over the FORA or microwell array surface. The washes can occur in any time period after the starting material has reacted with the reagent to form a product in each reaction chamber but before the reagent delivered to any one reaction chamber has diffused out of that reaction chamber into any other reaction chamber. In one embodiment, any one reaction chamber is independent of the product formed in any other reaction chamber, but is generated using one or more common reagents.
An embodiment of a complete apparatus is illustrated in FIG. 2 . The apparatus includes an inlet conduit 200 in communication with a detachable perfusion chamber 226. The inlet conduit 200 allows for entry of sequencing reagents via a plurality of tubes 202-212, which are each in communication with a plurality of sequencing dispensing reagent vessels 214-224.
Reagents are introduced through the conduit 200 into the perfusion chamber 226 using either a pressurized system or pumps to drive positive flow. Typically, the reagent flow rates are from 0.05 to 50 ml/minute (e.g., 1 to 50 ml/minute) with volumes from 0.100 ml to continuous flow (for washing). Valves are under computer control to allow cycling of nucleotides and wash reagents. Sequencing reagents, e.g., polymerase can be either pre-mixed with nucleotides or added in stream. A manifold brings all six tubes 202-212 together into one for feeding the perfusion chamber. Thus several reagent delivery ports allow access to the perfusion chamber. For example, one of the ports may be utilized to allow the input of the aqueous sequencing reagents, while another port allows these reagents (and any reaction products) to be withdrawn from the perfusion chamber.
The perfusion chamber 226 contains the substrate comprising the plurality of reaction chambers. The perfusion chamber allows for a uniform, linear flow of the required sequencing reagents, in aqueous solution, over the amplified nucleic acids and allows for the rapid and complete exchange of these reagents. Thus, it is suitable for performing pyrophosphate-based sequencing reactions. The perfusion chamber can also be used to prepare the anchor primers and perform amplification reactions, e.g., the RCA reactions described herein.
The invention also provides a method for delivering nucleic acid sequencing enzymes to an array. In some embodiments, one of the nucleic acid sequencing enzymes can be a polypeptide with sulfurylase activity or the nucleic acid sequencing enzyme can be a polypeptide with luciferase activity. In another embodiment, one of the nucleic acid sequencing enzymes can be a polypeptide with both sulfurylase and luciferase activity. In a more preferred embodiment, the reagent can be suitable for use in a nucleic acid sequencing reaction.
In a preferred embodiment, one or more reagents are delivered to an array immobilized or attached to a population of mobile solid supports, e.g., a bead or microsphere. The bead or microsphere need not be spherical, irregular shaped beads may be used. They are typically constructed from numerous substances, e.g., plastic, glass or ceramic and bead sizes ranging from nanometers to millimeters depending on the width of the reaction chamber. Preferably, the diameter of each mobile solid support can be between 0.01 and 0.1 times the width of each cavity. Various bead chemistries can be used e.g., methylstyrene, polystyrene, acrylic polymer, latex, paramagnetic, thoria sol, carbon graphite and titanium dioxide. The construction or chemistry of the bead can be chosen to facilitate the attachment of the desired reagent.
In another embodiment, the bioactive agents are synthesized first, and then covalently attached to the beads. As is appreciated by someone skilled in the art, this will be done depending on the composition of the bioactive agents and the beads. The functionalization of solid support surfaces such as certain polymers with chemically reactive groups such as thiols, amines, carboxyls, etc. is generally known in the art. Accordingly, âblankâ beads may be used that have surface chemistries that facilitate the attachment of the desired functionality by the user. Additional examples of these surface chemistries for blank beads include, but are not limited to, amino groups including aliphatic and aromatic amines, carboxylic acids, aldehydes, amides, chloromethyl groups, hydrazide, hydroxyl groups, sulfonates and sulfates.
These functional groups can be used to add any number of different candidate agents to the beads, generally using known chemistries. For example, candidate agents containing carbohydrates may be attached to an amino-functionalized support; the aldehyde of the carbohydrate is made using standard techniques, and then the aldehyde is reacted with an amino group on the surface. In an alternative embodiment, a sulfhydryl linker may be used. There are a number of sulfhydryl reactive linkers known in the art such as SPDP, maleimides, α-haloacetyls, and pyridyl disulfides (see for example the 1994 Pierce Chemical Company catalog, technical section on cross-linkers, pages 155-200, incorporated here by reference) which can be used to attach cysteine containing proteinaceous agents to the support. Alternatively, an amino group on the candidate agent may be used for attachment to an amino group on the surface. For example, a large number of stable bifunctional groups are well known in the art, including homobifunctional and heterobifunctional linkers (see Pierce Catalog and Handbook, pages 155-200). In an additional embodiment, carboxyl groups (either from the surface or from the candidate agent) may be derivatized using well known linkers (see Pierce catalog). For example, carbodiimides activate carboxyl groups for attack by good nucleophiles such as amines (see Torchilin et al., Critical Rev. Thereapeutic Drug Carrier Systems, 7(4):275-308 (1991)). Proteinaceous candidate agents may also be attached using other techniques known in the art, for example for the attachment of antibodies to polymers; see Slinkin et al., Bioconj. Chem. 2:342-348 (1991); Torchilin et al., supra; Trubetskoy et al., Bioconj. Chem. 3:323-327 (1992); King et al., Cancer Res. 54:6176-6185 (1994); and Wilbur et al., Bioconjugate Chem. 5:220-235 (1994). It should be understood that the candidate agents may be attached in a variety of ways, including those listed above. Preferably, the manner of attachment does not significantly alter the functionality of the candidate agent; that is, the candidate agent should be attached in such a flexible manner as to allow its interaction with a target.
Specific techniques for immobilizing enzymes on beads are known in the prior art. In one case, NH2 surface chemistry beads are used. Surface activation is achieved with a 2.5% glutaraldehyde in phosphate buffered saline (10 mM) providing a pH of 6-9 (138 mM NaCl, 2.7 mM KCl). This mixture is stirred on a stir bed for approximately 2 hours at room temperature. The beads are then rinsed with ultrapure water plus 0.01% Tween 20 (surfactant) â0.02%, and rinsed again with a pH 7.7 PBS plus 0.01 % tween 20. Finally, the enzyme is added to the solution, preferably after being prefiltered using a 0.45 μm amicon micropure filter.
The population of mobile solid supports are disposed in the reaction chambers. In some embodiments, 5% to 20% of the reaction chambers can have a mobile solid support with at least one reagent immobilized thereon, 20% to 60% of the reaction chambers can have a mobile solid support with at least one reagent immobilized thereon or 50% to 100% of the reaction chambers can have a mobile solid support with at least one reagent immobilized thereon. Preferably, at least one reaction chamber has a mobile solid support having at least one reagent immobilized thereon and the reagent is suitable for use in a nucleic acid sequencing reaction.
In some embodiments, the reagent immobilized to the mobile solid support can be a polypeptide with sulfurylase activity, a polypeptide with luciferase activity or a chimeric polypeptide having both sulfurylase and luciferase activity. In one embodiment, it can be a ATP sulfurylase and luciferase fusion protein. Since the product of the sulfurylase reaction is consumed by luciferase, proximity between these two enzymes may be achieved by covalently linking the two enzymes in the form of a fusion protein. This invention would be useful not only in substrate channeling but also in reducing production costs and potentially doubling the number of binding sites on streptavidin-coated beads.
In another embodiment, the sulfurylase is a thermostable ATP sulfurylase. In a preferred embodiment, the thermostable sulfurylase is active at temperatures above ambient (to at least 50° C.). In one embodiment, the ATP sulfurylase is from a thermophile. In an additional embodiment, the mobile solid support can have a first reagent and a second reagent immobilized thereon, the first reagent is a polypeptide with sulfurylase activity and the second reagent is a polypeptide with luciferase activity.
In another embodiment, the reagent immobilized to the mobile solid support can be a nucleic acid; preferably the nucleic acid is a single stranded concatamer. In a preferred embodiment, the nucleic acid can be used for sequencing a nucleic acid, e.g., a pyrosequencing reaction.
The invention also provides a method for detecting or quantifying ATP activity using a mobile solid support; preferably the ATP can be detected or quantified as part of a nucleic acid sequencing reaction.
A FORA that has been âcarpetedâ with mobile solid supports with either nucleic acid or reagent enzymes attached thereto is shown as FIG. 7 .
The solid support is optically linked to an imaging system 230, which includes a CCD system in association with conventional optics or a fiber optic bundle. In one embodiment the perfusion chamber substrate includes a fiber optic array wafer such that light generated near the aqueous interface is transmitted directly through the optical fibers to the exterior of the substrate or chamber. When the CCD system includes a fiber optic connector, imaging can be accomplished by placing the perfusion chamber substrate in direct contact with the connector. Alternatively, conventional optics can be used to image the light, e.g., by using a 1-1 magnification high numerical aperture lens system, from the exterior of the fiber optic substrate directly onto the CCD sensor. When the substrate does not provide for fiber optic coupling, a lens system can also be used as described above, in which case either the substrate or the perfusion chamber cover is optically transparent. An exemplary CCD imaging system is described above.
The imaging system 230 is used to collect light from the reactors on the substrate surface. Light can be imaged, for example, onto a CCD using a high sensitivity low noise apparatus known in the art. For fiber-optic based imaging, it is preferable to incorporate the optical fibers directly into the cover slip or for a FORA to have the optical fibers that form the microwells also be the optical fibers that convey light to the detector.
The imaging system is linked to a computer control and data collection system 240. In general, any commonly available hardware and software package can be used. The computer control and data collection system is also linked to the conduit 200 to control reagent delivery.
The photons generated by the pyrophosphate sequencing reaction are captured by the CCD only if they pass through a focusing device (e.g., an optical lens or optical fiber) and are focused upon a CCD element. However, the emitted photons will escape equally in all directions. In order to maximize their subsequent âcaptureâ and quantitation when utilizing a planar array (e.g., a DNA chip), it is preferable to collect the photons as close as possible to the point at which they are generated, e.g. immediately at the planar solid support. This is accomplished by either: (i) utilizing optical immersion oil between the cover slip and a traditional optical lens or optical fiber bundle or, preferably, (ii) incorporating optical fibers directly into the cover slip itself. Similarly, when a thin, optically transparent planar surface is used, the optical fiber bundle can also be placed against its back surface, eliminating the need to âimageâ through the depth of the entire reaction/perfusion chamber.
Detection Means
The reaction event, e.g., photons generated by luciferase, may be detected and quantified using a variety of detection apparatuses, e.g., a photomultiplier tube, a CCD, CMOS, absorbance photometer, a luminometer, charge injection device (CID), or other solid state detector, as well as the apparatuses described herein. In a preferred embodiment, the quantitation of the emitted photons is accomplished by the use of a CCD camera fitted with a fused fiber optic bundle. In another preferred embodiment, the quantitation of the emitted photons is accomplished by the use of a CCD camera fitted with a microchannel plate intensifier. A back-thinned CCD can be used to increase sensitivity. CCD detectors are described in, e.g., Bronks, et al., 1995. Anal. Chem. 65: 2750-2757.
An exemplary CCD system is a Spectral Instruments, Inc. (Tucson, Ariz.) Series 600 4-port camera with a Lockheed-Martin LM485 CCD chip and a 1-1 fiber optic connector (bundle) with 6-8 μm individual fiber diameters. This system has 4096Ã4096, or greater than 16 million pixels and has a quantum efficiency ranging from 10% to >40%. Thus, depending on wavelength, as much as 40% of the photons imaged onto the CCD sensor are converted to detectable electrons.
In other embodiments, a fluorescent moiety can be used as a label and the detection of a reaction event can be carried out using a confocal scanning microscope to scan the surface of an array with a laser or other techniques such as scanning near-field optical microscopy (SNOM) are available which are capable of smaller optical resolution, thereby allowing the use of âmore denseâ arrays. For example, using SNOM, individual polynucleotides may be distinguished when separated by a distance of less than 100 nm, e.g., 10 nmÃ10 nm. Additionally, scanning tunneling microscopy (Binning et al., Helvetica Physica Acta, 55:726-735, 1982) and atomic force microscopy (Hanswa et al., Annu Rev Biophys Biomol Struct, 23:115-139, 1994) can be used.
The invention provides an apparatus for simultaneously monitoring an array of reaction chambers for light indicating that a reaction is taking place at a particular site. The apparatus can include an array of reaction chambers formed from a planar substrate having a plurality of cavitated surfaces, each cavitated surface forming a reaction chamber adapted to contain analytes. The reaction chambers can have a center-to-center spacing of between 5 to 200 μm and the array can have more than 400,000 discrete reaction chambers. The apparatus can also include an optically sensitive device arranged so that in use the light from a particular reaction chamber will impinge upon a particular predetermined region of said optically sensitive device. The apparatus can further include a means for determining the light level impinging upon each predetermined region and a means to record the variation of said light level with time for each of said reaction chamber.
The invention also provides an analytic sensor, which can include an array formed from a first bundle of optical fibers with a plurality of cavitated surfaces at one end thereof, each cavitated surface forming a reaction chamber adapted to contain analytes. The reaction chambers can have a center-to-center spacing of between 5 to 200 μm and the array can have more than 400,000 discrete reaction chambers. The analytic sensor can also include an enzymatic or fluorescent means for generating light in the reaction chambers. The analytic sensor can further include a light detection means comprising a light capture means and a second fiber optic bundle for transmitting light to the light detecting means. The second fiber optic bundle can be in optical contact with the array, such that light generated in an individual reaction chamber is captured by a separate fiber or groups of separate fibers of the second fiber optic bundle for transmission to the light capture means. The light capture means can be a CCD camera as described herein. The reaction chambers can contain one or more mobile solid supports with a bioactive agent immobilized thereon. In some embodiments, the analytic sensor is suitable for use in a biochemical assay or suitable for use in a cell-based assay.
Methods of Sequencing Nucleic Acids
The invention also provides a method for sequencing nucleic acids which generally comprises (a) providing one or more nucleic acid anchor primers and a plurality of single-stranded circular nucleic acid templates disposed within a plurality of reaction chambers or cavities; (b) annealing an effective amount of the nucleic acid anchor primer to at least one of the single-stranded circular templates to yield a primed anchor primer-circular template complex; (c) combining the primed anchor primer-circular template complex with a polymerase to form an extended anchor primer covalently linked to multiple copies of a nucleic acid complementary to the circular nucleic acid template; (d) annealing an effective amount of a sequencing primer to one or more copies of said covalently linked complementary nucleic acid; (e) extending the sequencing primer with a polymerase and a predetermined nucleotide triphosphate to yield a sequencing product and, if the predetermined nucleotide triphosphate is incorporated onto the 3â² end of said sequencing primer, a sequencing reaction byproduct; and (f) identifying the sequencing reaction byproduct, thereby determining the sequence of the nucleic acid. In one embodiment, the sequencing byproduct is PPi. In another embodiment, a dATP or ddATP analogue is used in place of deoxy- or dideoxy adenosine triphosphate. This analogue is capable of acting as a substrate for a polymerase but incapable of acting as a substrate for a PPi-detection enzyme. This method can be carried out in separate parallel common reactions in an aqueous environment.
In another aspect, the invention includes a method of determining the base sequence of a plurality of nucleotides on an array, which generally comprises (a) providing a plurality of sample DNAs, each disposed within a plurality of cavities on a planar surface; (b) adding an activated nucleotide 5â²-triphosphate precursor of one known nitrogenous base to a reaction mixture in each reaction chamber, each reaction mixture comprising a template-directed nucleotide polymerase and a single-stranded polynucleotide template hybridized to a complementary oligonucleotide primer strand at least one nucleotide residue shorter than the templates to form at least one unpaired nucleotide residue in each template at the 3â²-end of the primer strand, under reaction conditions which allow incorporation of the activated nucleoside 5â²-triphosphate precursor onto the 3â²-end of the primer strands, provided the nitrogenous base of the activated nucleoside 5â²-triphosphate precursor is complementary to the nitrogenous base of the unpaired nucleotide residue of the templates; (c) detecting whether or not the nucleoside 5â²-triphosphate precursor was incorporated into the primer strands in which incorporation of the nucleoside 5â²-triphosphate precursor indicates that the unpaired nucleotide residue of the template has a nitrogenous base composition that is complementary to that of the incorporated nucleoside 5â²-triphosphate precursor; and (d) sequentially repeating steps (b) and (c), wherein each sequential repetition adds and, detects the incorporation of one type of activated nucleoside 5â²-triphosphate precursor of known nitrogenous base composition; and (e) determining the base sequence of the unpaired nucleotide residues of the template in each reaction chamber from the sequence of incorporation of said nucleoside precursors.
In one embodiment of the invention, the anchor primer is linked to a particle. The anchor primer could be linked to the particle prior to or after formation of the extended anchor primer. The sequencing reaction byproduct could be PPi and a coupled sulfurylase/luciferase reaction is used to generate light for detection. Either or both of the sulfurylase and luciferase could be immobilized on one or more mobile solid supports disposed at each reaction site.
In another aspect, the invention involves, a method of determining the base sequence of a plurality of nucleotides on an array. The method includes providing a plurality of sample DNAs, each disposed within a plurality of cavities on a planar surface, each cavity forming an analyte reaction chamber, wherein the reaction chambers have a center to center spacing of between 5 to 200 μm. Then an activated nucleotide 5â²-triphosphate precursor of one known nitrogenous base is added to a reaction mixture in each reaction chamber. Each reaction mixture includes a template-directed nucleotide polymerase and a single-stranded polynucleotide template hybridized to a complementary oligonucleotide primer strand at least one nucleotide residue shorter than the templates to form at least one unpaired nucleotide residue in each template at the 3â²-end of the primer strand, under reaction conditions which allow incorporation of the activated nucleoside 5â²-triphosphate precursor onto the 3â²-end of the primer strands, provided the nitrogenous base of the activated nucleoside 5â²-triphosphate precursor is complementary to the nitrogenous base of the unpaired nucleotide residue of the templates. Then it is detected whether or not the nucleoside 5â²-triphosphate precursor was incorporated into the primer strands in which incorporation of the nucleoside 5â²-triphosphate precursor indicates that the unpaired nucleotide residue of the template has a nitrogenous base composition that is complementary to that of the incorporated nucleoside 5â²-triphosphate precursor. Then these steps are sequentially repeated, wherein each sequential repetition adds and, detects the incorporation of one type of activated nucleoside 5â²-triphosphate precursor of known nitrogenous base composition. The base sequence of the unpaired nucleotide residues of the template in each reaction chamber is then determined from the sequence of incorporation of the nucleoside precursors.
In another aspect, the invention involves a method for determining the nucleic acid sequence in a template nucleic acid polymer. The method includes introducing a plurality of template nucleic acid polymers into a plurality of cavities on a planar surface, each cavity forming an analyte reaction chamber, wherein the reaction chambers have a center to center spacing of between 5 to 200 μm. Each reaction chamber also has a polymerization environment in which the nucleic acid polymer will act as a template polymer for the synthesis of a complementary nucleic acid polymer when nucleotides are added. A series of feedstocks is successively provided to the polymerization environment, each feedstock having a nucleotide selected from among the nucleotides from which the complementary nucleic acid polymer will be formed, such that if the nucleotide in the feedstock is complementary to the next nucleotide in the template polymer to be sequenced the nucleotide will be incorporated into the complementary polymer and inorganic pyrophosphate will be released. Then the formation of inorganic pyrophosphate is detected to determine the identity of each nucleotide in the complementary polymer and thus the sequence of the template polymer.
In another aspect, the invention involves, a method of identifying the base in a target position in a DNA sequence of sample DNA. The method includes providing a sample of DNA disposed within a plurality of cavities on a planar surface, each cavity forming an analyte reaction chamber, wherein the reaction chambers have a center to center spacing of between 5 to 200 μm, the DNA being rendered single stranded either before or after being disposed in the reaction chambers. An extension primer is then provided which hybridizes to the immobilized single-stranded DNA at a position immediately adjacent to the target position. The immobilized single-stranded DNA is subjected to a polymerase reaction in the presence of a predetermined nucleotide triphosphate, wherein if the predetermined nucleotide triphosphate is incorporated onto the 3Ⲡend of the sequencing primer then a sequencing reaction byproduct is formed. The sequencing reaction byproduct is then identified, thereby determining the nucleotide complementary to the base at the target position.
In another aspect, the invention involves a method of identifying a base at a target position in a sample DNA sequence. The method includes providing sample DNA disposed within a plurality of cavities on a planar surface, each cavity forming an analyte reaction chamber, wherein the reaction chambers have a center to center spacing of between 5 to 200 μm, the DNA being rendered single stranded either before or after being disposed in the reaction chambers and providing an extension primer which hybridizes to the sample DNA immediately adjacent to the target position. The sample DNA sequence and the extension primer are then subjected to a polymerase reaction in the presence of a nucleotide triphosphate whereby the nucleotide triphosphate will only become incorporated and release pyrophosphate (PPi) if it is complementary to the base in the target position, the nucleotide triphosphate being added either to separate aliquots of sample-primer mixture or successively to the same sample-primer mixture. The release of PPi is then detected to indicate which nucleotide is incorporated.
In another aspect, the invention involves a method of identifying a base at a target position in a single-stranded sample DNA sequence. The method includes providing an extension primer which hybridizes to sample DNA immediately adjacent to the target position, the sample DNA disposed within a plurality of cavities on a planar surface, each cavity forming an analyte reaction chamber, wherein the reaction chambers have a center to center spacing of between 5 to 200 um, the DNA being rendered single stranded either before or after being disposed in the reaction chambers. The sample DNA and extension primer is subjected to a polymerase reaction in the presence of a predetermined deoxynucleotide or dideoxynucleotide whereby the deoxynucleotide or dideoxynucleotide will only become incorporated and release pyrophosphate (PPi) if it is complementary to the base in the target position, the predetermined deoxynucleotides or dideoxynucleotides being added either to separate aliquots of sample-primer mixture or successively to the same sample-primer mixture. Any release of PPi is detected enzymatically to indicate which deoxynucleotide or dideoxynucleotide is incorporated. Characterized in that, the PPi-detection enzyme(s) are included in the polymerase reaction step and in that in place of deoxy- or dideoxy adenosine triphosphate (ATP) a dATP or ddATP analogue is used which is capable of acting as a substrate for a polymerase but incapable of acting as a substrate for a the PPi-detection enzyme.
In another aspect, the invention involves a method for sequencing a nucleic acid. The method includes providing one or more nucleic acid anchor primers; and a plurality of nucleic acid templates disposed within a plurality of cavities on the above described arrays. An effective amount of the nucleic acid anchor primer is annealed to at least one of the single-stranded circular templates to yield a primed anchor primer-circular template complex. The primed anchor primer-circular template complex is then combined with a polymerase to form an extended anchor primer covalently linked to multiple copies of a nucleic acid complementary to the circular nucleic acid template; followed by annealing of an effective amount of a sequencing primer to one or more copies of the covalently linked complementary nucleic acid. The sequencing primer is then extended with a polymerase and a predetermined nucleotide triphosphate to yield a sequencing product and, if the predetermined nucleotide triphosphate is incorporated onto the 3â² end of the sequencing primer, a sequencing reaction byproduct. Then the sequencing reaction byproduct is identified, thereby determining the sequence of the nucleic acid.
Structure of Anchor Primers
The anchor primers of the invention generally comprise a stalk region and at least one adaptor region. In a preferred embodiment the anchor primer contains at least two contiguous adapter regions. The stalk region is present at the 5â² end of the anchor primer and includes a region of nucleotides for attaching the anchor primer to the solid substrate.
The adaptor region(s) comprise nucleotide sequences that hybridize to a complementary sequence present in one or more members of a population of nucleic acid sequences. In some embodiments, the anchor primer includes two adjoining adaptor regions, which hybridize to complementary regions ligated to separate ends of a target nucleic acid sequence. This embodiment is illustrated in FIG. 1 , which is discussed in more detail below. In additional embodiments, the adapter regions in the anchor primers are complementary to non-contiguous regions of sequence present in a second nucleic acid sequence. Each adapter region, for example, can be homologous to each terminus of a fragment produced by digestion with one or more restriction endonucleases. The fragment can include, e.g., a sequence known or suspected to contain a sequence polymorphism. Additionally, the anchor primer may contain two adapter regions that are homologous to a gapped region of a target nucleic acid sequence, i.e., one that is non-contiguous because of a deletion of one or more nucleotides. When adapter regions having these sequences are used, an aligning oligonucleotide corresponding to the gapped sequence may be annealed to the anchor primer along with a population of template nucleic acid molecules.
The anchor primer may optionally contain additional elements such as one or more restriction enzyme recognition sites, RNA polymerase binding sites, e.g., a T7 promoter site, or sequences present in identified DNA sequences, e.g., sequences present in known genes. The adapter region(s) may also include sequences known to flank sequence polymorphisms. Sequence polymorphisms include nucleotide substitutions, insertions, deletions, or other rearrangements which result in a sequence difference between two otherwise identical nucleic acid sequences. An example of a sequence polymorphism is a single nucleotide polymorphism (SNP).
In general, any nucleic acid capable of base-pairing can be used as an anchor primer. In some embodiments, the anchor primer is an oligonucleotide. As utilized herein the term oligonucleotide includes linear oligomers of natural or modified monomers or linkages, e.g., deoxyribonucleosides, ribonucleosides, anomeric forms thereof, peptide nucleic acids (PNAs), and the like, that are capable of specifically binding to a target polynucleotide by way of a regular pattern of monomer-to-monomer interactions. These types of interactions can include, e.g., Watson-Crick type of base-pairing, base stacking, Hoogsteen or reverse-Hoogsteen types of base-pairing, or the like. Generally, the monomers are linked by phosphodiester bonds, or analogs thereof, to form oligonucleotides ranging in size from, e.g., 3-200, 8-150, 10-100, 20-80, or 25-50 monomeric units. Whenever an oligonucleotide is represented by a sequence of letters, it is understood that the nucleotides are oriented in the 5â²â3â² direction, from left-to-right, and that the letter âAâ donates deoxyadenosine, the letter âTâ denotes thymidine, the letter âCâ denotes deoxycytosine, and the letter âGâ denotes deoxyguanosine, unless otherwise noted herein. The oligonucleotides of the present invention can include non-natural nucleotide analogs. However, where, for example, processing by enzymes is required, or the like, oligonucleotides comprising naturally occurring nucleotides are generally required for maintenance of biological function.
Linking Primers to Solid Substrates
Anchor primers are linked to the solid substrate at the sensitized sites. A region of a solid substrate containing a linked primer is referred to herein as an anchor pad. Thus, by specifying the sensitized states on the solid support, it is possible to form an array or matrix of anchor pads. The anchor pads can be, e.g., small diameter spots etched at evenly spaced intervals on the solid support. The anchor pads can be located at the bottoms of the cavitations or wells if the substrate has been cavitated, etched, or otherwise micromachined as discussed above.
In one embodiment, the anchor primer is linked to a particle. The anchor primer can be linked to the particle prior to formation of the extended anchor primer or after formation of the extended anchor primer.
The anchor primer can be attached to the solid support via a covalent or non-covalent interaction. In general, any linkage recognized in the art can be used. Examples of such linkages common in the art include any suitable metal (e.g., Co2+, Ni2+)-hexahistidine complex, a biotin binding protein, e.g., NEUTRAVIDIN⢠modified avidin (Pierce Chemicals, Rockford, Ill.), streptavidin/biotin, avidin/biotin, glutathione S-transferase (GST)/glutathione, monoclonal antibody/antigen, and maltose binding protein/maltose, and pluronic coupling technologies. Samples containing the appropriate tag are incubated with the sensitized substrate so that zero, one, or multiple molecules attach at each sensitized site.
One biotin-(strept-)avidin-based anchoring method uses a thin layer of a photoactivatable biotin analog dried onto a solid surface. (Hengsakul and Cass, 1996. Bioconjugate Chem. 7: 249-254). The biotin analog is then exposed to white light through a mask, so as to create defined areas of activated biotin. Avidin (or streptavidin) is then added and allowed to bind to the activated biotin. The avidin possesses free biotin binding sites which can be utilized to âanchorâ the biotinylated oligonucleotides through a biotin-(strept-)avidin linkage.
Alternatively, the anchor primer can be attached to the solid support with a biotin derivative possessing a photo-removable protecting group. This moiety is covalently bound to bovine serum albumin (BSA), which is attached to the solid support, e.g., a glass surface. See Pirrung and Huang, 1996. Bioconjugate Chem. 7: 317-321. A mask is then used to create activated biotin within the defined irradiated areas. Avidin may then be localized to the irradiated area, with biotinylated DNA subsequently attached through a BSA-biotin-avidin-biotin link. If desired, an intermediate layer of silane is deposited in a self-assembled monolayer on a silicon dioxide silane surface that can be patterned to localize BSA binding in defined regions. See e.g., Mooney, et al., 1996. Proc. Natl. Acad. Sci. USA 93: 12287-12291.
In pluronic based attachment, the anchor primers are first attached to the termini of a polyethylene oxide-polypropylene oxide-polyethylene oxide triblock copolymer, which is also known as a pluronic compound. The pluronic moiety can be used to attach the anchor primers to a solid substrate. Pluronics attach to hydrophobic surfaces by virtue of the reaction between the hydrophobic surface and the polypropylene oxide. The remaining polyethylene oxide groups extend off the surface, thereby creating a hydrophilic environment. Nitrilotriacetic acid (NTA) can be conjugated to the terminal ends of the polyethylene oxide chains to allow for hexahistidine tagged anchor primers to be attached. In another embodiment, pyridyl disulfide (PDS) can be conjugated to the ends of the polyethylene chains allowing for attachment of a thiolated anchor primer via a disulfide bond. In one preferred embodiment, Pluronic F108 (BASF Corp.) is used for the attachment.
Each sensitized site on a solid support is potentially capable of attaching multiple anchor primers. Thus, each anchor pad may include one or more anchor primers. It is preferable to maximize the number of pads that have only a single productive reaction center (e.g., the number of pads that, after the extension reaction, have only a single sequence extended from the anchor primer). This can be accomplished by techniques which include, but are not limited to: (i) varying the dilution of biotinylated anchor primers that are washed over the surface; (ii) varying the incubation time that the biotinylated primers are in contact with the avidin surface; (iii) varying the concentration of open- or closed-circular template so that, on average, only one primer on each pad is extended to generate the sequencing template; or (iv) reducing the size of the anchor pad to approach single-molecule dimensions (<1 μm) such that binding of one anchor inhibits or blocks the binding of another anchor (e.g. by photoactivation of a small spot); or (v) reducing the size of the anchor pad such that binding of one circular template inhibits or blocks the binding of a second circular template.
In some embodiments, each individual pad contains just one linked anchor primer. Pads having only one anchor primer can be made by performing limiting dilutions of a selected anchor primer on to the solid support such that, on average, only one anchor primer is deposited on each pad. The concentration of anchor primer to be applied to a pad can be calculated utilizing, for example, a Poisson distribution model.
In order to maximize the number of reaction pads that contain a single anchor primer, a series of dilution experiments are performed in which a range of anchor primer concentrations or circular template concentrations are varied. For highly dilute concentrations of primers, primers and circular templates binding to the same pad will be independent of each other, and a Poisson distribution will characterize the number of anchor primers extended on any one pad. Although there will be variability in the number of primers that are actually extended, a maximum of 37% of the pads will have a single extended anchor primer (the number of pads with a single anchor oligonucleotide). This number can be obtained as follows.
Let Np be the average number of anchor primers on a pad and f be the probability that an anchor primer is extended with a circular template. Then the average number of extended anchor primers per pad is Npf, which is defined as the quantity a. There will be variability in the number of primers that are actually extended. In the low-concentration limit, primers and circular templates binding to the same pad will be independent of each other, and a Poisson distribution P(n) will characterize the number of anchor primers n extended on any pad. This distribution may be mathematically defined by: P(n)=(an/n!)exp(âa), with P(1)=a exp(âa). The probability P(1) assumes its maximum value exp(â1) for a=1, with 37% of pads having a single extended anchor primer.
A range of anchor primer concentrations and circular template concentrations may be subsequently scanned to find a value of Npf closest to 1. A preferable method to optimize this distribution is to allow multiple anchor primers on each reaction pad, but use a limiting dilution of circular template so that, on average, only one primer on each pad is extended to generate the sequencing template.
Alternatively, at low concentrations of anchor primers, at most one anchor primer will likely be bound on each reaction pad. A high concentration of circular template may be used so that each primer is likely to be extended.
Where the reaction pads are arrayed on a planar surface or a fiber optic array, the individual pads are approximately 10 μm on a side, with a 100 μm spacing between adjacent pads. Hence, on a 1 cm2 surface a total of approximately 10,000 microreactors could be deposited, and, according to the Poisson distribution, approximately 3700 of these will contain a single anchor primer. In certain embodiments, after the primer oligonucleotide has been attached to the solid support, modified, e.g., biotinylated, enzymes are deposited to bind to the remaining, unused avidin binding sites on the surface.
In other embodiments multiple anchor primers are attached to any one individual pad in an array. Limiting dilutions of a plurality of circular nucleic acid templates (described in more detail below) may be hybridized to the anchor primers so immobilized such that, on average, only one primer on each pad is hybridized to a nucleic acid template. Library concentrations to be used may be calculated utilizing, for example, limiting dilutions and a Poisson distribution model.
Nucleic Acid Templates
The nucleic acid templates that can be sequenced according to the invention, e.g., a nucleic acid library, in general can include open circular or closed circular nucleic acid molecules. A âclosed circleâ is a covalently closed circular nucleic acid molecule, e.g., a circular DNA or RNA molecule. An âopen circleâ is a linear single-stranded nucleic acid molecule having a 5â² phosphate group and a 3â² hydroxyl group. In one embodiment, the single stranded nucleic acid contains at least 100 copies of nucleic acid sequence, each copy covalently linked end to end. In some embodiments, the open circle is formed in situ from a linear double-stranded nucleic acid molecule. The ends of a given open circle nucleic acid molecule can be ligated by DNA ligase. Sequences at the 5â² and 3â² ends of the open circle molecule are complementary to two regions of adjacent nucleotides in a second nucleic acid molecule, e.g., an adapter region of an anchor primer, or to two regions that are nearly adjoining in a second DNA molecule. Thus, the ends of the open-circle molecule can be ligated using DNA ligase, or extended by DNA polymerase in a gap-filling reaction. Open circles are described in detail in Lizardi, U.S. Pat. No. 5,854,033. An open circle can be converted to a closed circle in the presence of a DNA ligase (for DNA) or RNA ligase following, e.g., annealing of the open circle to an anchor primer.
If desired, nucleic acid templates can be provided as padlock probes. Padlock probes are linear oligonucleotides that include target-complementary sequences located at each end, and which are separated by a linker sequence. The linkers can be ligated to ends of members of a library of nucleic acid sequences that have been, e.g., physically sheared or digested with restriction endonucleases. Upon hybridization to a target-sequence, the 5â²- and 3â²-terminal regions of these linear oligonucleotides are brought in juxtaposition. This juxtaposition allows the two probe segments (if properly hybridized) to be covalently-bound by enzymatic ligation (e.g., with T4 DNA ligase), thus converting the probes to circularly-closed molecules which are catenated to the specific target sequences (see e.g., Nilsson, et al., 1994. Science 265: 2085-2088). The resulting probes are suitable for the simultaneous analysis of many gene sequences both due to their specificity and selectivity for gene sequence variants (see e.g., Lizardi, et al., 1998. Nat. Genet. 19: 225-232; Nilsson, et al., 1997. Natl. Genet. 16: 252-255) and due to the fact that the resulting reaction products remain localized to the specific target sequences. Moreover, intramolecular ligation of many different probes is expected to be less susceptible to non-specific cross-reactivity than multiplex PCR-based methodologies where non-cognate pairs of primers can give rise to irrelevant amplification products (see e.g., Landegren and Nilsson, 1997. Ann. Med. 29: 585-590).
A starting library can be constructed comprising either single-stranded or double-stranded nucleic acid molecules, provided that the nucleic acid sequence includes a region that, if present in the library, is available for annealing, or can be made available for annealing, to an anchor primer sequence. For example, when used as a template for rolling circle amplification, a region of a double-stranded template needs to be at least transiently single-stranded in order to act as a template for extension of the anchor primer.
Library templates can include multiple elements, including, but not limited to, one or more regions that are complementary to the anchor primer. For example, the template libraries may include a region complementary to a sequencing primer, a control nucleotide region, and an insert sequence comprised of the sequencing template to be subsequently characterized. As is explained in more detail below, the control nucleotide region is used to calibrate the relationship between the amount of byproduct and the number of nucleotides incorporated. As utilized herein the term âcomplementâ refers to nucleotide sequences that are able to hybridize to a specific nucleotide sequence to form a matched duplex.
In one embodiment, a library template includes: (i) two distinct regions that are complementary to the anchor primer, (ii) one region homologous to the sequencing primer, (iii) one optional control nucleotide region, (iv) an insert sequence of, e.g., 30-500, 50-200, or 60-100 nucleotides, that is to be sequenced. The template can, of course, include two, three, or all four of these features.
The template nucleic acid can be constructed from any source of nucleic acid, e.g., any cell, tissue, or organism, and can be generated by any art-recognized method. Suitable methods include, e.g., sonication of genomic DNA and digestion with one or more restriction endonucleases (RE) to generate fragments of a desired range of lengths from an initial population of nucleic acid molecules. Preferably, one or more of the restriction enzymes have distinct four-base recognition sequences. Examples of such enzymes include, e.g., Sau3A1, MspI, and TaqI. Preferably, the enzymes are used in conjunction with anchor primers having regions containing recognition sequences for the corresponding restriction enzymes. In some embodiments, one or both of the adapter regions of the anchor primers contain additional sequences adjoining known restriction enzyme recognition sequences, thereby allowing for capture or annealing to the anchor primer of specific restriction fragments of interest to the anchor primer. In other embodiments, the restriction enzyme is used with a type IIS restriction enzyme.
Alternatively, template libraries can be made by generating a complementary DNA (cDNA) library from RNA, e.g., messenger RNA (mRNA). The cDNA library can, if desired, be further processed with restriction endonucleases to obtain a 3â² end characteristic of a specific RNA, internal fragments, or fragments including the 3â² end of the isolated RNA. Adapter regions in the anchor primer may be complementary to a sequence of interest that is thought to occur in the template library, e.g., a known or suspected sequence polymorphism within a fragment generated by endonuclease digestion.
In one embodiment, an indexing oligonucleotide can be attached to members of a template library to allow for subsequent correlation of a template nucleic acid with a population of nucleic acids from which the template nucleic acid is derived. For example, one or more samples of a starting DNA population can be fragmented separately using any of the previously disclosed methods (e.g., restriction digestion, sonication). An indexing oligonucleotide sequence specific for each sample is attached to, e.g., ligated to, the termini of members of the fragmented population. The indexing oligonucleotide can act as a region for circularization, amplification and, optionally, sequencing, which permits it to be used to index, or code, a nucleic acid so as to identify the starting sample from which it is derived.
Distinct template libraries made with a plurality of distinguishable indexing primers can be mixed together for subsequent reactions. Determining the sequence of the member of the library allows for the identification of a sequence corresponding to the indexing oligonucleotide. Based on this information, the origin of any given fragment can be inferred.
Annealing and Amplification of Primer-Template Nucleic Acid Complexes
Libraries of nucleic acids are annealed to anchor primer sequences using recognized techniques (see, e.g., Hatch, et al., 1999. Genet. Anal. Biomol. Engineer. 15: 35-40; Kool, U.S. Pat. No. 5,714,320 and Lizardi, U.S. Pat. No. 5,854,033). In general, any procedure for annealing the anchor primers to the template nucleic acid sequences is suitable as long as it results in formation of specific, i.e., perfect or nearly perfect, complementarity between the adapter region or regions in the anchor primer sequence and a sequence present in the template library.
A number of in vitro nucleic acid amplification techniques may be utilized to extend the anchor primer sequence. The size of the amplified DNA preferably is smaller than the size of the anchor pad and also smaller than the distance between anchor pads.
The amplification is typically performed in the presence of a polymerase, e.g., a DNA or RNA-directed DNA polymerase, and one, two, three, or four types of nucleotide triphosphates, and, optionally, auxiliary binding proteins. In general, any polymerase capable of extending a primed 3â²-OH group can be used a long as it lacks a 3â² to 5â² exonuclease activity. Suitable polymerases include, e.g., the DNA polymerases from Bacillus stearothermophilus, Thermus acquaticus, Pyrococcus furiosis, Thermococcus litoralis, and Thermus thermophilus, bacteriophage T4 and T7, and the E. coli DNA polymerase I Klenow fragment. Suitable RNA-directed DNA polymerases include, e.g., the reverse transcriptase from the Avian Myeloblastosis Virus, the reverse transcriptase from the Moloney Murine Leukemia Virus, and the reverse transcriptase from the Human Immunodeficiency Virus-1.
A number of in vitro nucleic acid amplification techniques have been described. These amplification methodologies may be differentiated into those methods: (i) which require temperature cycling-polymerase chain reaction (PCR) (see e.g., Saiki, et al., 1995. Science 230: 1350-1354), ligase chain reaction (see e.g., Barany, 1991. Proc. Natl. Acad. Sci. USA 88: 189-193; Barringer, et al., 1990. Gene 89: 117-122) and transcription-based amplification (see e.g., Kwoh, et al., 1989. Proc. Natl. Acad. Sci. USA 86: 1173-1177) and (ii) isothermal amplification systemsâself-sustaining, sequence replication (see e.g., Guatelli, et al., 1990. Proc. Natl. Acad. Sci. USA 87: 1874-1878); the Qβ replicase system (see e.g., Lizardi, et al., 1988. BioTechnology 6: 1197-1202); strand displacement amplification Nucleic Acids Res. Apr. 11, 1992; 20(7):1691-6.; and the methods described in PNAS Jan. 1, 1992; 89(1):392-6; and NASBA J Virol Methods. December 1991; 35(3):273-86.
Isothermal amplification also includes rolling circle-based amplification (RCA). RCA is discussed in, e.g., Kool, U.S. Pat. No. 5,714,320 and Lizardi, U.S. Pat. No. 5,854,033; Hatch, et al., 1999. Genet. Anal Biomol. Engineer. 15: 35-40. The result of the RCA is a single DNA strand extended from the 3â² terminus of the anchor primer (and thus is linked to the solid support matrix) and including a concatamer containing multiple copies of the circular template annealed to a primer sequence. Typically, 1,000 to 10,000 or more copies of circular templates, each having a size of, e.g., approximately 30-500, 50-200, or 60-100 nucleotides size range, can be obtained with RCA.
The product of RCA amplification following annealing of a circular nucleic acid molecule to an anchor primer is shown schematically in FIG. 1A . A circular template nucleic acid 102 is annealed to an anchor primer 104, which has been linked to a surface 106 at its 5â² end and has a free 3â² OH available for extension. The circular template nucleic acid 102 includes two adapter regions 108 and 110 which are complementary to regions of sequence in the anchor primer 104. Also included in the circular template nucleic acid 102 is an insert 112 and a region 114 homologous to a sequencing primer, which is used in the sequencing reactions described below.
Upon annealing, the free 3â²-OH on the anchor primer 104 can be extended using sequences within the template nucleic acid 102. The anchor primer 102 can be extended along the template multiple times, with each iteration adding to the sequence extended from the anchor primer a sequence complementary to the circular template nucleic acid. Four iterations, or four rounds of rolling circle replication, are shown in FIG. 1A as the extended anchor primer amplification product 114. Extension of the anchor primer results in an amplification product covalently or otherwise physically attached to the substrate 106.
Additional embodiments of circular templates and anchor primers are shown in more detail in FIGS. 1B-1D . FIG. 1B illustrates an annealed open circle linear substrate that can serve, upon ligation, as a template for extension of an anchor primer. A template molecule having the sequence 5â²-TCg TgT gAg gTC TCA gCA TCT TAT gTA TAT TTA CTT CTA TTC TCA gTT gCC TAA gCT gCA gCC A-3â² (SEQ ID NO:1) is annealed to an anchor primer having a biotin linker at its 5â² terminus and the sequence 5â²-gAC CTC ACA CgA Tgg CTg CAg CTT-3â² (SEQ ID NO:2). Annealing of the template results in juxtaposition of the 5â² and 3â² ends of the template molecule. The 3â²OH of the anchor primer can be extended using the circular template.
The use of a circular template and an anchor primer for identification of single nucleotide polymorphisms is shown in FIG. 1C . Shown is a generic anchor primer having the sequence 5â²-gAC CTC ACA CgA Tgg CTg CAg CTT-3â² (SEQ ID NO:3). The anchor primer anneals to an SNP probe having the sequence 5â²-TTT ATA TgT ATT CTA CgA CTC Tgg AgT gTg CTA CCg ACg TCg AAt CCg TTg ACT CTT ATC TTC A-3â² (SEQ ID NO:4). The SNP probe in turn hybridizes to a region of a SNP-containing region of a gene having the sequence 5â²-CTA gCT CgT ACA TAT AAA TgA AgA TAA gAT CCT g -3â² (SEQ ID NO:5). Hybridization of a nucleic acid sequence containing the polymorphism to the SNP probe complex allows for subsequent ligation and circularization of the SNP probe. The SNP probe is designed so that its 5â² and 3â² termini anneal to the genomic region so as to abut in the region of the polymorphic site, as is indicated in FIG. 1C . The circularized SNP probe can be subsequently extended and sequenced using the methods described herein. A nucleic acid lacking the polymorphism does not hybridize so as to result in juxtaposition of the 5â² and 3â² termini of the SNP probe. In this case, the SNP probe cannot be ligated to form a circular substrate needed for subsequent extension.
FIG. 1D illustrates the use of a gap oligonucleotide to along with a circular template molecule. An anchor primer having the sequence 5â²-gAC CTC ACA CgA gTA gCA Tgg CTg CAg CTT-3â² (SEQ ID NO:6) is attached to a surface through a biotin linker. A template molecule having the sequence 5â²-TCg TgT gAg gTC TCA gCA TCT TAT gTA TAT TTA CTT CTA TTC TCA gTT gCC TAA gCT gCA gCC A-3â² (SEQ ID NO:7) is annealed to the anchor primer to result in partially single stranded, or gapped region, in the anchor primer flanked by a double-stranded region. A gapping molecule having the sequence 5â²-TgC TAC-3â² then anneals to the anchor primer. Ligation of both ends of the gap oligonucleotide to the template molecule results in formation of a circular nucleic acid molecule that can act as a template for rolling circle amplification.
Circular oligonucleotides that are generated during polymerase-mediated DNA replication are dependent upon the relationship between the template and the site of replication initiation. In double-stranded DNA templates, the critical features include whether the template is linear or circular in nature, and whether the site of initiation of replication (i.e., the replication âforkâ) is engaged in synthesizing both strands of DNA or only one. In conventional double-stranded DNA replication, the replication fork is treated as the site at which the new strands of DNA are synthesized. However, in linear molecules (whether replicated unidirectionally or bidirectionally), the movement of the replication fork(s) generate a specific type of structural motif. If the template is circular, one possible spatial orientation of the replicating molecule takes the form of a θ structure.
Alternatively, RCA can occur when the replication of the duplex molecule begins at the origin. Subsequently, a nick opens one of the strands, and the free 3â²-terminal hydroxyl moiety generated by the nick is extended by the action of DNA polymerase. The newly synthesized strand eventually displaces the original parental DNA strand. This aforementioned type of replication is known as rolling-circle replication (RCR) because the point of replication may be envisaged as ârolling aroundâ the circular template strand and, theoretically, it could continue to do so indefinitely. Additionally, because the newly synthesized DNA strand is covalently-bound to the original template, the displaced strand possesses the original genomic sequence (e.g., gene or other sequence of interest) at its 5â²-terminus. In RCR, the original genomic sequence is followed by any number of âreplication unitsâ complementary to the original template sequence, wherein each replication unit is synthesized by continuing revolutions of said original template sequence. Hence, each subsequent revolution displaces the DNA which is synthesized in the previous replication cycle.
In vivo, RCR is utilized in several biological systems. For example, the genome of several bacteriophage are single-stranded, circular DNA. During replication, the circular DNA is initially converted to a duplex form, which is then replicated by the aforementioned rolling-circle replication mechanism. The displaced terminus generates a series of genomic units that can be cleaved and inserted into the phage particles. Additionally, the displaced single-strand of a rolling-circle can be converted to duplex DNA by synthesis of a complementary DNA strand. This synthesis can be used to generate the concatemeric duplex molecules required for the maturation of certain phage DNAs. For example, this provides the principle pathway by which λ bacteriophage matures. RCR is also used in vivo to generate amplified rDNA in Xenopus oocytes, and this fact may help explain why the amplified rDNA is comprised of a large number of identical repeating units. In this case, a single genomic repeating unit is converted into a rolling-circle. The displaced terminus is then converted into duplex DNA which is subsequently cleaved from the circle so that the two termini can be ligated together so as to generate the amplified circle of rDNA.
Through the use of the RCA reaction, a strand may be generated which represents many tandem copies of the complement to the circularized molecule. For example, RCA has recently been utilized to obtain an isothermal cascade amplification reaction of circularized padlock probes in vitro in order to detect single-copy genes in human genomic DNA samples (see Lizardi, et al., 1998. Nat. Genet. 19: 225-232). In addition, RCA has also been utilized to detect single DNA molecules in a solid phase-based assay, although difficulties arose when this technique was applied to in situ hybridization (see Lizardi, et al., 1998. Nat. Genet. 19: 225-232).
If desired, RCA can be performed at elevated temperatures, e.g., at temperatures greater than 37° C., 42° C., 45° C., 50° C., 60° C., or 70° C. In addition, RCA can be performed initially at a lower temperature, e.g., room temperature, and then shifted to an elevated temperature. Elevated temperature RCA is preferably performed with thermostable nucleic acid polymerases and with primers that can anneal stably and with specificity at elevated temperatures.
RCA can also be performed with non-naturally occurring oligonucleotides, e.g., peptide nucleic acids. Further, RCA can be performed in the presence of auxiliary proteins such as single-stranded binding proteins.
The development of a method of amplifying short DNA molecules which have been immobilized to a solid support, termed RCA has been recently described in the literature (see e.g., Hatch, et al., 1999. Genet. Anal. Biomol. Engineer. 15: 3540; Zhang, et al., 1998. Gene 211: 277-85; Baner, et al., 1998. Nucl. Acids Res. 26: 5073-5078; Liu, et al., 1995. J. Am. Chem. Soc. 118: 1587-1594; Fire and Xu, 1995. Proc. Natl. Acad. Sci. USA 92: 4641-4645; Nilsson, et al., 1994. Science 265: 2085-2088). RCA targets specific DNA sequences through hybridization and a DNA ligase reaction. The circular product is then subsequently used as a template in a rolling circle replication reaction.
RCA driven by DNA polymerase can replicate circularized oligonucleotide probes with either linear or geometric kinetics under isothermal conditions. In the presence of two primers (one hybridizing to the + strand, and the other, to the â strand of DNA), a complex pattern of DNA strand displacement ensues which possesses the ability to generate 1Ã109 or more copies of each circle in a short period of time (i.e., less-than 90 minutes), enabling the detection of single-point mutations within the human genome. Using a single primer, RCA generates hundreds of randomly-linked copies of a covalently closed circle in several minutes. If solid support matrix-associated, the DNA product remains bound at the site of synthesis, where it may be labeled, condensed, and imaged as a point light source. For example, linear oligonucleotide probes, which can generate RCA signals, have been bound covalently onto a glass surface. The color of the signal generated by these probes indicates the allele status of the target, depending upon the outcome of specific, target-directed ligation events. As RCA permits millions of individual probe molecules to be counted and sorted, it is particularly amenable for the analysis of rare somatic mutations. RCA also shows promise for the detection of padlock probes bound to single-copy genes in cytological preparations.
In addition, a solid-phase RCA methodology has also been developed to provide an effective method of detecting constituents within a solution. Initially, a recognition step is used to generate a complex h a circular template is bound to a surface. A polymerase enzyme is then used to amplify the bound complex. RCA uses small DNA probes that are amplified to provide an intense signal using detection methods, including the methods described in more detail below.
Other examples of isothermal amplification systems include, e.g., (i) self-sustaining, sequence replication (see e.g., Guatelli, et al., 1990. Proc. Natl. Acad. Sci. USA 87: 1874-1878), (ii) the Qβ replicase system (see e.g., Lizardi, et al., 1988. BioTechnology 6: 1197-1202), and (iii) nucleic acid sequence-based amplification (NASBAâ¢; see Kievits, et al., 1991. J. Virol. Methods 35: 273-286).
Methods for Determining the Nucleotide Sequence of the Amplified Product
Amplification of a nucleic acid template as described above results in multiple copies of a template nucleic acid sequence covalently linked to an anchor primer. In one embodiment, a region of the sequence product is determined by annealing a sequencing primer to a region of the template nucleic acid, and then contacting the sequencing primer with a DNA polymerase and a known nucleotide triphosphate, i.e., dATP, dCTP, dGTP, dTFP, or an analog of one of these nucleotides. The sequence can be determined by detecting a sequence reaction byproduct, as is described below.
The sequence primer can be any length or base composition, as long as it is capable of specifically annealing to a region of the amplified nucleic acid template. No particular structure for the sequencing primer is required so long as it is able to specifically prime a region on the amplified template nucleic acid. Preferably, the sequencing primer is complementary to a region of the template that is between the sequence to be characterized and the sequence hybridizable to the anchor primer. The sequencing primer is extended with the DNA polymerase to form a sequence product. The extension is performed in the presence of one or more types of nucleotide triphosphates, and if desired, auxiliary binding proteins.
Incorporation of the dNTP is preferably determined by assaying for the presence of a sequencing byproduct. In a preferred embodiment, the nucleotide sequence of the sequencing product is determined by measuring inorganic pyrophosphate (PPi) liberated from a nucleotide triphosphate (dNTP) as the dNMP is incorporated into an extended sequence primer. This method of sequencing, termed Pyrosequencing⢠technology (PyroSequencing AB, Stockholm, Sweden) can be performed in solution (liquid phase) or as a solid phase technique. PPi-based sequencing methods are described generally in, e.g., WO9813523A1, Ronaghi, et al., 1996. Anal. Biochem. 242: 84-89, and Ronaghi, et al., 1998. Science 281: 363-365 (1998). These disclosures of PPi sequencing are incorporated herein in their entirety, by reference.
Pyrophosphate released under these conditions can be detected enzymatically (e.g., by the generation of light in the luciferase-luciferin reaction). Such methods enable a nucleotide to be identified in a given target position, and the DNA to be sequenced simply and rapidly while avoiding the need for electrophoresis and the use of potentially dangerous radiolabels.
PPi can be detected by a number of different methodologies, and various enzymatic methods have been previously described (see e.g., Reeves, et al., 1969. Anal. Biochem. 28: 282-287; Guillory, et al, 1971. Anal. Biochem. 39: 170-180; Johnson, et al., 1968. Anal. Biochem. 15: 273; Cook, et al., 1978. Anal. Biochem. 91: 557-565; and Drake, et al., 1979. Anal. Biochem. 94: 117-120).
PPi liberated as a result of incorporation of a dNTP by a polymerase can be converted to ATP using, e.g., an ATP sulfurylase. This enzyme has been identified as being involved in sulfur metabolism. Sulfur, in both reduced and oxidized forms, is an essential mineral nutrient for plant and animal growth (see e.g., Schmidt and Jager, 1992. Ann. Rev. Plant Physiol. Plant Mol. Biol. 43: 325-349). In both plants and microorganisms, active uptake of sulfate is followed by reduction to sulfide. As sulfate has a very low oxidation/reduction potential relative to available cellular reductants, the primary step in assimilation requires its activation via an ATP-dependent reaction (see e.g., Leyh, 1993. Crit. Rev. Biochem. Mol. Biol. 28: 515-542). ATP sulfurylase (ATP: sulfate adenylyltransferase; EC 2.7.7.4) catalyzes the initial reaction in the metabolism of inorganic sulfate (SO4 â2); see e.g., Robbins and Lipmann, 1958. J. Biol. Chem. 233: 686-690; Hawes and Nicholas, 1973. Biochem. J. 133: 541-550). In this reaction SO4 â2 is activated to adenosine 5â²-phosphosulfate (APS).
ATP sulfurylase has been highly purified from several sources such as Saccharomyces cereisiae (see e.g., Hawes and Nicholas, 1973. Biochem. J. 133: 541-550); Penicillium chrysogenum (see e.g., Renosto, et al., 1990. J. Biol. Chem. 265: 10300-10308); rat liver (see e.g.. Yu. et al., 1989. Arch. Biochem. Biophys. 269: 165-174); and plants (see e.g., Shaw and Anderson, 1972. Biochem. J. 127: 237-247; Osslund, et al, 1982. Plant Physiol. 70: 39-45). Furthermore, ATP sulfurylase genes have been cloned from prokaryotes (see e.g., Leyh, et al., 1992. J. Biol. Chem. 267: 10405-10410: Schwedock and Long, 1989. Mol. Plant Microbe Interaction 2: 181-194; Laue and Nelson, 1994. J. Bacteriol. 176: 3723-3729); eukaryotes (see e.g., Cherest, et al., 1987. Mol. Gen. Genet. 210: 307-313; Mountain and Korch, 1991. Yeast 7: 873-880; Foster, et al., 1994. J. Biol. Chem. 269: 19777-19786); plants (see e.g., Leustek, et al., 1994. Plant Physiol. 105: 897-90216); and animals (see e.g., Li, et al., 1995. J. Biol. Chem. 270: 29453-29459). The enzyme is a homo-oligomer or heterodimer, depending upon the specific source (see e.g., Leyh and Suo, 1992. J. Biol. Chem. 267: 542-545).
In some embodiments, a thermostable sulfurylase is used. Thermostable sulfurylases can be obtained from, e.g., Archaeoglobus or Pyrococcus spp. Sequences of thermostable sulfurylases are available at database Acc. No. 028606, Acc. No. Q9YCR4, and Acc. No. P56863.
ATP sulfurylase has been used for many different applications, for example, bioluminometric detection of ADP at high concentrations of ATP (see e.g., Schultz, et al., 1993. Anal. Biochem. 215: 302-304); continuous monitoring of DNA polymerase activity (see e.g., Nyrbn, 1987. Anal. Biochem. 167: 235-238); and DNA sequencing (see e.g., Ronaghi, et al., 1996. Anal. Biochem. 242: 84-89; Ronaghi, et al., 1998. Science 281: 363-365; Ronaghi, et al., 1998. Anal. Biochem. 267: 65-71).
Several assays have been developed for detection of the forward ATP sulfurylase reaction. The colorimetric molybdolysis assay is based on phosphate detection (see e.g., Wilson and Bandurski, 1958. J. Biol. Chem. 233: 975-981), whereas the continuous spectrophotometric molybdolysis assay is based upon the detection of NADH oxidation (see e.g., Seubert, et al., 1983. Arch. Biochem. Biophys. 225: 679-691; Seubert, et al., 1985. Arch. Biochem. Biophys. 240: 509-523). The later assay requires the presence of several detection enzymes. In addition, several radioactive assays have also been described in the literature (see e.g., Daley, et al., 1986. Anal. Biochem. 157: 385-395). For example, one assay is based upon the detection of 32PPi released from 32P-labeled ATP (see e.g., Scubert, et al., 1985. Arch. Biochem. Biophys. 240: 509-523) and another on the incorporation of 35S into [35S]-labeled APS (this assay also requires purified APS kinase as a coupling enzyme; see e.g., Scubert, et al., 1983. Arch. Biochem. Biophys. 225: 679-691); and a third reaction depends upon the release of 35SO4 â2 from [35S]-labeled APS (see e.g., Daley, et al., 1986. Anal. Biochem. 157: 385-395).
For detection of the reversed ATP sulfurylase reaction a continuous spectrophotometric assay (see e.g., Segel, et al., 1987. Methods Enzymol. 143: 334-349); a bioluminometric assay (see e.g., Balharry and Nicholas, 1971. Anal. Biochem. 40: 1-17); an 35SO4 â2 release assay (see e.g., Seubert, et al., 1985. Arch. Biochem. Biophys. 240: 509-523); and a 32PPi incorporation assay (see e.g., Osslund, et al., 1982. Plant Physiol. 70: 39-45) have been previously described.
ATP produced by an ATP sulfurylase can be hydrolyzed using enzymatic reactions to generate light. Light-emitting chemical reactions (i.e., chemiluminescence) and biological reactions (i.e., bioluminescence) are widely used in analytical biochemistry for sensitive measurements of various metabolites. In bioluminescent reactions, the chemical reaction that leads to the emission of light is enzyme-catalyzed. For example, the luciferin-luciferase system allows for specific assay of ATP and the bacterial luciferase-oxidoreductase system can be used for monitoring of NAD(P)H. Both systems have been extended to the analysis of numerous substances by means of coupled reactions involving the production or utilization of ATP or NAD(P)H (see e.g., Kricka, 1991. Chemiluminescent and bioluminescent techniques. Clin. Chem. 37: 1472-1281).
The development of new reagents have made it possible to obtain stable light emission proportional to the concentrations of ATP (see e.g., Lundin, 1982. Applications of firefly luciferase In; Luminescent Assays (Raven Press, New York) or NAD(P)H (see e.g., Lovgren, et al., Continuous monitoring of NADH-converting reactions by bacterial luminescence. J. Appl. Biochem. 4: 103-111). With such stable light emission reagents, it is possible to make endpoint assays and to calibrate each individual assay by addition of a known amount of ATP or NAD(P)H. In addition, a stable light-emitting system also allows continuous monitoring of ATP- or NAD(P)H-converting systems.
Suitable enzymes for converting ATP into light include luciferases, e.g., insect luciferases. Luciferases produce light as an end-product of catalysis. The best known light-emitting enzyme is that of the firefly, Photinus pyralis (Coleoptera). The corresponding gene has been cloned and expressed in bacteria (see e.g., de Wet, et al., 1985. Proc. Natl. Acad. Sci. USA 80: 7870-7873) and plants (see e.g., Ow, et al., 1986. Science 234: 856-859), as well as in insect (see e.g., Jha, et al., 1990. FEBS Lett. 274: 24-26) and mammalian cells (see e.g., de Wet, et al., 1987. Mol. Cell. Biol. 7: 725-7373; Keller, et al., 1987. Proc. Natl. Acad. Sci. USA 82: 3264-3268). In addition, a number of luciferase genes from the Jamaican click beetle Pyroplorus plagiophihalamus (Coleoptera), have recently been cloned and partially characterized (see e.g., Wood, et al., 1989. J. Biolumin. Chemilumin. 4: 289-301; Wood, et al., 1989. Science 244: 700-702). Distinct luciferases can sometimes produce light of different wavelengths, which may enable simultaneous monitoring of light emissions at different wavelengths. Accordingly, these aforementioned characteristics are unique, and add new dimensions with respect to the utilization of current reporter systems.
Firefly luciferase catalyzes bioluminescence in the presence of luciferin, adenosine 5â²-triphosphate (ATP), magnesium ions, and oxygen, resulting in a quantum yield of 0.88 (see e.g., McElroy and Selinger, 1960. Arch. Biochem. Biophys. 88: 136-145). The firefly luciferase bioluminescent reaction can be utilized as an assay for the detection of ATP with a detection limit of approximately 1Ã10â13 M (see e.g., Leach, 1981. J. Appl. Biochem. 3: 473-517). In addition, the overall degree of sensitivity and convenience of the luciferase-mediated detection systems have created considerable interest in the development of firefly luciferase-based biosensors (see e.g., Green and Kricka, 1984. Talanta 31: 173-176; Blum, et al., 1989. J. Biolumin. Chemilumin. 4: 543-550).
Using the above-described enzymes, the sequence primer is exposed to a polymerase and a known dNTP. If the dNTP is incorporated onto the 3â² end of the primer sequence, the dNTP is cleaved and a PPi molecule is liberated. The PPi is then converted to ATP with ATP sulfurylase. Preferably, the ATP sulfurylase is present at a sufficiently high concentration that the conversion of PPi proceeds with first-order kinetics with respect to PPi. In the presence of luciferase, the ATP is hydrolyzed to generate a photon. The reaction preferably has a sufficient concentration of luciferase present within the reaction mixture such that the reaction, ATPâADP+PO4 3â+photon (light), proceeds with first-order kinetics with respect to ATP. The photon can be measured using methods and apparatuses described below. In one embodiment, the PPi and a coupled sulfurylase/luciferase reaction is used to generate light for detection. In some embodiments, either or both the sulfurylase and luciferase are immobilized on one or more mobile solid supports disposed at each reaction site.
The present invention thus permits PPi release to be detected during the polymerase reaction giving a real-time signal. The sequencing reactions may be continuously monitored in real-time. A procedure for rapid detection of PPi release is thus enabled by the present invention. The reactions have been estimated to take place in less than 2 seconds (Nyren and Lundin, supra). The rate limiting step is the conversion of PPi to ATP by ATP sulfurylase, while the luciferase reaction is fast and has been estimated to take less than 0.2 seconds Incorporation rates for polymerases have also been estimated by various methods and it has been found, for example, that in the case of Klenow polymerase, complete incorporation of one base may take less than 0.5 seconds. Thus, the estimated total time for incorporation of one base and detection by this enzymatic assay is approximately 3 seconds. It will be seen therefore that very fast reaction times are possible, enabling real-time detection. The reaction times could further be decreased by using a more thermostable luciferase.
For most applications it is desirable to use reagents free of contaminants like ATP and PPi. These contaminants may be removed by flowing the reagents through a pre-column containing apyrase and/-or pyrophosphatase bound to resin. Alternatively, the apyrase or pyrophosphatase can be bound to magnetic beads and used to remove contaminating ATP and PPi present in the reagents. In addition it is desirable to wash away diffusible sequencing reagents, e.g., unincorporated dNTPs, with a wash buffer. Any wash buffer used in pyrophosphate sequencing can be used.
In some embodiments, the concentration of reactants in the sequencing reaction include 1 pmol DNA, 3 pmol polymerase, 40 pmol dNTP in 0.2 ml buffer. See Ronaghi, et al., Anal. Biochem. 242: 84-89 (1996).
The sequencing reaction can be performed with each of four predetermined nucleotides, if desired. A âcompleteâ cycle generally includes sequentially administering sequencing reagents for each of the nucleotides dATP, dGTP, dCTP and dTTP (or dUTP), in a predetermined order. Unincorporated dNTPs are washed away between each of the nucleotide additions. Alternatively, unincorporated dNTPs are degraded by apyrase (see below). The cycle is repeated as desired until the desired amount of sequence of the sequence product is obtained. In some embodiments, about 10-1000, 10-100, 10-75, 20-50, or about 30 nucleotides of sequence information is obtained from extension of one annealed sequencing primer.
In some embodiments, the nucleotide is modified to contain a disulfide-derivative of a hapten such as biotin. The addition of the modified nucleotide to the nascent primer annealed to the anchored substrate is analyzed by a post-polymerization step that includes i) sequentially binding of, in the example where the modification is a biotin, an avidin- or streptavidin-conjugated moiety linked to an enzyme molecule, ii) the washing away of excess avidin- or streptavidin-linked enzyme, iii) the flow of a suitable enzyme substrate under conditions amenable to enzyme activity, and iv) the detection of enzyme substrate reaction product or products. The hapten is removed in this embodiment through the addition of a reducing agent. Such methods enable a nucleotide to be identified in a given target position, and the DNA to be sequenced simply and rapidly while avoiding the need for electrophoresis and the use of potentially dangerous radiolabels.
A preferred enzyme for detecting the hapten is horse-radish peroxidase. If desired, a wash buffer, can be used between the addition of various reactants herein. Apyrase can be used to remove unreacted dNTP used to extend the sequencing primer. The wash buffer can optionally include apyrase.
Example haptens, e.g., biotin, digoxygenin, the fluorescent dye molecules cy3 and cy5, and fluorescein, are incorporated at various efficiencies into extended DNA molecules. The attachment of the hapten can occur through linkages via the sugar, the base, and via the phosphate moiety on the nucleotide. Example means for signal amplification include fluorescent, electrochemical and enzymatic. In a preferred embodiment using enzymatic amplification, the enzyme, e.g. alkaline phosphatase (AP), horse-radish peroxidase (HRP), beta-galactosidase, luciferase, can include those for which light-generating substrates are known, and the means for detection of these light-generating (chemiluminescent) substrates can include a CCD camera.
In a preferred mode, the modified base is added, detection occurs, and the hapten-conjugated moiety is removed or inactivated by use of either a cleaving or inactivating agent. For example, if the cleavable-linker is a disulfide, then the cleaving agent can be a reducing agent, for example dithiothreitol (DTT), beta-mercaptoethanol, etc. Other embodiments of inactivation include heat, cold, chemical denaturants, surfactants, hydrophobic reagents, and suicide inhibitors.
Luciferase can hydrolyze dATP directly with concomitant release of a photon. This results in a false positive signal because the hydrolysis occurs independent of incorporation of the dATP into the extended sequencing primer. To avoid this problem, a dATP analog can be used which is incorporated into DNA, i.e., it is a substrate for a DNA polymerase, but is not a substrate for luciferase. One such analog is α-thio-dATP. Thus, use of α-thio-dATP avoids the spurious photon generation that can occur when dATP is hydrolyzed without being incorporated into a growing nucleic acid chain.
Typically, the PPi-based detection is calibrated by the measurement of the light released following the addition of control nucleotides to the sequencing reaction mixture immediately after the addition of the sequencing primer. This allows for normalization of the reaction conditions. Incorporation of two or more identical nucleotides in succession is revealed by a corresponding increase in the amount of light released. Thus, a two-fold increase in released light relative to control nucleotides reveals the incorporation of two successive dNTPs into the extended primer.
If desired, apyrase may be âwashedâ or âflowedâ over the surface of the solid support so as to facilitate the degradation of any remaining, non-incorporated dNTPs within the sequencing reaction mixture. Apyrase also degrades the generated ATP and hence âturns offâ the light generated from the reaction. Upon treatment with apyrase, any remaining reactants are washed away in preparation for the following dNTP incubation and photon detection steps. Alternatively, the apyrase may be bound to the solid or mobile solid support.
When the support is planar, the pyrophosphate sequencing reactions preferably take place in a thin reaction chamber that includes one optically transparent solid support surface and an optically transparent cover. In some embodiments, the array has a planar top surface and a planar bottom surface, the planar top surface has at least 1,000 cavities thereon each cavity forming a reaction chamber. In additional embodiments, the planar bottom surface is optically conductive such that optical signals from the reaction chambers can be detected through the bottom planar surface. In a preferred embodiment, the distance between the top surface and the bottom surface is no greater than 10 cm. Sequencing reagents may then be delivered by flowing them across the surface of the substrate. More preferably, the cavities contain reagents for analyzing a nucleic acid or protein. In an additional embodiment, the array has a second surface spaced apart from the planar array and in opposing contact therewith such that a flow chamber is formed over the array. When the support is not planar, the reagents may be delivered by dipping the solid support into baths of any given reagents.
In a preferred embodiment, an array can be used to carry out separate parallel common reactions in an aqueous environment. The array can have a substrate having at least 1,000 discrete reaction chambers containing a starting material that is capable of reacting with a reagent, each of the reaction chambers being dimensioned such that when one or more fluids containing at least one reagent is delivered into each reaction chamber, the diffusion time for the reagent to diffuse out of the well exceeds the time required for the starting material to react with the reagent to form a product. The reaction chambers can be formed by generating a plurality of cavities on the substrate. The plurality of cavities can be formed in the substrate via etching, molding or micromaching. The cavities can have a planar bottom or a concave bottom. In a preferred embodiment, the substrate is a fiber optic bundle. In an additional embodiment, the reaction chambers are formed by generating discrete patches on a planar surface. The patches can have a different surface chemistry than the surrounding planar surface.
In various embodiments, some components of the reaction are immobilized, while other components are provided in solution. For example, in some embodiments, the enzymes utilized in the pyrophosphate sequencing reaction (e.g., sulfurylase, luciferase) may be immobilized if desired onto the solid support. Similarly, one or more or of the enzymes utilized in the pyrophosphate sequencing reaction, e.g., sulfurylase, luciferase may be immobilized at the termini of a fiber optic reactor array. When luciferase is immobilized, it is preferably less than 50 μm from an anchored primer. Other components of the reaction, e.g., a polymerase (such as Klenow fragment), nucleic acid template, and nucleotides can be added by flowing, spraying, or rolling. In still further embodiments, one more of the reagents used in the sequencing reactions is delivered on beads.
In some embodiments, reagents are dispensed using an expandable, flexible membrane to dispense reagents and seal reactors on FORA surface during extension reactions. Reagents can be sprayed or rolled onto either the FORA surface or onto the flexible membrane. The flexible membrane could then be either rapidly expanded or physically moved into close proximity with the FORA thereby sealing the wells such that PPi would be unable to diffuse from well to well. Preferably, data acquisition takes place at a reasonable time after reaction initiation to allow maximal signal to generate.
A sequence in an extended anchor primer can also be identified using sequencing methods other than by detecting a sequence byproduct. For example, sequencing can be performed by measuring incorporation of labeled nucleotides or other nucleotide analogs. These methods can be used in conjunction with fluorescent or electrochemiluminescent-based methods.
Alternatively, sequence byproducts can be generated using dideoxynucleotides having a label on the 3â² carbon. Preferably, the label can be cleaved to reveal a 3â² hydroxyl group. In this method, addition of a given nucleotide is scored as positive or negative, and one base is determined at each trial. In this embodiment, solid phase enzymes are not required and multiple measurements can be made.
In another embodiment, the identity of the extended anchor primer product is determined using labeled deoxynucleotides. The labeled deoxynucleotides can be, e.g., fluorescent nucleotides. Preferably the fluorescent nucleolides can be detected following laser-irradiation. Preferably, the fluorescent label is not stable for long periods of exposure. If desired, the fluorescent signal can be quenched, e.g., photobleached, to return signal to background levels prior to addition of the next base. A preferred electrochemiluminescent label is ruthenium-tris-bi-pyridyl.
In one embodiment, a single stranded circular nucleic acid is immobilized in the reaction chamber; preferably each reaction chamber has no more than one single stranded circular nucleic acid disposed therein. More preferably, a single stranded circular nucleic acid is immobilized on a mobile solid support disposed in the reaction chamber. In another embodiment, each single stranded circular nucleic acid contains at least 100 copies of a nucleic acid sequence, each copy covalently linked end to end.
The invention also comprises kits for use in methods of the invention which could include one or more of the following components: (a) a test specific primer which hybridizes to sample DNA so that the target position is directly adjacent to the 3â² end of the primer; (b) a polymerase; (c) detection enzyme means for identifying PPi release; (d) deoxynucleotides including, in place of dATP, a dATP analogue which is capable of acting as a substrate for a polymerase but incapable of acting as a substrate for a said PPi-detection enzyme; and (e) optionally dideoxynucleotides, optionally ddATP being replaced by a ddATP analogue which is capable of acting as a substrate for a polymerase but incapable of acting as a substrate for a said PPi-detection enzyme. If the kit is for use with initial PCR amplification then it could also include the following components: (i) a pair of primers for PCR, at least one primer having means permitting immobilization of said primer; (ii) a polymerase which is preferably heat stable, for example Taq1 polymerase; (iii) buffers for the PCR reaction; and (iv) deoxynucleotides. Where an enzyme label is used to evaluate PCR, the kit will advantageously contain a substrate for the enzyme and other components of a detection system.
Mathematical Analysis Underlying Optimization of the Pyrophosphate Sequencing Reaction
While not wishing to be bound by theory, it is believed that optimization of reaction conditions can be performed using assumptions underlying the following analyses.
Solid-phase pyrophosphate sequencing was initially developed by combining a solid-phase technology and a sequencing-by-synthesis technique utilizing bioluminescence (see e.g., Ronaghi, et al., 1996. Real-time DNA sequencing using detection of pyrophosphate release. Anal. Biochem. 242: 84-89). In the solid-phase methodology, an immobilized, primed DNA strand is incubated with DNA polymerase, ATP sulfurylase, and luciferase. By stepwise nucleotide addition with intermediate washing, the event of sequential polymerization can be followed. The signal-to-noise ratio was increased by the use of α-thio dATP in the system. This dATP analog is efficiently incorporated by DNA polymerase but does not serve as a substrate for luciferase. This reduces background bioluminescence and facilitates performance of the sequencing reaction in real-time. In these early studies, sequencing of a PCR product using streptavidin-coated magnetic beads as a solid support was presented. However, it was found that the loss of the beads during washing, which was performed between each nucleotide and enzyme addition, limited the technique to short sequences.
Currently, pyrophosphate sequencing methodologies have a reasonably well-established history for ascertaining the DNA sequence from many identical copies of a single DNA sequencing template (see e.g., Ronaghi, et al., 1996. Real-Time DNA Sequencing Using Detection of Pyrophosphate Release, Anal. Biochem. 242: 84-89; Nyrén, et al., Method of Sequencing DNA, patent WO9813523A1 (issued Apr. 2, 1998; filed Sep. 26, 1997); Ronaghi, et al., 1998. A Sequencing Method Based on Real-Time Pyrophosphate Science 281: 363-365 (1998). Pyrophosphate (PPi)-producing reactions can be monitored by a very sensitive technique based on bioluminescence (see e.g., Nyrén, et al., 1996. pp. 466-496 (Proc. 9th Inter. Symp. Biolumin. Chemilumin.). These bioluminometric assays rely upon the detection of the PPi released in the different nucleic acid-modifying reactions. In these assays, the PPi which is generated is subsequently converted to ATP by ATP sulfurylase and the ATP production is continuously monitored by luciferase. For example, in polymerase-mediated reactions, the PPi is generated when a nucleotide is incorporated into a growing nucleic acid chain being synthesized by the polymerase. While generally, a DNA polymerase is utilized to generate PPi during a pyrophosphate sequencing reaction (see e.g., Ronaghi, et al., 1998. Doctoral Dissertation, The Royal Institute of Technology, Dept. of Biochemistry (Stockholm, Sweden)), it is also possible to use reverse transcriptase (see e.g., Karamohamamed, et al., 1996. pp. 319-329 (Proc. 9th Inter. Symp. Biolumin. Chemilumin.) or RNA polymerase (see e.g., Karamohamamed, et al., 1998. BioTechniques 24: 302-306) to follow the polymerization event.
For example, a bioluminometric primer extension assay has been utilized to examine single nucleotide mismatches at the 3â²-terminus (see e.g., Nyrén, et al., 1997. Anal. Biochem. 244: 367-373). A phage promoter is typically attached onto at least one of the arbitrary primers and, following amplification, a transcriptional unit may be obtained which can then be subjected to stepwise extension by RNA polymerase. The transcription-mediated PPi-release can then be detected by a bioluminometric assay (e.g., ATP sulfurylase-luciferase). By using this strategy, it is likely to be possible to sequence double-stranded DNA without any additional specific sequencing primer. In a series of ârun-offâ assays. the extension by T7 phage RNA polymerase has been examined and was found to be rather slow (see e.g., Kwok, et al., 1990. Nucl. Acids Res. 18: 999-1005). The substitution of an α-thio nucleotide analogs for the subsequent, correct natural deoxynucleotide after the 3â²-mismatch termini, could decrease the rate of polymerization by 5-fold to 13-fold. However, after incorporation of a few bases, the rate of DNA synthesis is comparable with the rate observed for a normal template/primer.
Single-base detection by this technique has been improved by incorporation of apyrase to the system, which catalyzes NTP hydrolysis and reduces the nucleotide concentration far below the Km of DNA polymerase, effectively removing dNTP from a preceding step before proceeding to addition of the subsequent dNTP. The above-described technique provides a rapid and real-time analysis for applications in the areas of mutation detection and single-nucleotide polymorphism (SNP) analysis.
The pyrophosphate sequencing system uses reactions catalyzed sequentially by several enzymes to monitor DNA synthesis. Enzyme properties such as stability, specificity, sensitivity, K
Mand k
CATare important for the optimal performance of the system. In the pyrophosphate sequencing system, the activity of the detection enzymes (i.e., sulfurylase and luciferase) generally remain constant during the sequencing reaction, and are only very slightly inhibited by high amounts of products (see e.g., Ronaghi, et al., 1998.
Doctoral Dissertation,The Royal Institute of Technology, Dept. of Biochemistry (Stockholm, Sweden)). Sulfurylase converts each PPi to ATP in approximately 2.0 seconds (see e.g., Nyrén and Lundin, 1985.
Anal. Biochem.151: 504-509). The reported reaction conditions for 1 pmol PPi in 0.2 ml buffer (5 nM) are 0.3 U/ml ATP sulfurylase (ATP:sulfate adenylyltransferase; Prod. No. A8957; Sigma Chemical Co., St. Louis, Mo.) and 5 μM APS (see e.g., Ronaghi, et al., 1996. Real-Time DNA Sequencing Using Detection of Pyrophosphate Release,
Anal. Biochem.242: 84-89). The manufacturer's information (Sigma Chemical Co., St. Louis, Mo.) for sulfurylase sports an activity of 5-20 units per mg protein (i.e., one unit will produce 1.0 μmole of ATP from APS and PPi per minute at pH 8.0 at 30 C). whereas the specific activity has been reported elsewhere as 140 units per mg (see Karamohamed, et al., 1999. Purification, and Luminometric Analysis of Recombinant
Saccharomyces cerevisiaeMET3 Adenosine Triphosphate Sulfurylase Expressed in
Escherichia coli. Prot. Express. Purification15: 381-388). Due to the fact that the reaction conditions utilized in the practice of the present invention are similar to those reaction conditions reported in the aforementioned reference, the sulfurylase concentration within the assay was estimated as 4.6 nM. The K
Mvalues for sulfurylase are [APS]=0.5 μM and [PPi]=7 μM. The generation of light by luciferase takes place in less than 0.2 seconds. The most critical reactions are the DNA polymerization and the degradation of nucleotides. The value of constants characterizing the enzymes utilized in the pyrophosphate sequencing methodology are listed below for reference:
Enzyme KM (μM) kCAT (Sâ1) Klenow 0.18 (dTTP) 0.92 T7 DNA Polymerase 0.36 (dTTP) 0.52 ATP Sulfurylase 0.56 (APS); 7.0 (PPi) 38 Firefly Luciferase ââ20 (ATP) 0.015 Apyrase â120 (ATP); 260 (ADP) 500 (ATP)The enzymes involved in these four reactions compete for the same substrates. Therefore, changes in substrate concentrations are coupled. The initial reaction is the binding of a dNTP to a polymerase/DNA complex for chain elongation. For this step to be rapid, the nucleotide triphosphate concentration must be above the KM of the DNA polymerase. If the concentration of the nucleotide triphosphates is too high, however, lower fidelity of the polymerase may be observed (see e.g., Cline, et al., 1996. PCR fidelity of Pfu DNA polymerase and other thermostable DNA polymerases. Nucl. Acids Res. 24: 3546-3551). A suitable range of concentrations is established by the KM for the misincorporation, which is usually much higher (see e.g., Capson, et al., 1992. Kinetic characterization of the polymerase and exonuclease activity of the gene 43 protein of bacteriophage T4. Biochemistry 31: 10984-10994). Although a very high fidelity can be achieved by using polymerases with inherent exonuclease activity, their use also holds the disadvantage that primer degradation may occur.
Although the exonuclease activity of the Klenow fragment of DNA polymerase I (Klenow) is low, it has been demonstrated that the 3â²-terminus of a primer may be degraded with longer incubations in the absence of nucleotide triphosphates (see e.g., Ronaghi, et al., 1998. Doctoral Dissertation, The Royal Institute of Technology, Dept. of Biochemistry (Stockholm, Sweden)). Fidelity is maintained without exonuclease activity because an induced-fit binding mechanism in the polymerization step provides a very efficient selectivity for the correct dNTP. Fidelities of 1Ã105 to 1Ã106 have been reported (see e.g., Wong, et al., 1991. An induced-fit kinetic mechanism for DNA replication fidelity. Biochemistry 30: 526-537). In pyrophosphate sequencing, exonuclease-deficient (exo-) polymerases, such as exo-Klenow or Sequenase®, have been confirmed to have high fidelity.
Estimates for the spatial and temporal constraints on the pyrophosphate sequencing methodology of the present invention have been calculated, wherein the system possesses a 1 cm2 area with height approximately 50 μm, for a total volume of 5 μl. With respect to temporal constraints, the molecular species participating in the cascade of reactions are initially defined, wherein:
It is further specified that N(0) is the DNA with no nucleotides added, N(1) has 1 nucleotide added, N(2) has 2 nucleotides added, and so on. The pseudo-first-order rate constants which relate the concentrations of molecular species are:
N(n)âN(n+1)+PPi ââkN
PPiâATP ââkP
ATPâL ââkA
In addition, the diffusion constants D
Pfor PPi and D
Afor ATP must also be specified. These values may be estimated from the following exemplar diffusion constants for biomolecules in a dilute water solution (see Weisiger, 1997. Impact of Extracellular and Intracellular Diffusion on Hepatic Uptake Kinetics).
Molecule D/10â5 cm2/sec Method Original Reference Albumin 0.066 lag time 1 Albumin 0.088 light scattering 2 Water 1.940 NMR 3wherein,
Original Reference1 is: Longsworth, 1954. Temperature dependence of diffusion in aqueous solutions,
J. Phys. Chem.58: 770-773;
Original Reference2 is: Gaigalas, et al., 1992. Diffusion of bovine serum albumin in aqueous solutions,
J. Phys. Chem.96: 2355-2359; and
Original Reference3 is: Cheng, 1993. Quantitation of non-Einstein diffusion behavior of water in biological tissues by proton NMR diffusion imaging: Synthetic image calculations,
Magnet. Reson. Imaging11: 569-583.
In order to estimate the diffusion constant of PPi, the following exemplar values may be utilized (see
CRC Handbook of Chemistry and Physics,1983. (W. E. Weast. Ed.) CRC Press, Inc., Boca Raton, Fla.):
Molecule D/10â5 cm2/sec Molecular Weight/amu sucrose 0.5226 342.30 mannitol 0.682 182.18 penta-erythritol 0.761 136.15 glycolamide 1.142 N/A glycine 1.064 75.07The molecular weight of PPi is 174 amu. Based upon the aforementioned exemplar values, a diffusion constant of approximately 0.7Ã10â5 cm2/sec for PPi is expected.
Enzymes catalyzing the three pyrophosphate sequencing reactions are thought to approximate Michaelis-Menten kinetics (see e.g. Stryer, 1988. Biochemistry, W. H. Freeman and Company, New York), which may be described:
K M =[E][S]/[ES],
velocity=V max [S]/(K M +[S]),
V max =k CAT [E T]
where [S] is the concentration of substrate, [E] is the concentration of free enzyme, [ES] is the concentration of the enzyme-substrate complex, and [ET] is the total concentration of enzyme=[E]+[ES].
It is preferable that the reaction times are at least as fast as the solution-phase pyrophosphate-based sequencing described in the literature. That rate that a substrate is converted into product is
âd[S]/dt=k CAT [E T ][S]/(K M +[S])
The effective concentration of substrate may be estimated from the size of a replicated DNA molecule, at most (10 μm)3 and the number of copies (approximately 10,000), yielding a concentration of approximately 17 nM. This is this is smaller than the KM for the enzymes described previously, and therefore the rate can be estimated to be
âd[S]/dt=(k CAT /K M)[E T ][S].
Thus, with pseudo first-order kinetics, the rate constant for disappearance of substrate depends on kCAT and KM, which are constants for a given enzyme, and [ET]. Using the same enzyme concentrations reported in the literature will therefore produce similar rates.
The first step in the pyrophosphate sequencing reaction (i.e., incorporation of a new nucleotide and release of PPi) will now be examined in detail. The preferred reaction conditions are: 1 pmol DNA, 3 pmol polymerase, 40 pmol dNTP in 0.2 ml buffer. Under the aforementioned, preferred reaction conditions, the KM for nucleotide incorporation for the Klenow fragment of DNA polymerase I is 0.2 μM and for Sequenase 2.0⢠(US Biochemicals, Cleveland, Ohio) is 0.4 μM, and complete incorporation of 1 base is less than 0.2 sec (see e.g., Ronaghi, et al., 1996. Real-Time DNA Sequencing Using Detection of Pyrophosphate Release, Anal. Biochem. 242: 84-89) with a polymerase concentration of 15 nM.
In a 5 μl reaction volume, there are a total of 10,000 anchor primers with 10,000 sequencing primer sites each, or 1Ã108 total extension sites=0.17 fmol. Results which have been previously published in the literature suggest that polymerase should be present at 3-times abundance, or 0.5 fmol, within the reaction mixture. The final concentration of polymerase is then 0.1 nM. It should be noted that these reaction conditions are readily obtained in the practice of tic present invention.
As previously stated, the time required for the nucleotide addition reaction is no greater than 0.2 sec per nucleotide. Hence, if tie reaction is allowed to proceed for a total of T seconds, then nucleotide addition should be sufficiently rapid that stretches of up to (T/0.2) identical nucleotides should be completely filled-in by the action of the polymerase. As discussed previously, the rate-limiting step of the pyrophosphate sequencing reaction is the sulfurylase reaction, which requires a total of approximately 2 sec to convert one PPi to ATP. Accordingly, a total reaction time which allows completion of the sulfurylase reaction, should be sufficient to allow the polymerase to âfill-inâ stretches of up to 10 identical nucleotides. In random DNA species, regions of 10 or more identical nucleotides have been demonstrated to occur with a per-nucleotide probability of approximately 4â10, which is approximately 1Ã10â6. In the 10,000 sequences which are extended from anchor primers in a preferred embodiment of the present invention, each of which will be extended at least 30 nucleotides and preferably 100 nucleotides, it is expected that approximately one run of 10 identical nucleotides will be present. Thus, it may be concluded that runs of identical nucleotides should not pose a difficulty in the practice of the present invention.
The overall size of the resulting DNA molecule is, preferably, smaller than the size of the anchoring pads (i.e., 10 μm) and must be smaller than the distance between the individual anchoring pads (i.e., 100 μm). The radius of gyration of a single-stranded DNA concatamer with N total nucleotides may be mathematically-estimated by the following equation: radius=b (N/N0)0.6, where b is the persistence length and N0 is the number of nucleotides per persistence length; the exponent 0.6 is characteristic of a self-avoiding walk (see e.g., Doi, 1986. The Theory of Polymer Dynamics (Clarendon Press, New York); Flory, 1953. Principles of Polymer Chemistry (Cornell University Press, New York)). Using single-stranded DNA as an example, b is 4 nm and N0 is 13.6 nucleotides. (see e.g., Grosberg, 1994. Statistical Physics of Macromolecules (AIP Press, New York)). Using 10,000 copies of a 100-mer, N=1Ã106 and the radius of gyration is 3.3 μm.
The diffusion of PPi will now be discussed in detail. In the reaction conditions utilized in the present invention, [PPi] is approximately 0.17 fmol in 5 μl, or 0.03 nM, and [sulfurylase] is 4.6 nM as described previously. In the first 2 sec of the reaction, about 7% (0.002 nM) of PPi is consumed by sulfurylase, using GEPASI simulation software (see Mendes, P. (1993) GEPASI: a software package for modeling the dynamics, steady states and control of biochemical and other systems. Comput. Appl. Biosci. 9, 563-571.). The parameters used in simulation were KM(PPi)=7 μM, kCATâ38 sâ1, and [sulfurylase]=4.6 nM. Therefore, it may be concluded that at least 93% of PPi molecules may diffuse away before being converted to ATP (during the 2 sec reaction time.
The mean time for each PPi to react is 1/kP=2 seconds. The mean square distance it diffuses in each direction is approximately 2DP/kP, or 2.8Ã103 μm2. The RMS distance in each direction is 53 μm. This value indicates that each of the individual anchor primers must be more than 50 μm apart, or PPi which is released from one anchor could diffuse to the next, and be detected.
Another method which may be used to explain the aforementioned phenomenon is to estimate the amount of PPi over a first anchor pad that was generated at said first anchor pad relative to the amount of PPi that was generated at a second anchor pad and subsequently diffused over to the location of said first anchor pad. When these two quantities approach each other in magnitude, it becomes difficult to distinguish the âtrueâ signal from that of the background. This may be mathematically-described by defining a as the radius of an anchor pad and 1/b2as the density of an anchor pad. Based upon previously published data, a is approximately equal to 10 μm and b is approximately equal to 100 μm. The amount of PPi which is present over said first anchor pad may be described by: exp(âkPt)[1âexp(âa2/2DPt)] and the amount of PPi present over the second anchor pads may be mathematically approximated by:
(1/3)exp(âkPt)[pa2/b2]exp(âb2/2DPt). The prefactor 1/3 assumes that ¼ of the DNA sequences will incorporate 1 nucleotide, ¼ of these will then incorporate a second nucleotide, etc., and thus the sum of the series is 1/3. The amounts of PPi over the first and second anchor pads become similar in magnitude when 2DPt is approximately equal to b2, thus indicating that the RMS distance a molecule diffuses is equal to the distance between adjacent anchor pads. In accord, based upon the assay conditions utilized in the practice of the present invention, the anchor pads must be placed no closer than approximately 50 μm apart, and preferable are at least 3-times further apart (i.e., 150 μm).
Although the aforementioned findings set a limit on the surface density of anchor pads, it is possible to decrease the distance requirements, while concomitantly increasing the overall surface density of the anchor pads, by the use of a number of different approaches. One approach is to detect only the early light, although this has the disadvantage of losing signal, particularly from DNA sequences which possess a number of contiguous, identical nucleotides.
A second approach to decrease the distance between anchor pads is to increase the concentration of sulfurylase in the reaction mixture. The reaction rate kP is directly proportional to the sulfurylase concentration, and the diffusion distance scales as kP â1/2. Therefore, if the sulfurylase enzyme concentration is increased by a factor of 4-times, the distance between individual anchor pads may be concomitantly reduced by a factor of 2-times.
A third approach is to increase the effective concentration of sulfurylase (which will also work for other enzymes described herein) by binding the enzyme to the surface of (he anchor pads. The anchor pad can be approximated as one wall of a cubic surface enclosing a sequencing reaction center. Assuming a 10 μmÃ10 μm surface for the pad, the number of molecules bound to the pad to produce a concentration of a 1 μM is approximately 600,000 molecules.
The sulfurylase concentration in the assay is estimated as 5 nM. The number of bound molecules to reach this effective concentration is about 3000 molecules. Thus, by binding more enzyme molecules, a greater effective concentration will be attained. For example, 10,000 molecules could be bound per anchor pad.
As previously estimated, each sulfurylase molecule occupies a total area of 65 nm2 on a surface. Accordingly, anchoring a total of 10,000 sulfurylase enzyme molecules on a surface (i.e., so as to equal the 10,000 PPi released) would require 1.7 μm2. This value is only approximately 2% of the available surface area on a 10 μmÃ10 μm anchor pad. Hence, the concentration of the enzyme may be readily increased to a much higher value.
A fourth approach to allow a decrease in the distance between individual anchor pads, is to utilize one or more agents to increase the viscosity of the aqueous-based, pyrophosphate sequencing reagents (e.g., glycerol, polyethylene glycol (PEG), and the like) so as to markedly increase the time it takes for the PPi to diffuse. However, these agents will also concomitantly increase the diffusion time for other non-immobilized components within the sequencing reaction, thus slowing the overall reaction kinetics. Additionally, the use of these agents may also function to chemically-interfere with the sequencing reaction itself.
A fifth, and preferred, methodology to allow a decrease in the distance between individual anchor pads, is to conduct the pyrophosphate sequencing reaction in a spatial-geometry which physically-prevents the released PPi from diffusing laterally. For example, uniform cavities or microwells, such as those generated by acid-etching the termini of optical fiber bundles, may be utilized to prevent such lateral diffusion of PPi (see Michael, et al., 1998. Randomly Ordered Addressable High-Density Optical Sensor Arrays, Anal. Chem. 70: 1242-1248). In this embodiment, the important variable involves the total diffusion time for the PPi to exit a cavity of height h, wherein h is the depth of the etched cavity. This diffusion time may be calculated utilizing the equation: 2DPt=h2. By use of the preferred pyrophosphate sequencing reaction conditions of the present invention in the aforementioned calculations, it may be demonstrated that a cavity 50 μm in depth would be required for the sequencing reaction to proceed to completion before complete diffusion of the PPi from said cavity. Moreover, this type of geometry has the additional advantage of concomitantly reducing background signal from the PPi released from adjacent anchor pads.
Additionally, to prevent background generated by diffusion of PPi from one pad to another, the region of substrate between the pads can be coated with immobilized phosphatase.
Subsequently, once ATP has been formed by use of the preferred reaction conditions of the present invention, the reaction time, 1/kA, has been shown to be 0.2 seconds. Because this reaction time is much lower than the time which the PPi is free to diffuse, it does not significantly alter any of the aforementioned conclusions regarding the assay geometry and conditions utilized in the present invention.
In order to mitigate the generation of background light, it is preferable to âlocalizeâ (e.g., by anchoring or binding) the luciferase in the region of the DNA sequencing templates. It is most preferable to localize the luciferase to a region that is delineated by the distance a PPi molecule can diffuse before it forms ATP. Methods for binding luciferase to a solid support matrix are well-known in the literature (see e.g., Wang, et al, 1997. Specific Immobilization of Firefly Luciferase through a Biotin Carboxyl Carrier Protein Domain, Analytical Biochem. 246: 133-139). Thus, for a 2 second diffusion time, the luciferase is anchored within a 50 μm distance of the DNA strand. It should be noted, however, that it would be preferable to decrease the diffusion time and thus to further limit the surface area which is required for luciferase binding.
Additionally, to prevent background generated by diffusion of ATP from one pad to another, the region of substrate between the pads can be coated with immobilized ATPase, especially one that hydrolyzes ATP to ADP, e.g. alkaline phosphatase.
In order to determine the concentration of luciferase which it is necessary to bind, previously published conditions were utilized in which luciferase is used at a concentration which gives a response of 200 mV for 0.1 μm ATP (see Ronaghi, et al., 1996. Real-Time DNA Sequencing Using Detection of Pyrphosphate Release, Analytical Biochem. 242: 84-89). More specifically, it is known from the literature that, in a 0.2 ml reaction volume, 2 ng of luciferase gives a response of 10 mV for 0.1 μM ATP (see Karamohamed and Nyrén, 1999. Real-Time Detection and Quantification of Adenosine Triphosphate Sulfurylase Activity by a Bioluminometric Approach, Analytical Biochem. 271: 81-85). Accordingly, a concentration of 20 ng of luciferase within a 0.2 ml total reaction volume would be required to reproduce these previously-published literature conditions. In the volume of a 10 μm cube around each of the individual anchor pads of the present invention, a luciferase concentration of 1Ã10â16 grams would be required, and based upon the 71 kDa molecular weight of luciferase, this concentration would be equivalent to approximately 1000 luciferase molecules. As previously stated, the surface area of luciferase has been computed at 50 nm2. Thus, assuming the luciferase molecules were biotinylated and bound to the anchor pad, 1000 molecules would occupy a total area of 0.05 μm2. From these calculations it becomes readily apparent that a plethora of luciferase molecules may be bound to the anchor pad, as the area of each anchor pad area is 100 μm2.
Again, based upon previously published results in the literature, each nucleotide takes approximately 3 seconds to sequence (i.e., 0.2 second to add a nucleotide; 2 seconds to make ATP; 0.2 seconds to get bioluminescence). Accordingly, a cycle time of approximately 60 seconds per nucleotide is reasonable, requiring approximately 30 minutes per experiment to generate 30 nucleotides of information per sequencing template.
In an alternative embodiment to the aforementioned sequencing methodology (i.e., polymeraseâPPiâsulfurylaseâATPâluciferaseâlight), a polymerase may be developed (e.g., through the use of protein fusion and the like) which possesses the ability to generate light when it incorporates a nucleotide into a growing DNA chain. In yet another alternative embodiment, a sensor may be developed which directly measures the production of PPi in the sequencing reaction. As the production of PPi changes the electric potential of the surrounding buffer, this change could be measured and calibrated to quantify the concentration of PPi produced.
As previously discussed, the polymerase-mediated incorporation of dNTPs into the nucleotide sequence in the pyrophosphate sequencing reaction causes the release of a photon (i.e., light). The photons generated by the pyrophosphate sequencing reaction may subsequently be âcapturedâ and quantified by a variety of methodologies including, but not limited to: a photomultiplier tube, CCD, absorbance photometer, a luminometer, and the like.
The photons generated by the pyrophosphate sequencing reaction are captured by the CCD. The efficiency of light capture increases if they pass through a focusing device (e.g., an optical lens or optical fiber) and are focused upon a CCD element. The fraction of these photons which are captured may be estimated by the following calculations. First, it is assumed that the lens that focuses the emitted photons is at a distance r from the surface of the solid surface (i.e., DNA chip or etched fiber optic well), where r=1 cm and that the photons must pass through a region of diameter b (area=Ïb2/4) so as to be focused upon the array element, where b=100 μm. (This produces an optical system with numerical aperture of approximately 0.01 in air.) It should also be noted that the emitted photons should escape equally in all directions. At distance r, the photons are dispersed over an area of which is equal to 4Ïr2. Thus, the fraction of photons which pass through the lens is described by: (1/2)[1â(1+b2/4r2)â1/2]. When the value of r is much larger than that of b, the fraction which pass through the lens may then be described by: b2/16r2. For the aforementioned values of r and b, this fraction of photons is 6Ã10â6. Note that the fraction of captured photons increases as b increases or r decreases (i.e. as the numerical aperture of the imaging system increases). Use of FORA in which the microwells are etched into the termini of optical fibers, which then also serve to focus the light onto a CCD, greatly increases the numerical aperture from the example given above, with the numerical aperture of many fiber optics being in the range of 0.7. For each nucleotide addition, it is expected that approximately 10,000 PPi molecules will be generated and, if all are converted by sulfurylase and luciferase, these PPi will result in the emission of approximately 1Ã104 photons. In order to maximize their subsequent âcaptureâ and quantitation when utilizing a planar array (e.g., a DNA chip), it is preferable to collect the photons immediately at the planar solid support (e.g., the cover slip). This may be accomplished by either: (i) utilizing optical immersion oil between the cover slip and a traditional optical lens or optical fiber bundle or, preferably, (ii) incorporating optical fibers directly into the cover slip itself. Performing the previously described calculations (where in this case, b=100 μm and r=50 μm), the fraction collected is found to be 0.15, which equates to the capture of approximately 1Ã103 photons. This value would be sufficient to provide an adequate signal.
The following examples are meant to illustrate, not limit, the invention.
The termini of a thin wafer fiber optic array are cavitated by inserting the termini into acid as described by Healey et al., Anal Chem. 69: 2213-2216 (1997).
A thin layer of a photoactivatable biotin analog is dried onto the cavitated surface as described in Hengsakul and Cass (Bioconjugate Chem. 7: 249-254, 1996) and exposed to white light through a mask to create defined pads, or areas of active biotin. Next, avidin is added and allowed to bind to the biotin. Biotinylated oligonucleotides are then added. The avidin has free biotin binding sites that can anchor biotinylated oligonucleotides through a biotin-avidin-biotin link.
The pads are approximately 10 μm on a side with a 100 μm spacing. Oligonucleotides are added so that approximately 37% of the pads include one anchored primer. On a 1 cm2 surface are deposited 10,000 pads, yielding approximately 3700 pads with a single anchor primer.
A library of open circle library templates is prepared from a population of nucleic acids suspected of containing a single nucleotide polymorphism on a 70 bp Sau3A1-MspI fragment. The templates include adapters that are complementary to the anchor primer, a region complementary to a sequencing primer, and an insert sequence that is to be characterized. The library is generated using Sau3A1 and MspI to digest the genomic DNA. Inserts approximately 65-75 nucleotides are selected and ligated to adapter oligonucleotides 12 nucleotides in length. The adapter oligonucleotides have sequences complementary to sequences to an anchor primers linked to a substrate surface as described in Example 1.
The library is annealed to the array of anchor primers. A DNA polymerase is added, along with dNTPs, and rolling circle replication is used to extend the anchor primer. The result is a single DNA strand, still anchored to the solid support, that is a concatenation of multiple copies of the circular template. 10,000 or more copies of circular templates in the hundred nucleotide size range.
The fiber optic array wafer containing amplified nucleic acids as described in Example 2 is placed in a perfusion chamber and attached to a bundle of fiber optic arrays, which are themselves linked to a 16 million pixel CCD camera. A sequencing primer is delivered into the perfusion chamber and allowed to anneal to the amplified sequences. Then sulfurylase, apyrase, and luciferase are attached to the cavitated substrate using biotin-avidin.
The sequencing primer primes DNA synthesis extending into the insert suspected of having a polymorphism, as shown in FIG. 1 . The sequencing primer is first extended by delivering into the perfusion chamber, in succession, a wash solution, a DNA polymerase, and one of dTTP, dGTP, dCTP, or α thio dATP (a dATP analog). The sulfurylase, luciferase, and apyrase, attached to the termini convert any PPi liberated as part of the sequencing reaction to detectable light. The apyrase present degrades any unreacted dNTP. Light is typically allowed to collect for 3 seconds (although 1-100, e.g., 2-10 seconds is also suitable) by a CCD camera linked to the fiber imaging bundle, after which additional wash solution is added to the perfusion chamber to remove excess nucleotides and byproducts. The next nucleotide is then added, along with polymerase, thereby repeating the cycle.
During the wash the collected light image is transferred from the CCD camera to a computer. Light emission is analyzed by the computer and used to determine whether the corresponding dNTP has been incorporated into the extended sequence primer. Addition of dNTPs and pyrophosphate sequencing reagents is repeated until the sequence of the insert region containing the suspected polymorphism is obtained.
A primer having the sequence 5â²-gAC CTC ACA CgA Tgg CTg CAg CTT-3â² (SEQ ID NO:2) was annealed to a 88 nucleotide template molecule having the sequence 5â²-TCg TgT gAg gTC TCA gCA TCT TAT gTA TAT TTA CTT CTA TTC TCA gTT gCC TAA gCT gCA gCC A-3â² (SEQ ID NO:1). Annealing of the template to the primer resulted in juxtaposition of the 5â² and 3â² ends of the template molecule. The annealed template was exposed to ligase, which resulted in ligation of the 5â² and 3â² ends of the template to generate a circular molecule.
The annealed primer was extended using Klenow fragment and nucleotides in rolling circle amplification for 12 hours at 37° C. The product was purified using the SPRI technique (Seradyn, Indianapolis, Ind.). Rolling circle amplification resulted in formation of tandem repeats of a sequence complementary to the circular template sequence.
The tandem repeat product in the extended sequence was identified by annealing a sequencing primer having the sequence 5â²-AAgCTgCAgCCATCgTgTgAgg-3â² (SEQ ID NO:8) and subjecting the annealed primer to 40 alternating cycles of 95° C., 1 minute, 20 seconds, 60° C. using ET terminator chemistry (Amersham-Pharmacia) in the presence of 1M betaine.
The sequencing product was then diluted to 1/5 volume and purified on a G-50 Sephadex column prior to injection into a MegaBACE sequencing system with linear polyacrylamide (Amersham-Pharmacia).
An electropherogram of the sequencing analysis is shown in FIG. 5 . The tracing demonstrates that multiple copies of the 88 bp circular template molecule are generated tandemly, and that these copies can be detected in a DNA sequencing reaction.
DNA beads: DeoxyoligonucleotideâggggAATTCAAAATTTggC (SEQ ID NO:9) were annealed to capture probes, which were biotinylated at the 5â² end, and then immobilized on either Dynal M-280 (Dynal) or MPG beads (CPG) (bead concentration was 1 mg/ml). The immobilization was carried out by incubating the beads, with a fixed amount of oligonucleotide for 30 minutes. Different loadings of oligonucleotide were obtained by changing amount of oligonucleotide used during incubation. After incubation, the beads were washed in respective volumes of TE buffer and resuspened in same volumes of TE.
Enzyme beads: A mixture of 1:1 (vol/vol) of sulfurylase (1 mg/mL) and luciferase (3 mg/mL) with BBCP domains on their N-termini were incubated with equal volume of Dynal M-280 (Dynal) (concentration: 10 mg/mL) for one hour at 4° C. After an hour of incubation the beads were washed with assay buffer (25 mM Tricine, 5 mM MgOAc and 1 mg/mL BSA) four times and then resuspended in same volume of assay buffer.
FORA Preparation: The DNA beads were diluted 10 times to a final concentration of 0.1 mg/mL before use. The enzyme beads were used at 10 mg/mL concentration. The FORA was placed in jig which has 10 spots created by O-rings (3 mm in diameter). 5 uL of DNA beads were delivered, in 9 spots. The first spot on the inlet was a control spot, with no DNA, to detect any background in the reagents. The jig was placed in a centrifuge and spun at 2000 rpm for five minutes. The centrifugal force, forces the beads to the bottom of the wells (approximately 5-10 beads/well) The jig is removed from the centrifuge and 5 uL of SL beads are added and the jig is placed in the centrifuged and the spun at 2000 rpm for five minutes. The process is repeated with 5 uL of SL beads. The FORA is removed from the jig, placed in a falcon tube containing assay buffer and washed by a gentle rocking motion three to four times. The FORA thus prepared is ready for sequence analysis by pyrophosphate sequencing.
Reagents: Reagents used for sequence analysis and as controls were the four nucleotides and 0.1 μM Pyrophosphate (PPi) were made in substrate solution, where substrate refers to a mixture of 300 μM Luciferin and 4 μM adenosine 5â²-phosphosulfate, APS, which are the substrates for the cascade of reactions involving PPi, Luciferase and Sulfurylase. The substrate was made in assay buffer. The concentration of PPi used to test the enzymes and determine the background levels of reagents passing through the chamber was 0.1 μM. The concentration of the nucleotides, dTTP, dGTP, dCTP was 6.5 μM and that of αdATP was 50 μM. Each of the nucleotides was mixed with DNA polymerase, Klenow at a concentration of 100 U/mL.
The FORA was placed in the flow chamber of the embodied instrument, and the flow chamber was attached to the faceplate of the CCD camera. The FORA was washed by flowing substrate (3 ml per min, 2 min) through the chamber. Subsequently, a sequence of reagents was flown through the chamber by the pump connected to an actuator, which was programmed to switch positions, which had tubes inserted in the different reagents. The camera was set up in a fast acquisition mode, with exposure time=2.5 s.
The signal output from the pad is the average of counts on all the pixels within the pad. The frame number is equivalent of the time passed during the experiment. The graph indicates the flow of the different reagents.
It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.
. A method for sequencing a nucleic acid, the method comprising:
(a) providing one or more nucleic acid anchor primers;
(b) providing a plurality of single-stranded nucleic acid templates disposed within a plurality of cavities on a planar surface, each cavity forming an analyte reaction chamber, wherein the reaction chambers have a center to center spacing of between 5 to 200 μm;
(c) annealing an effective amount of the nucleic acid anchor primer to at least one of the single-stranded templates to yield a primed anchor primer-template complex;
(d) combining the primed anchor primer-template complex with a polymerase to form an extended anchor primer covalently linked to multiple copies of a nucleic acid complementary to the nucleic acid template;
(e) annealing an effective amount of a sequencing primer to one or more copies of said covalently linked complementary nucleic acid;
(f) extending the sequencing primer with a polymerase and a predetermined nucleotide triphosphate to yield a sequencing product and, if the predetermined nucleotide triphosphate is incorporated onto the 3â² end of said sequencing primer, a sequencing reaction byproduct; and
(g) identifying the sequencing reaction byproduct, thereby determining the sequence of the nucleic acid.
2. The method of claim 1 wherein each single stranded nucleic acid contains at least 100 copies of a nucleic acid sequence, each copy covalently linked end to end.
3. The method of claim 1 wherein said single stranded nucleic acid template is a circular single stranded nucleic acid template.
4. The method of claim 1 wherein each reaction chamber has a width in at least one dimension of between 0.3 μm and 100 μm.
5. The method of claim 1 wherein each reaction chamber has a width in at least one dimension of between 0.3 μm and 20 μm.
6. The method of claim 1 wherein each reaction chamber has a width in at least one dimension of between 0.3 μm and 10 μm.
7. The method of claim 1 wherein each reaction chamber has a width in at least one dimension of between 20 μm and 70 μm.
8. The method of claim 1 wherein the cavities number greater than 400,000.
9. The method of claim 1 wherein the cavities number between 400,000 and 20,000,000.
10. The method of claim 1 wherein the cavities number between 1,000,000 and 16,000,000.
11. The method of claim 1 wherein the center to center spacing is between 10 to 150 μm.
12. The method of claim 1 wherein the center to center spacing is between 50 to 100 μm.
13. The method of claim 1 wherein each cavity has a depth of between 10 μm and 100 μm.
14. The method of claim 1 wherein each cavity has a depth that is between 0.25 and 5 times the size of the width of the cavity.
15. The method of claim 1 wherein each cavity has a depth that is between 0.3 and 1 times the size of the width of the cavity.
16. The method of claim 1 wherein the nucleic acid sequence is further amplified to produce multiple copies of said nucleic acid sequence after being disposed in the reaction chamber.
17. The method of claim 16 wherein the nucleic acid sequence is amplified using an amplification technology selected from the group consisting of polymerase chain reaction, ligase chain reaction and isothermal DNA amplification.
18. The method of claim 1 wherein the single stranded nucleic acid is immobilized in the reaction chamber.
19. The method of claim 1 wherein the single stranded nucleic acid is immobilized on one or more mobile solid supports disposed in the reaction chamber.
20. A method for sequencing a nucleic acid, the method comprising:
(a) providing at least one nucleic acid anchor primer;
(b) providing a plurality of single-stranded nucleic acid templates in an array having at least 400,000 discrete reaction sites;
(c) annealing a first amount of the nucleic acid anchor primer to at least one of the single-stranded templates to yield a primed anchor primer-template complex;
(d) combining the primed anchor primer-template complex with a polymerase to form an extended anchor primer covalently linked to multiple copies of a nucleic acid complementary to the nucleic acid template;
(e) annealing a second amount of a sequencing primer to one or more copies of the covalently linked complementary nucleic acid;
(f) extending the sequencing primer with a polymerase and a predetermined nucleotide triphosphate to yield a sequencing product and, when the predetermined nucleotide triphosphate is incorporated onto the 3â² end of the sequencing primer, to yield a sequencing reaction byproduct; and
(g) identifying the sequencing reaction byproduct, thereby determining the sequence of the nucleic acid at each reaction site that contains a nucleic acid template.
21. The method of claim 20 wherein said single-stranded nucleic acid template is a circular single-stranded nucleic acid template.
22. The method of claim 20 wherein the anchor primer is linked to a particle.
23. The method of claim 20 wherein the anchor primer is linked to the particle prior to formation of the extended anchor primer.
24. The method of claim 20 wherein the anchor primer is linked to the particle after formation of the extended anchor primer.
25. The method of claim 20 wherein the sequencing reaction byproduct is PPi and a coupled sulfurylase/luciferase reaction is used to generate light for detection.
26. The method of claim 25 wherein either or both of the sulfurylase and luciferase are immobilized on one or more mobile solid supports disposed at each reaction site.
27. A method of determining the base sequence of a plurality of nucleotides on an array, the method comprising:
(a) providing a plurality of sample DNAs, each disposed within a plurality of cavities on a planar surface, each cavity forming an analyte reaction chamber, wherein the reaction chambers have a center to center spacing of between 5 to 200 μm;
(b) adding an activated nucleotide 5â²-triphosphate precursor of one known nitrogenous base to a reaction mixture in each reaction chamber, each reaction mixture comprising a template-directed nucleotide polymerase and a single-stranded polynucleotide template hybridized to a complementary oligonucleotide primer strand at least one nucleotide residue shorter than the templates to form at least one unpaired nucleotide residue in each template at the 3â²-end of the primer strand, under reaction conditions which allow incorporation of the activated nucleoside 5â²-triphosphate precursor onto the 3â²-end of the primer strands, provided the nitrogenous base of the activated nucleoside 5â²-triphosphate precursor is complementary to the nitrogenous base of the unpaired nucleotide residue of the templates;
(c) detecting whether or not the nucleoside 5â²-triphosphate precursor was incorporated into the primer strands in which incorporation of the nucleoside 5â²-triphosphate precursor indicates that the unpaired nucleotide residue of the template has a nitrogenous base composition that is complementary to that of the incorporated nucleoside 5â²-triphosphate precursor;
(d) sequentially repeating steps (b) and (c), wherein each sequential repetition adds and, detects the incorporation of one type of activated nucleoside 5â²-triphosphate precursor of known nitrogenous base composition; and
(e) determining the base sequence of the unpaired nucleotide residues of the template in each reaction chamber from the sequence of incorporation of said nucleoside precursors.
28. A method for determining the nucleic acid sequence in a template nucleic acid polymer, comprising:
(a) introducing a plurality of template nucleic acid polymers into a plurality of cavities on a planar surface, each cavity forming an analyte reaction chamber, wherein the reaction chambers have a center to center spacing of between 5 to 200 μm, each reaction chamber having a polymerization environment in which the nucleic acid polymer will act as a template polymer for the synthesis of a complementary nucleic acid polymer when nucleotides are added;
(b) successively providing to the polymerization environment a series of feedstocks, each feedstock comprising a nucleotide selected from among the nucleotides from which the complementary nucleic acid polymer will be formed, such that if the nucleotide in the feedstock is complementary to the next nucleotide in the template polymer to be sequenced said nucleotide will be incorporated into the complementary polymer and inorganic pyrophosphate will be released; and
(c) detecting the formation of inorganic pyrophosphate to determine the identify of each nucleotide in the complementary polymer and thus the sequence of the template polymer.
29. A method of identifying the base in a target position in a DNA sequence of sample DNA, wherein:
(a) disposing sample DNA within a plurality of cavities on a planar surface, each cavity forming an analyte reaction chamber, wherein the reaction chambers have a center to center spacing of between 5 to 200 μm, said DNA being rendered single stranded either before or after being disposed in the reaction chambers,
(b) providing an extension primer which hybridizes to said immobilized single-stranded DNA at a position immediately adjacent to said target position;
(c) subjecting said immobilized single-stranded DNA to a polymerase reaction in the presence of a predetermined nucleotide triphosphate, wherein if the predetermined nucleotide triphosphate is incorporated onto the 3â² end of said sequencing primer then a sequencing reaction byproduct is formed; and
(d) identifying the sequencing reaction byproduct, thereby determining the nucleotide complementary to the base at said target position.
30. A method of identifying a base at a target position in a sample DNA sequence comprising:
(a) providing sample DNA disposed within a plurality of cavities on a planar surface, each cavity forming an analyte reaction chamber, wherein the reaction chambers have a center to center spacing of between 5 to 200 μm, said DNA being rendered single stranded either before or after being disposed in the reaction chambers;
(b) providing an extension primer which hybridizes to the sample DNA immediately adjacent to the target position;
(c) subjecting the sample DNA sequence and the extension primer to a polymerase reaction in the presence of a nucleotide triphosphate whereby the nucleotide triphosphate will only become incorporated and release pyrophosphate (PPi) if it is complementary to the base in the target position, said nucleotide triphosphate being added either to separate aliquots of sample-primer mixture or successively to the same sample-primer mixture; and
(d) detecting the release of PPi to indicate which nucleotide is incorporated.
31. A method of identifying a base at a target position in a single-stranded sample DNA sequence, the method comprising:
(a) providing an extension primer which hybridizes to sample DNA immediately adjacent to the target position, said sample DNA disposed within a plurality of cavities on a planar surface, each cavity forming an analyte reaction chamber, wherein the reaction chambers have a center to center spacing of between 5 to 200 um, said DNA being rendered single stranded either before or after being disposed in the reaction chambers;
(b) subjecting the sample DNA and extension primer to a polymerase reaction in the presence of a predetermined deoxynucleotide or dideoxynucleotide whereby the deoxynucleotide or dideoxynucleotide will only become incorporated and release pyrophosphate (PPi) if it is complementary to the base in the target position, said predetermined deoxynucleotides or dideoxynucleotides being added either to separate aliquots of sample-primer mixture or successively to the same sample-primer mixture; and
(c) detecting any release of PPi enzymatically to indicate which deoxynucleotide or dideoxynucleotide is incorporated; characterized in that, the PPi-detection enzyme(s) are included in the polymerase reaction step and in that in place of deoxy- or dideoxy adenosine triphosphate (ATP) a dATP or ddATP analogue is used which is capable of acting as a substrate for a polymerase but incapable of acting as a substrate for a said PPi-detection enzyme.
32. A method of determining the base sequence of a plurality of nucleotides on an array, the method comprising:
(a) providing a plurality of sample DNAs, each disposed within a plurality of cavities on a planar surface, each cavity forming an analyte reaction chamber, wherein the reaction chambers have a center to center spacing of between 5 to 200 μm;
(b) detecting the light level emitted from a plurality of reaction sites on respective portions of an optically sensitive device;
(c) converting the light impinging upon each of said portions of said optically sensitive device into an electrical signal which is distinguishable from the signals from all of said other regions;
(d) determining a light intensity for each of said discrete regions from the corresponding electrical signal; and
(e) recording the variations of said electrical signals with time.
33. Method for sequencing a nucleic acid, the method comprising:
(a) providing one or more nucleic acid anchor primers;
(b) providing a plurality of single-stranded nucleic acid templates disposed within a plurality of cavities on a planar surface, each cavity forming an analyte reaction chamber, wherein the reaction chambers have a center to center spacing of between 5 to 200 μm;
(c) detecting the light level emitted from a plurality of reaction sites on respective portions of an optically sensitive device;
(d) converting the light impinging upon each of said portions of said optically sensitive device into an electrical signal which is distinguishable from the signals from all of said other regions;
(e) determining a light intensity for each of said discrete regions from the corresponding electrical signal; and
(f) recording the variations of said electrical signals with time.
34. A method for sequencing a nucleic acid, the method comprising:
(a) providing at least one nucleic acid anchor primer;
(b) providing a plurality of single-stranded nucleic acid templates in an array having at least 400,000 discrete reaction sites;
(c) detecting the light level emitted from a plurality of reaction sites on respective portions of an optically sensitive device;
(d) converting the light impinging upon each of said portions of said optically sensitive device into an electrical signal which is distinguishable from the signals from all of said other regions;
(e) determining a light intensity for each of said discrete regions from the corresponding electrical signal; and
(f) recording the variations of said electrical signals with time.
US10/222,298 1999-09-16 2002-08-15 Apparatus and method for sequencing a nucleic acid Abandoned US20070092872A1 (en) Priority Applications (1) Application Number Priority Date Filing Date Title US10/222,298 US20070092872A1 (en) 1999-09-16 2002-08-15 Apparatus and method for sequencing a nucleic acid Applications Claiming Priority (5) Application Number Priority Date Filing Date Title US09/398,833 US6274320B1 (en) 1999-09-16 1999-09-16 Method of sequencing a nucleic acid US66419700A 2000-09-18 2000-09-18 US09/814,338 US7244559B2 (en) 1999-09-16 2001-03-21 Method of sequencing a nucleic acid US10/104,280 US20030068629A1 (en) 2001-03-21 2002-03-21 Apparatus and method for sequencing a nucleic acid US10/222,298 US20070092872A1 (en) 1999-09-16 2002-08-15 Apparatus and method for sequencing a nucleic acid Related Parent Applications (1) Application Number Title Priority Date Filing Date US10/104,280 Continuation US20030068629A1 (en) 1999-09-16 2002-03-21 Apparatus and method for sequencing a nucleic acid Publications (1) Family ID=25214762 Family Applications (5) Application Number Title Priority Date Filing Date US09/814,338 Expired - Fee Related US7244559B2 (en) 1999-09-16 2001-03-21 Method of sequencing a nucleic acid US10/104,280 Abandoned US20030068629A1 (en) 1999-09-16 2002-03-21 Apparatus and method for sequencing a nucleic acid US10/222,592 Expired - Fee Related US7335762B2 (en) 1999-09-16 2002-08-15 Apparatus and method for sequencing a nucleic acid US10/222,298 Abandoned US20070092872A1 (en) 1999-09-16 2002-08-15 Apparatus and method for sequencing a nucleic acid US10/299,180 Expired - Fee Related US7264929B2 (en) 1999-09-16 2002-11-19 Method of sequencing a nucleic acid Family Applications Before (3) Application Number Title Priority Date Filing Date US09/814,338 Expired - Fee Related US7244559B2 (en) 1999-09-16 2001-03-21 Method of sequencing a nucleic acid US10/104,280 Abandoned US20030068629A1 (en) 1999-09-16 2002-03-21 Apparatus and method for sequencing a nucleic acid US10/222,592 Expired - Fee Related US7335762B2 (en) 1999-09-16 2002-08-15 Apparatus and method for sequencing a nucleic acid Family Applications After (1) Application Number Title Priority Date Filing Date US10/299,180 Expired - Fee Related US7264929B2 (en) 1999-09-16 2002-11-19 Method of sequencing a nucleic acid Country Status (9) Cited By (65) * Cited by examiner, â Cited by third party Publication number Priority date Publication date Assignee Title US20080318796A1 (en) * 2006-10-27 2008-12-25 Complete Genomics,Inc. Efficient arrays of amplified polynucleotides US20090005252A1 (en) * 2006-02-24 2009-01-01 Complete Genomics, Inc. High throughput genome sequencing on DNA arrays US20090011943A1 (en) * 2005-06-15 2009-01-08 Complete Genomics, Inc. High throughput genome sequencing on DNA arrays US20090127589A1 (en) * 2006-12-14 2009-05-21 Ion Torrent Systems Incorporated Methods and apparatus for measuring analytes using large scale FET arrays US20100137143A1 (en) * 2008-10-22 2010-06-03 Ion Torrent Systems Incorporated Methods and apparatus for measuring analytes US20100192032A1 (en) * 2006-02-16 2010-07-29 Yi-Ju Chen System and Method for Correcting Primer Extension Errors in Nucleic Acid Sequence Data US20100282617A1 (en) * 2006-12-14 2010-11-11 Ion Torrent Systems Incorporated Methods and apparatus for detecting molecular interactions using fet arrays US20100301398A1 (en) * 2009-05-29 2010-12-02 Ion Torrent Systems Incorporated Methods and apparatus for measuring analytes US20110213563A1 (en) * 2007-02-15 2011-09-01 454 Life Sciences Corporation System and method to correct out of phase errors in dna sequencing data by use of a recursive algorithm US8217433B1 (en) 2010-06-30 2012-07-10 Life Technologies Corporation One-transistor pixel array US8262900B2 (en) 2006-12-14 2012-09-11 Life Technologies Corporation Methods and apparatus for measuring analytes using large scale FET arrays US20130053281A1 (en) * 2010-02-02 2013-02-28 Arash Zarrine-Afsar Fluid sampling device and method of use thereof US8470164B2 (en) 2008-06-25 2013-06-25 Life Technologies Corporation Methods and apparatus for measuring analytes using large scale FET arrays US8552771B1 (en) 2012-05-29 2013-10-08 Life Technologies Corporation System for reducing noise in a chemical sensor array US8653567B2 (en) 2010-07-03 2014-02-18 Life Technologies Corporation Chemically sensitive sensor with lightly doped drains US8666678B2 (en) 2010-10-27 2014-03-04 Life Technologies Corporation Predictive model for use in sequencing-by-synthesis US8673627B2 (en) 2009-05-29 2014-03-18 Life Technologies Corporation Apparatus and methods for performing electrochemical reactions US8685324B2 (en) 2010-09-24 2014-04-01 Life Technologies Corporation Matched pair transistor circuits US8747748B2 (en) 2012-01-19 2014-06-10 Life Technologies Corporation Chemical sensor with conductive cup-shaped sensor surface US8776573B2 (en) 2009-05-29 2014-07-15 Life Technologies Corporation Methods and apparatus for measuring analytes US8821798B2 (en) 2012-01-19 2014-09-02 Life Technologies Corporation Titanium nitride as sensing layer for microwell structure US8841217B1 (en) 2013-03-13 2014-09-23 Life Technologies Corporation Chemical sensor with protruded sensor surface US8858782B2 (en) 2010-06-30 2014-10-14 Life Technologies Corporation Ion-sensing charge-accumulation circuits and methods US8963216B2 (en) 2013-03-13 2015-02-24 Life Technologies Corporation Chemical sensor with sidewall spacer sensor surface US8962366B2 (en) 2013-01-28 2015-02-24 Life Technologies Corporation Self-aligned well structures for low-noise chemical sensors US9080968B2 (en) 2013-01-04 2015-07-14 Life Technologies Corporation Methods and systems for point of use removal of sacrificial material US9109251B2 (en) 2004-06-25 2015-08-18 University Of Hawaii Ultrasensitive biosensors US9116117B2 (en) 2013-03-15 2015-08-25 Life Technologies Corporation Chemical sensor with sidewall sensor surface US9128044B2 (en) 2013-03-15 2015-09-08 Life Technologies Corporation Chemical sensors with consistent sensor surface areas US9416413B2 (en) 2010-06-11 2016-08-16 Life Technologies Corporation Alternative nucleotide flows in sequencing-by-synthesis methods US9428807B2 (en) 2011-04-08 2016-08-30 Life Technologies Corporation Phase-protecting reagent flow orderings for use in sequencing-by-synthesis US9594870B2 (en) 2010-12-29 2017-03-14 Life Technologies Corporation Time-warped background signal for sequencing-by-synthesis operations US9618475B2 (en) 2010-09-15 2017-04-11 Life Technologies Corporation Methods and apparatus for measuring analytes US9671363B2 (en) 2013-03-15 2017-06-06 Life Technologies Corporation Chemical sensor with consistent sensor surface areas US9777323B2 (en) 2012-06-18 2017-10-03 Siemens Aktiengesellschaft Assembly and method for analyzing nucleic acid sequences by way of so-called sequencing-by-synthesis US9823217B2 (en) 2013-03-15 2017-11-21 Life Technologies Corporation Chemical device with thin conductive element US9835585B2 (en) 2013-03-15 2017-12-05 Life Technologies Corporation Chemical sensor with protruded sensor surface US9841398B2 (en) 2013-01-08 2017-12-12 Life Technologies Corporation Methods for manufacturing well structures for low-noise chemical sensors US9926597B2 (en) 2013-07-26 2018-03-27 Life Technologies Corporation Control nucleic acid sequences for use in sequencing-by-synthesis and methods for designing the same US9944984B2 (en) 2005-06-15 2018-04-17 Complete Genomics, Inc. High density DNA array US9970984B2 (en) 2011-12-01 2018-05-15 Life Technologies Corporation Method and apparatus for identifying defects in a chemical sensor array US10077472B2 (en) 2014-12-18 2018-09-18 Life Technologies Corporation High data rate integrated circuit with power management US10100357B2 (en) 2013-05-09 2018-10-16 Life Technologies Corporation Windowed sequencing US10146906B2 (en) 2010-12-30 2018-12-04 Life Technologies Corporation Models for analyzing data from sequencing-by-synthesis operations US10241075B2 (en) 2010-12-30 2019-03-26 Life Technologies Corporation Methods, systems, and computer readable media for nucleic acid sequencing US10273540B2 (en) 2010-10-27 2019-04-30 Life Technologies Corporation Methods and apparatuses for estimating parameters in a predictive model for use in sequencing-by-synthesis US10329608B2 (en) 2012-10-10 2019-06-25 Life Technologies Corporation Methods, systems, and computer readable media for repeat sequencing US10379079B2 (en) 2014-12-18 2019-08-13 Life Technologies Corporation Methods and apparatus for measuring analytes using large scale FET arrays US10410739B2 (en) 2013-10-04 2019-09-10 Life Technologies Corporation Methods and systems for modeling phasing effects in sequencing using termination chemistry US10451585B2 (en) 2009-05-29 2019-10-22 Life Technologies Corporation Methods and apparatus for measuring analytes US10458942B2 (en) 2013-06-10 2019-10-29 Life Technologies Corporation Chemical sensor array having multiple sensors per well US10605767B2 (en) 2014-12-18 2020-03-31 Life Technologies Corporation High data rate integrated circuit with transmitter configuration US10612017B2 (en) 2009-05-29 2020-04-07 Life Technologies Corporation Scaffolded nucleic acid polymer particles and methods of making and using US10619205B2 (en) 2016-05-06 2020-04-14 Life Technologies Corporation Combinatorial barcode sequences, and related systems and methods US10679724B2 (en) 2012-05-11 2020-06-09 Life Technologies Corporation Models for analyzing data from sequencing-by-synthesis operations US10676787B2 (en) 2014-10-13 2020-06-09 Life Technologies Corporation Methods, systems, and computer-readable media for accelerated base calling US10704164B2 (en) 2011-08-31 2020-07-07 Life Technologies Corporation Methods, systems, computer readable media, and kits for sample identification US10978174B2 (en) 2015-05-14 2021-04-13 Life Technologies Corporation Barcode sequences, and related systems and methods US11231451B2 (en) 2010-06-30 2022-01-25 Life Technologies Corporation Methods and apparatus for testing ISFET arrays US11307166B2 (en) 2010-07-01 2022-04-19 Life Technologies Corporation Column ADC US11339430B2 (en) 2007-07-10 2022-05-24 Life Technologies Corporation Methods and apparatus for measuring analytes using large scale FET arrays US11474070B2 (en) 2010-12-30 2022-10-18 Life Technologies Corporation Methods, systems, and computer readable media for making base calls in nucleic acid sequencing US11636919B2 (en) 2013-03-14 2023-04-25 Life Technologies Corporation Methods, systems, and computer readable media for evaluating variant likelihood US12146189B2 (en) 2011-08-31 2024-11-19 Life Technologies Corporation Methods, systems, computer readable media, and kits for sample identification US12338492B2 (en) 2019-08-08 2025-06-24 Life Technologies Corporation Alternative nucleotide flows in sequencing-by-synthesis methods Families Citing this family (516) * Cited by examiner, â Cited by third party Publication number Priority date Publication date Assignee Title WO1994003624A1 (en) * 1992-08-04 1994-02-17 Auerbach Jeffrey I Methods for the isothermal amplification of nucleic acid molecules US6261808B1 (en) * 1992-08-04 2001-07-17 Replicon, Inc. Amplification of nucleic acid molecules via circular replicons US7875440B2 (en) 1998-05-01 2011-01-25 Arizona Board Of Regents Method of determining the nucleotide sequence of oligonucleotides and DNA molecules US6780591B2 (en) 1998-05-01 2004-08-24 Arizona Board Of Regents Method of determining the nucleotide sequence of oligonucleotides and DNA molecules US6818395B1 (en) 1999-06-28 2004-11-16 California Institute Of Technology Methods and apparatus for analyzing polynucleotide sequences US7244559B2 (en) * 1999-09-16 2007-07-17 454 Life Sciences Corporation Method of sequencing a nucleic acid US7211414B2 (en) 2000-12-01 2007-05-01 Visigen Biotechnologies, Inc. Enzymatic nucleic acid synthesis: compositions and methods for altering monomer incorporation fidelity EP1368497A4 (en) 2001-03-12 2007-08-15 California Inst Of Techn METHOD AND DEVICE FOR ANALYZING POLYNUCLEOTIDE SEQUENCES BY ASYNCHRONOUS BASE EXTENSION JP2002306180A (en) * 2001-04-16 2002-10-22 Hitachi Ltd Nucleotide base sequence analysis method, nucleobase sequence analysis reagent kit and nucleobase sequence analyzer US20030096268A1 (en) * 2001-07-06 2003-05-22 Michael Weiner Method for isolation of independent, parallel chemical micro-reactions using a porous filter US7595883B1 (en) 2002-09-16 2009-09-29 The Board Of Trustees Of The Leland Stanford Junior University Biological analysis arrangement and approach therefor CN101128601B (en) 2003-01-29 2011-06-08 454çå½ç§å¦å ¬å¸ Methods of amplifying and sequencing nucleic acids US7575865B2 (en) * 2003-01-29 2009-08-18 454 Life Sciences Corporation Methods of amplifying and sequencing nucleic acids JP2006516743A (en) * 2003-02-05 2006-07-06 㶠ã¸ã§ãã©ã« ãã¹ãã¿ã« ã³ã¼ãã¬ã¼ã·ã§ã³ Microchip-based system for HIV diagnostic related applications US8003374B2 (en) 2003-03-25 2011-08-23 The Regents Of The University Of California Reagentless, reusable, bioelectronic detectors US20040191801A1 (en) * 2003-03-25 2004-09-30 Heeger Alan J. Reagentless, reusable bioelectronic detectors and their use as authentication devices CA2531105C (en) 2003-07-05 2015-03-17 The Johns Hopkins University Method and compositions for detection and enumeration of genetic variations US7169560B2 (en) 2003-11-12 2007-01-30 Helicos Biosciences Corporation Short cycle methods for sequencing polynucleotides WO2005054431A2 (en) * 2003-12-01 2005-06-16 454 Corporation Method for isolation of independent, parallel chemical micro-reactions using a porous filter WO2005080606A1 (en) * 2004-02-18 2005-09-01 Xiaochuan Zhou Fluidic devices and methods for multiplex chemical and biochemical reactions EP1716254B1 (en) 2004-02-19 2010-04-07 Helicos Biosciences Corporation Methods for analyzing polynucleotide sequences GB2413796B (en) * 2004-03-25 2006-03-29 Global Genomics Ab Methods and means for nucleic acid sequencing US7622281B2 (en) 2004-05-20 2009-11-24 The Board Of Trustees Of The Leland Stanford Junior University Methods and compositions for clonal amplification of nucleic acid JP2008512084A (en) 2004-05-25 2008-04-24 ããªã³ã¹ ãã¤ãªãµã¤ã¨ã³ã·ã¼ãº ã³ã¼ãã¬ã¤ã·ã§ã³ Methods and devices for nucleic acid sequencing US7476734B2 (en) 2005-12-06 2009-01-13 Helicos Biosciences Corporation Nucleotide analogs US20060024711A1 (en) * 2004-07-02 2006-02-02 Helicos Biosciences Corporation Methods for nucleic acid amplification and sequence determination EP3415641B1 (en) * 2004-09-17 2023-11-01 Pacific Biosciences Of California, Inc. Method for analysis of molecules US7170050B2 (en) 2004-09-17 2007-01-30 Pacific Biosciences Of California, Inc. Apparatus and methods for optical analysis of molecules US7785785B2 (en) 2004-11-12 2010-08-31 The Board Of Trustees Of The Leland Stanford Junior University Charge perturbation detection system for DNA and other molecules US7629173B2 (en) * 2004-12-29 2009-12-08 Corning Incorporated Optical reader system and method for monitoring and correcting lateral and angular misalignments of label independent biosensors US7482120B2 (en) 2005-01-28 2009-01-27 Helicos Biosciences Corporation Methods and compositions for improving fidelity in a nucleic acid synthesis reaction CA2596496A1 (en) 2005-02-01 2006-08-10 Agencourt Bioscience Corp. Reagents, methods, and libraries for bead-based sequencing EP2236628A3 (en) 2005-02-01 2010-10-13 AB Advanced Genetic Analysis Corporation Reagents, methods and libraries for bead-based sequencing CA2967430C (en) * 2005-03-10 2018-05-08 Gen-Probe Incorporated Systems and methods to perform assays for detecting or quantifying analytes within samples US7785862B2 (en) 2005-04-07 2010-08-31 454 Life Sciences Corporation Thin film coated microwell arrays US7682816B2 (en) * 2005-04-07 2010-03-23 454 Life Sciences Corporation Thin film coated microwell arrays and methods of using same US20060228721A1 (en) * 2005-04-12 2006-10-12 Leamon John H Methods for determining sequence variants using ultra-deep sequencing US9410889B2 (en) * 2005-06-10 2016-08-09 Applied Biosystem, Llc Method and system for multiplex genetic analysis US7666593B2 (en) 2005-08-26 2010-02-23 Helicos Biosciences Corporation Single molecule sequencing of captured nucleic acids US7981365B2 (en) * 2005-09-15 2011-07-19 The United States Of America As Represented By The Secretary Of The Navy Electrospray coating of aerosols for labeling and identification WO2007044245A2 (en) * 2005-10-07 2007-04-19 Callida Genomics, Inc. Self-assembled single molecule arrays and uses thereof US7960104B2 (en) 2005-10-07 2011-06-14 Callida Genomics, Inc. Self-assembled single molecule arrays and uses thereof WO2007120208A2 (en) * 2005-11-14 2007-10-25 President And Fellows Of Harvard College Nanogrid rolling circle dna sequencing DE102005056016A1 (en) * 2005-11-17 2007-05-31 Leibniz-Institut Für Polymerforschung Dresden E.V. Structurable polymer layer composite, process for its preparation and application US7803542B2 (en) * 2005-11-29 2010-09-28 The Regents Of The University Of California Signal-on architecture for electronic, oligonucleotide-based detectors US20070168197A1 (en) * 2006-01-18 2007-07-19 Nokia Corporation Audio coding WO2008030631A2 (en) 2006-02-03 2008-03-13 Microchip Biotechnologies, Inc. Microfluidic devices US8460879B2 (en) 2006-02-21 2013-06-11 The Trustees Of Tufts College Methods and arrays for target analyte detection and determination of target analyte concentration in solution US11237171B2 (en) 2006-02-21 2022-02-01 Trustees Of Tufts College Methods and arrays for target analyte detection and determination of target analyte concentration in solution US7397546B2 (en) 2006-03-08 2008-07-08 Helicos Biosciences Corporation Systems and methods for reducing detected intensity non-uniformity in a laser beam US8071296B2 (en) 2006-03-13 2011-12-06 Agency For Science, Technology And Research Nucleic acid interaction analysis WO2007108432A1 (en) * 2006-03-20 2007-09-27 Mitsui Chemicals, Inc. Optical film and method for producing same WO2007121489A2 (en) * 2006-04-19 2007-10-25 Applera Corporation Reagents, methods, and libraries for gel-free bead-based sequencing US10522240B2 (en) 2006-05-03 2019-12-31 Population Bio, Inc. Evaluating genetic disorders US7702468B2 (en) 2006-05-03 2010-04-20 Population Diagnostics, Inc. Evaluating genetic disorders CN101501088B (en) 2006-06-06 2011-11-30 é康å®å ¬å¸ A silicone acrylate hybrid composition US8614278B2 (en) 2006-06-06 2013-12-24 Dow Corning Corporation Silicone acrylate hybrid composition and method of making same US8569416B2 (en) 2006-06-06 2013-10-29 Dow Corning Corporation Single phase silicone acrylate formulation EP2366801B1 (en) 2006-06-14 2016-10-19 Verinata Health, Inc Methods for the diagnosis of fetal abnormalities US20080050739A1 (en) 2006-06-14 2008-02-28 Roland Stoughton Diagnosis of fetal abnormalities using polymorphisms including short tandem repeats US8137912B2 (en) 2006-06-14 2012-03-20 The General Hospital Corporation Methods for the diagnosis of fetal abnormalities US20080070792A1 (en) * 2006-06-14 2008-03-20 Roland Stoughton Use of highly parallel snp genotyping for fetal diagnosis EP2589668A1 (en) 2006-06-14 2013-05-08 Verinata Health, Inc Rare cell analysis using sample splitting and DNA tags JP2008109864A (en) * 2006-10-30 2008-05-15 Hitachi Ltd Genetic sequence analysis system US8338109B2 (en) 2006-11-02 2012-12-25 Mayo Foundation For Medical Education And Research Predicting cancer outcome US20080221832A1 (en) * 2006-11-09 2008-09-11 Complete Genomics, Inc. Methods for computing positional base probabilities using experminentals base value distributions US20090111705A1 (en) 2006-11-09 2009-04-30 Complete Genomics, Inc. Selection of dna adaptor orientation by hybrid capture EP2518162B1 (en) 2006-11-15 2018-03-07 Biospherex LLC Multitag sequencing and ecogenomics analysis US7813013B2 (en) * 2006-11-21 2010-10-12 Illumina, Inc. Hexagonal site line scanning method and system US20080242560A1 (en) * 2006-11-21 2008-10-02 Gunderson Kevin L Methods for generating amplified nucleic acid arrays WO2008127455A2 (en) * 2006-12-05 2008-10-23 Liquidia Technologies, Inc. Nanoarrays and methods and materials for fabricating same US20100021915A1 (en) * 2006-12-11 2010-01-28 Thomas Jefferson University High throughput dna sequencing method and apparatus US7932034B2 (en) 2006-12-20 2011-04-26 The Board Of Trustees Of The Leland Stanford Junior University Heat and pH measurement for sequencing of DNA US8617816B2 (en) * 2007-03-16 2013-12-31 454 Life Sciences, A Roche Company System and method for detection of HIV drug resistant variants US20080243865A1 (en) * 2007-03-28 2008-10-02 Oracle International Corporation Maintaining global state of distributed transaction managed by an external transaction manager for clustered database systems CA2689356A1 (en) * 2007-06-01 2008-12-11 454 Life Sciences Corporation System and meth0d for identification of individual samples from a multiplex mixture US20110105366A1 (en) 2007-06-18 2011-05-05 Illumina, Inc. Microfabrication methods for the optimal patterning of substrates JP2010531664A (en) 2007-06-28 2010-09-30 ï¼ï¼ï¼ ã©ã¤ã ãµã¤ã¨ã³ã·ã¼ãº ã³ã¼ãã¬ã¤ã·ã§ã³ System and method for adaptive reagent control in nucleic acid sequencing JP5020734B2 (en) * 2007-07-31 2012-09-05 æ ªå¼ä¼ç¤¾æ¥ç«ãã¤ãã¯ããã¸ã¼ãº Nucleic acid analysis method and apparatus ES2556627T3 (en) 2007-08-30 2016-01-19 Trustees Of Tufts College Methods to determine the concentration of an analyte in solution MX2010003724A (en) * 2007-10-04 2010-09-14 Halcyon Molecular Sequencing nucleic acid polymers with electron microscopy. WO2009052214A2 (en) * 2007-10-15 2009-04-23 Complete Genomics, Inc. Sequence analysis using decorated nucleic acids US7767441B2 (en) * 2007-10-25 2010-08-03 Industrial Technology Research Institute Bioassay system including optical detection apparatuses, and method for detecting biomolecules US7811810B2 (en) 2007-10-25 2010-10-12 Industrial Technology Research Institute Bioassay system including optical detection apparatuses, and method for detecting biomolecules US8415099B2 (en) 2007-11-05 2013-04-09 Complete Genomics, Inc. Efficient base determination in sequencing reactions US8298768B2 (en) * 2007-11-29 2012-10-30 Complete Genomics, Inc. Efficient shotgun sequencing methods US7901890B2 (en) * 2007-11-05 2011-03-08 Complete Genomics, Inc. Methods and oligonucleotide designs for insertion of multiple adaptors employing selective methylation US7897344B2 (en) * 2007-11-06 2011-03-01 Complete Genomics, Inc. Methods and oligonucleotide designs for insertion of multiple adaptors into library constructs US8518640B2 (en) * 2007-10-29 2013-08-27 Complete Genomics, Inc. Nucleic acid sequencing and process CN101910399B (en) * 2007-10-30 2015-11-25 èå©è¾¾åºå ç»è¡ä»½æéå ¬å¸ For the device of high throughput sequencing of nucleic acids US9551026B2 (en) 2007-12-03 2017-01-24 Complete Genomincs, Inc. Method for nucleic acid detection using voltage enhancement US8592150B2 (en) 2007-12-05 2013-11-26 Complete Genomics, Inc. Methods and compositions for long fragment read sequencing WO2009076323A2 (en) 2007-12-06 2009-06-18 Genalyte Inc. Monitoring enzymatic process US8815576B2 (en) * 2007-12-27 2014-08-26 Lawrence Livermore National Security, Llc. Chip-based sequencing nucleic acids US20090181390A1 (en) * 2008-01-11 2009-07-16 Signosis, Inc. A California Corporation High throughput detection of micrornas and use for disease diagnosis ES2990227T3 (en) * 2008-01-17 2024-11-29 Sequenom Inc Single-sequence nucleic acid analysis compositions and methods WO2009094583A1 (en) * 2008-01-23 2009-07-30 Complete Genomics, Inc. Methods and compositions for preventing bias in amplification and sequencing reactions WO2009097368A2 (en) 2008-01-28 2009-08-06 Complete Genomics, Inc. Methods and compositions for efficient base calling in sequencing reactions US20090203086A1 (en) * 2008-02-06 2009-08-13 454 Life Sciences Corporation System and method for improved signal detection in nucleic acid sequencing EP3045542B1 (en) 2008-03-28 2016-11-16 Pacific Biosciences of California, Inc. Methods for nucleic acid sequencing GB0809069D0 (en) 2008-05-19 2008-06-25 Univ Leuven Kath Gene signatures WO2009132028A1 (en) * 2008-04-21 2009-10-29 Complete Genomics, Inc. Array structures for nucleic acid detection DE102008001322A1 (en) * 2008-04-22 2009-10-29 Linos Photonics Gmbh & Co. Kg Sample array analysis system for use in e.g. pharma research, has detector detecting luminescence radiation emitted by samples, and light conductor array arranged in front of sample plate for conducting light on samples US8039817B2 (en) 2008-05-05 2011-10-18 Illumina, Inc. Compensator for multiple surface imaging US20110105356A1 (en) * 2008-05-07 2011-05-05 Derosier Chad F Compositions and methods for providing substances to and from an array CA2725978A1 (en) 2008-05-28 2009-12-03 Genomedx Biosciences, Inc. Systems and methods for expression-based discrimination of distinct clinical disease states in prostate cancer US10407731B2 (en) 2008-05-30 2019-09-10 Mayo Foundation For Medical Education And Research Biomarker panels for predicting prostate cancer outcomes CN101368206B (en) * 2008-07-16 2012-08-22 æ·±å³åå 康åºå ç§ææéå ¬å¸ Sequencing reaction small chamber, gene sequencing reaction bench and gene sequencing device WO2010009426A2 (en) * 2008-07-17 2010-01-21 Life Technologies Corporation Devices and methods for reagent delivery US20100036110A1 (en) * 2008-08-08 2010-02-11 Xiaoliang Sunney Xie Methods and compositions for continuous single-molecule nucleic acid sequencing by synthesis with fluorogenic nucleotides US20100035252A1 (en) * 2008-08-08 2010-02-11 Ion Torrent Systems Incorporated Methods for sequencing individual nucleic acids under tension US20100227327A1 (en) * 2008-08-08 2010-09-09 Xiaoliang Sunney Xie Methods and compositions for continuous single-molecule nucleic acid sequencing by synthesis with fluorogenic nucleotides US8583380B2 (en) 2008-09-05 2013-11-12 Aueon, Inc. Methods for stratifying and annotating cancer drug treatment options US20110244448A1 (en) * 2008-09-08 2011-10-06 Masataka Shirai Dna detecting apparatus, dna detecting device and dna detecting method US8383345B2 (en) 2008-09-12 2013-02-26 University Of Washington Sequence tag directed subassembly of short sequencing reads into long sequencing reads SI2562268T1 (en) 2008-09-20 2017-05-31 The Board of Trustees of the Leland Stanford Junior University Office of the General Counsel Building 170 Noninvasive diagnosis of fetal aneuploidy by sequencing US8222047B2 (en) 2008-09-23 2012-07-17 Quanterix Corporation Ultra-sensitive detection of molecules on single molecule arrays US20100075862A1 (en) * 2008-09-23 2010-03-25 Quanterix Corporation High sensitivity determination of the concentration of analyte molecules or particles in a fluid sample US20100075439A1 (en) * 2008-09-23 2010-03-25 Quanterix Corporation Ultra-sensitive detection of molecules by capture-and-release using reducing agents followed by quantification EP2347247B1 (en) 2008-10-27 2019-06-26 Genalyte, Inc. Biosensors based on optical probing and sensing US8486630B2 (en) 2008-11-07 2013-07-16 Industrial Technology Research Institute Methods for accurate sequence data and modified base position determination US9495515B1 (en) 2009-12-09 2016-11-15 Veracyte, Inc. Algorithms for disease diagnostics US10236078B2 (en) 2008-11-17 2019-03-19 Veracyte, Inc. Methods for processing or analyzing a sample of thyroid tissue GB2507680B (en) * 2008-11-17 2014-06-18 Veracyte Inc Methods and compositions of molecular profiling for disease diagnostics WO2010068702A2 (en) * 2008-12-10 2010-06-17 Illumina, Inc. Methods and compositions for hybridizing nucleic acids EP2389453B1 (en) * 2009-01-20 2015-11-25 The Board of Trustees of The Leland Stanford Junior University Single cell gene expression for diagnosis, prognosis and identification of drug targets US9074258B2 (en) 2009-03-04 2015-07-07 Genomedx Biosciences Inc. Compositions and methods for classifying thyroid nodule disease US20100285970A1 (en) * 2009-03-31 2010-11-11 Rose Floyd D Methods of sequencing nucleic acids AU2010242073C1 (en) 2009-04-30 2015-12-24 Good Start Genetics, Inc. Methods and compositions for evaluating genetic markers US9085798B2 (en) 2009-04-30 2015-07-21 Prognosys Biosciences, Inc. Nucleic acid constructs and methods of use US12129514B2 (en) 2009-04-30 2024-10-29 Molecular Loop Biosolutions, Llc Methods and compositions for evaluating genetic markers US8669057B2 (en) 2009-05-07 2014-03-11 Veracyte, Inc. Methods and compositions for diagnosis of thyroid conditions US9524369B2 (en) 2009-06-15 2016-12-20 Complete Genomics, Inc. Processing and analysis of complex nucleic acid sequence data EP2280080A1 (en) * 2009-07-31 2011-02-02 Qiagen GmbH Method of normalized quantification of nucleic acids using anchor oligonucleotides and adapter oligonucleotides US10174368B2 (en) * 2009-09-10 2019-01-08 Centrillion Technology Holdings Corporation Methods and systems for sequencing long nucleic acids CN102858995B (en) 2009-09-10 2016-10-26 森ç¹çéææ¯æ§è¡å ¬å¸ Targeting sequence measurement US20110071033A1 (en) 2009-09-23 2011-03-24 Celmatix, Inc. Methods and devices for assessing infertility and/or egg quality EP2496720B1 (en) 2009-11-06 2020-07-08 The Board of Trustees of the Leland Stanford Junior University Non-invasive diagnosis of graft rejection in organ transplant patients JP5441035B2 (en) * 2009-11-06 2014-03-12 å½ç«å¤§å¦æ³äººåé¸å 端ç§å¦æè¡å¤§å¦é¢å¤§å¦ Sample analyzer US8772016B2 (en) 2009-11-13 2014-07-08 Pacific Biosciences Of California, Inc. Sealed chip package US10446272B2 (en) 2009-12-09 2019-10-15 Veracyte, Inc. Methods and compositions for classification of samples US20110312503A1 (en) 2010-01-23 2011-12-22 Artemis Health, Inc. Methods of fetal abnormality detection EP2537031A4 (en) 2010-02-18 2013-07-17 Anthony P Shuber Compositions and methods for treating cancer US8236574B2 (en) 2010-03-01 2012-08-07 Quanterix Corporation Ultra-sensitive detection of molecules or particles using beads or other capture objects US8415171B2 (en) 2010-03-01 2013-04-09 Quanterix Corporation Methods and systems for extending dynamic range in assays for the detection of molecules or particles EP2542890B1 (en) 2010-03-01 2015-05-06 Quanterix Corporation Methods for extending dynamic range in assays for the detection of molecules or particles US9678068B2 (en) * 2010-03-01 2017-06-13 Quanterix Corporation Ultra-sensitive detection of molecules using dual detection methods US10787701B2 (en) 2010-04-05 2020-09-29 Prognosys Biosciences, Inc. Spatially encoded biological assays US20190300945A1 (en) 2010-04-05 2019-10-03 Prognosys Biosciences, Inc. Spatially Encoded Biological Assays SI2556171T1 (en) 2010-04-05 2016-03-31 Prognosys Biosciences, Inc. Spatially encoded biological assays WO2011140433A2 (en) 2010-05-07 2011-11-10 The Board Of Trustees Of The Leland Stanford Junior University Measurement and comparison of immune diversity by high-throughput sequencing WO2011143361A2 (en) 2010-05-11 2011-11-17 Veracyte, Inc. Methods and compositions for diagnosing conditions US20110287432A1 (en) 2010-05-21 2011-11-24 454 Life Science Corporation System and method for tailoring nucleotide concentration to enzymatic efficiencies in dna sequencing technologies EP2601609B1 (en) 2010-08-02 2017-05-17 Population Bio, Inc. Compositions and methods for discovery of causative mutations in genetic disorders US9121058B2 (en) 2010-08-20 2015-09-01 Integenx Inc. Linear valve arrays US8483969B2 (en) 2010-09-17 2013-07-09 Illuminia, Inc. Variation analysis for multiple templates on a solid support EP2619329B1 (en) 2010-09-24 2019-05-22 The Board of Trustees of The Leland Stanford Junior University Direct capture, amplification and sequencing of target dna using immobilized primers WO2012050920A1 (en) 2010-09-29 2012-04-19 Illumina, Inc. Compositions and methods for sequencing nucleic acids US20120077716A1 (en) 2010-09-29 2012-03-29 454 Life Sciences Corporation System and method for producing functionally distinct nucleic acid library ends through use of deoxyinosine US9074251B2 (en) 2011-02-10 2015-07-07 Illumina, Inc. Linking sequence reads using paired code tags EP3733866B1 (en) 2010-11-05 2023-11-15 Genalyte, Inc. Optical analyte detection systems and methods of use US20130267443A1 (en) 2010-11-19 2013-10-10 The Regents Of The University Of Michigan ncRNA AND USES THEREOF EP2561102A1 (en) 2010-12-02 2013-02-27 Katholieke Universiteit Leuven K.U. Leuven R&D Irak-related interventions and diagnosis EP2652659B1 (en) 2010-12-14 2020-04-15 Life Technologies Corporation Systems and methods for run-time sequencing run quality monitoring US9163281B2 (en) 2010-12-23 2015-10-20 Good Start Genetics, Inc. Methods for maintaining the integrity and identification of a nucleic acid template in a multiplex sequencing reaction CN102146442B (en) * 2010-12-31 2014-10-22 æ·±å³åå 康åºå ç§ææéå ¬å¸ Method and system for controlling sequencing process of gene sequencer CN102174384B (en) * 2011-01-05 2014-04-02 æ·±å³åå 康åºå ç§ææéå ¬å¸ Method and system for controlling sequencing and signal processing of gene sequencer WO2012100194A1 (en) 2011-01-20 2012-07-26 Ibis Biosciences, Inc. Microfluidic transducer US9952237B2 (en) 2011-01-28 2018-04-24 Quanterix Corporation Systems, devices, and methods for ultra-sensitive detection of molecules or particles WO2012106385A2 (en) 2011-01-31 2012-08-09 Apprise Bio, Inc. Methods of identifying multiple epitopes in cells CN103443338B (en) 2011-02-02 2017-09-22 åç顿大å¦åä¸åä¸å¿ Massively parallel continguity mapping CN103842379A (en) 2011-03-09 2014-06-04 ç»èä¿¡å·ç§æå ¬å¸ Methods and reagents for creating monoclonal antibodies US20120252682A1 (en) 2011-04-01 2012-10-04 Maples Corporate Services Limited Methods and systems for sequencing nucleic acids US20140302532A1 (en) 2011-04-12 2014-10-09 Quanterix Corporation Methods of determining a treatment protocol for and/or a prognosis of a patient's recovery from a brain injury GB201106254D0 (en) 2011-04-13 2011-05-25 Frisen Jonas Method and product US9476095B2 (en) 2011-04-15 2016-10-25 The Johns Hopkins University Safe sequencing system US10373705B2 (en) 2011-06-07 2019-08-06 Koninklijke Philips N.V. Providing nucleotide sequence data EP2718465B1 (en) 2011-06-09 2022-04-13 Illumina, Inc. Method of making an analyte array DE102011054101A1 (en) 2011-09-30 2013-04-04 Albert-Ludwigs-Universität Freiburg Method for the spatial arrangement of sample fragments for amplification and immobilization for further derivatizations US10221454B2 (en) 2011-10-10 2019-03-05 The Hospital For Sick Children Methods and compositions for screening and treating developmental disorders WO2013058907A1 (en) 2011-10-17 2013-04-25 Good Start Genetics, Inc. Analysis methods US9206418B2 (en) 2011-10-19 2015-12-08 Nugen Technologies, Inc. Compositions and methods for directional nucleic acid amplification and sequencing US10865440B2 (en) 2011-10-21 2020-12-15 IntegenX, Inc. Sample preparation, processing and analysis systems US20150136604A1 (en) 2011-10-21 2015-05-21 Integenx Inc. Sample preparation, processing and analysis systems DE102011085473A1 (en) 2011-10-28 2013-05-02 Albert-Ludwigs-Universität Freiburg Method for the identification of aptamers CA2856163C (en) 2011-10-28 2019-05-07 Illumina, Inc. Microarray fabrication system and method US10837879B2 (en) 2011-11-02 2020-11-17 Complete Genomics, Inc. Treatment for stabilizing nucleic acid arrays DK2773779T3 (en) 2011-11-04 2020-11-23 Population Bio Inc METHODS AND COMPOSITIONS FOR DIAGNOSIS, FORECAST AND PREVENTION OF NEUROLOGICAL CONDITIONS WO2013070634A1 (en) 2011-11-07 2013-05-16 Ingenuity Systems, Inc. Methods and systems for identification of causal genomic variants JP6093498B2 (en) * 2011-12-13 2017-03-08 æ ªå¼ä¼ç¤¾æ¥ç«ãã¤ãã¯ããã¸ã¼ãº Nucleic acid amplification method US10513737B2 (en) 2011-12-13 2019-12-24 Decipher Biosciences, Inc. Cancer diagnostics using non-coding transcripts US9823246B2 (en) 2011-12-28 2017-11-21 The Board Of Trustees Of The Leland Stanford Junior University Fluorescence enhancing plasmonic nanoscopic gold films and assays based thereon CN104334739A (en) 2012-01-13 2015-02-04 Dataçç©æéå ¬å¸ Genotyping by next-generation sequencing WO2013112923A1 (en) 2012-01-26 2013-08-01 Nugen Technologies, Inc. Compositions and methods for targeted nucleic acid sequence enrichment and high efficiency library generation CA2863887C (en) 2012-02-09 2023-01-03 Population Diagnostics, Inc. Methods of screening low frequency gdna variation biomarkers for pervasive developmental disorder (pdd) or pervasive developmental disorder - not otherwise specified (pdd_nos) ES2855130T3 (en) 2012-02-17 2021-09-23 Hutchinson Fred Cancer Res Compositions and methods to accurately identify mutations US9803239B2 (en) 2012-03-29 2017-10-31 Complete Genomics, Inc. Flow cells for high density array chips US8209130B1 (en) 2012-04-04 2012-06-26 Good Start Genetics, Inc. Sequence assembly US8812422B2 (en) 2012-04-09 2014-08-19 Good Start Genetics, Inc. Variant database US20130274148A1 (en) 2012-04-11 2013-10-17 Illumina, Inc. Portable genetic detection and analysis system and method US10227635B2 (en) 2012-04-16 2019-03-12 Molecular Loop Biosolutions, Llc Capture reactions US8906320B1 (en) 2012-04-16 2014-12-09 Illumina, Inc. Biosensors for biological or chemical analysis and systems and methods for same EP2659977B1 (en) * 2012-05-02 2019-04-24 IMEC vzw Microfluidics system for sequencing CN104603287B (en) 2012-05-10 2018-01-19 éç¨å»çå ¬å¸ Method for determining nucleotide sequence US10192024B2 (en) 2012-05-18 2019-01-29 454 Life Sciences Corporation System and method for generation and use of optimal nucleotide flow orders CN102703311B (en) * 2012-05-24 2013-08-21 ä¸å½ç§å¦é¢å京åºå ç»ç 究æ Reaction bin for adaptive regulation of DNA (Deoxyribose Nucleic Acid) sequencer CN102703302B (en) * 2012-05-24 2013-11-27 ä¸å½ç§å¦é¢å京åºå ç»ç 究æ Mounting seat for automatic clamping of sequencing chips CN102703310B (en) * 2012-05-24 2013-10-16 ä¸å½ç§å¦é¢å京åºå ç»ç 究æ CCD (charge coupled device) camera directly coupled with sequencing chip CN102703301A (en) * 2012-05-24 2012-10-03 ä¸å½ç§å¦é¢å京åºå ç»ç 究æ Darkroom used between installing seat of sequencing chip and CCD (Charge Coupled Device) camera US9012022B2 (en) 2012-06-08 2015-04-21 Illumina, Inc. Polymer coatings US8895249B2 (en) 2012-06-15 2014-11-25 Illumina, Inc. Kinetic exclusion amplification of nucleic acid libraries WO2013191775A2 (en) 2012-06-18 2013-12-27 Nugen Technologies, Inc. Compositions and methods for negative selection of non-desired nucleic acid sequences US20150011396A1 (en) 2012-07-09 2015-01-08 Benjamin G. Schroeder Methods for creating directional bisulfite-converted nucleic acid libraries for next generation sequencing WO2014026032A2 (en) 2012-08-08 2014-02-13 Apprise Bio, Inc. Increasing dynamic range for identifying multiple epitopes in cells ES3015226T3 (en) 2012-08-16 2025-04-30 Veracyte Sd Inc Prostate cancer prognosis using biomarkers WO2014043519A1 (en) 2012-09-14 2014-03-20 Population Diagnostics Inc. Methods and compositions for diagnosing, prognosing, and treating neurological conditions CN103092090B (en) * 2012-09-14 2015-04-08 çå¸æ½¼ Sequencing control system US10233495B2 (en) 2012-09-27 2019-03-19 The Hospital For Sick Children Methods and compositions for screening and treating developmental disorders WO2014060483A1 (en) 2012-10-17 2014-04-24 Spatial Transcriptomics Ab Methods and product for optimising localised or spatial detection of gene expression in a tissue sample US9177098B2 (en) 2012-10-17 2015-11-03 Celmatix Inc. Systems and methods for determining the probability of a pregnancy at a selected point in time US10162800B2 (en) 2012-10-17 2018-12-25 Celmatix Inc. Systems and methods for determining the probability of a pregnancy at a selected point in time US9181583B2 (en) 2012-10-23 2015-11-10 Illumina, Inc. HLA typing using selective amplification and sequencing AU2013338393C1 (en) 2012-10-29 2024-07-25 The Johns Hopkins University Papanicolaou test for ovarian and endometrial cancers US9836577B2 (en) 2012-12-14 2017-12-05 Celmatix, Inc. Methods and devices for assessing risk of female infertility EP2746405B1 (en) 2012-12-23 2015-11-04 HS Diagnomics GmbH Methods and primer sets for high throughput PCR sequencing US9683230B2 (en) 2013-01-09 2017-06-20 Illumina Cambridge Limited Sample preparation on a solid support WO2014113502A1 (en) 2013-01-15 2014-07-24 Quanterix Corporation Detection of dna or rna using single molecule arrays and other techniques EP4414990A3 (en) 2013-01-17 2024-11-06 Personalis, Inc. Methods and systems for genetic analysis WO2014116729A2 (en) 2013-01-22 2014-07-31 The Board Of Trustees Of The Leland Stanford Junior University Haplotying of hla loci with ultra-deep shotgun sequencing US9411930B2 (en) 2013-02-01 2016-08-09 The Regents Of The University Of California Methods for genome assembly and haplotype phasing EP3885446A1 (en) 2013-02-01 2021-09-29 The Regents of The University of California Methods for genome assembly and haplotype phasing US9512422B2 (en) 2013-02-26 2016-12-06 Illumina, Inc. Gel patterned surfaces DK2970951T3 (en) 2013-03-13 2019-05-13 Illumina Inc PROCEDURES FOR NUCLEAR ACID SEQUENCE EP2971159B1 (en) 2013-03-14 2019-05-08 Molecular Loop Biosolutions, LLC Methods for analyzing nucleic acids US10421996B2 (en) 2013-03-14 2019-09-24 Illumina, Inc. Modified polymerases for improved incorporation of nucleotide analogues US11976329B2 (en) 2013-03-15 2024-05-07 Veracyte, Inc. Methods and systems for detecting usual interstitial pneumonia DK2970958T3 (en) 2013-03-15 2018-02-19 Lineage Biosciences Inc METHODS FOR SEQUENCING THE IMMUN REPERTOIR WO2014151117A1 (en) 2013-03-15 2014-09-25 The Board Of Trustees Of The Leland Stanford Junior University Identification and use of circulating nucleic acid tumor markers BR112015022448B1 (en) 2013-03-15 2020-12-08 Illumina Cambridge Limited modified nucleotide or nucleoside molecule, methods for preparing the growth of polynucleotide complementary to single-stranded target polynucleotide in sequencing reaction and to determine the sequence of single-stranded target polynucleotide and kit US20140274747A1 (en) * 2013-03-15 2014-09-18 Illumina, Inc. Super resolution imaging BR112015022490A2 (en) 2013-03-15 2017-07-18 Veracyte Inc methods and compositions for sample classification US9193998B2 (en) 2013-03-15 2015-11-24 Illumina, Inc. Super resolution imaging EP2971130A4 (en) 2013-03-15 2016-10-05 Nugen Technologies Inc Sequential sequencing WO2014143637A1 (en) 2013-03-15 2014-09-18 The Board Of Trustees Of The University Of Illinois Methods and compositions for enhancing immunoassays WO2014197377A2 (en) 2013-06-03 2014-12-11 Good Start Genetics, Inc. Methods and systems for storing sequence read data CA2916660C (en) 2013-06-25 2022-05-17 Prognosys Biosciences, Inc. Spatially encoded biological assays using a microfluidic device AU2014284584B2 (en) 2013-07-01 2019-08-01 Illumina, Inc. Catalyst-free surface functionalization and polymer grafting WO2015021080A2 (en) 2013-08-05 2015-02-12 Twist Bioscience Corporation De novo synthesized gene libraries EP3038834B1 (en) 2013-08-30 2018-12-12 Illumina, Inc. Manipulation of droplets on hydrophilic or variegated-hydrophilic surfaces EP3965111B8 (en) 2013-08-30 2025-06-11 Personalis, Inc. Methods for genomic analysis EP3296408A1 (en) 2013-09-05 2018-03-21 The Jackson Laboratory Compositions for rna-chromatin interaction analysis and uses thereof US9352315B2 (en) 2013-09-27 2016-05-31 Taiwan Semiconductor Manufacturing Company, Ltd. Method to produce chemical pattern in micro-fluidic structure GB2535066A (en) 2013-10-03 2016-08-10 Personalis Inc Methods for analyzing genotypes US11041203B2 (en) 2013-10-18 2021-06-22 Molecular Loop Biosolutions, Inc. Methods for assessing a genomic region of a subject US10851414B2 (en) 2013-10-18 2020-12-01 Good Start Genetics, Inc. Methods for determining carrier status GB2535382A (en) 2013-11-07 2016-08-17 Univ Leland Stanford Junior Cell-free nucleic acids for the analysis of the human microbiome and components thereof EP2891722B1 (en) 2013-11-12 2018-10-10 Population Bio, Inc. Methods and compositions for diagnosing, prognosing, and treating endometriosis CN105849264B (en) 2013-11-13 2019-09-27 纽äºææ¯å ¬å¸ For identifying the composition and method that repeat sequencing reading CN114471756B (en) 2013-11-18 2024-04-16 å°¹ç¹æ ¹åå æ¯æéå ¬å¸ Cartridge and instrument for sample analysis EP3077430A4 (en) 2013-12-05 2017-08-16 Centrillion Technology Holdings Corporation Modified surfaces US10597715B2 (en) 2013-12-05 2020-03-24 Centrillion Technology Holdings Methods for sequencing nucleic acids EP3628747B1 (en) 2013-12-05 2022-10-05 Centrillion Technology Holdings Corporation Fabrication of patterned arrays EP3540074A1 (en) 2013-12-11 2019-09-18 The Regents of the University of California Method of tagging internal regions of nucleic acid molecules WO2015095226A2 (en) 2013-12-20 2015-06-25 Illumina, Inc. Preserving genomic connectivity information in fragmented genomic dna samples CN106062212A (en) 2013-12-23 2016-10-26 ä¼é²ç±³é£è¡ä»½æéå ¬å¸ Structured substrates for improving detection of light emissions and methods relating to the same AU2014377537B2 (en) 2014-01-16 2021-02-25 Illumina, Inc. Amplicon preparation and sequencing on solid supports US9677132B2 (en) 2014-01-16 2017-06-13 Illumina, Inc. Polynucleotide modification on solid support WO2015112974A1 (en) 2014-01-27 2015-07-30 The General Hospital Corporation Methods of preparing nucleic acids for sequencing US20150211061A1 (en) * 2014-01-27 2015-07-30 The General Hospital Corporation Methods for determining a nucleotide sequence JP2015139373A (en) * 2014-01-27 2015-08-03 æ ªå¼ä¼ç¤¾æ¥ç«ãã¤ãã¯ããã¸ã¼ãº Biomolecule analytical device, and biomolecule analyzer WO2015127517A1 (en) 2014-02-27 2015-09-03 Katholieke Universiteit Leuven Oxidative stress and cardiovascular disease events US10704097B2 (en) 2014-02-27 2020-07-07 Katholieke Universiteit Leuven Oxidative stress and cardiovascular disease events US9745614B2 (en) 2014-02-28 2017-08-29 Nugen Technologies, Inc. Reduced representation bisulfite sequencing with diversity adaptors US11060139B2 (en) 2014-03-28 2021-07-13 Centrillion Technology Holdings Corporation Methods for sequencing nucleic acids US20170067097A1 (en) * 2014-04-11 2017-03-09 Redvault Biosciences, Lp Systems and methods for clonal replication and amplification of nucleic acid molecules for genomic and therapeutic applications WO2015175530A1 (en) 2014-05-12 2015-11-19 Gore Athurva Methods for detecting aneuploidy GB2544198B (en) 2014-05-21 2021-01-13 Integenx Inc Fluidic cartridge with valve mechanism US10443087B2 (en) 2014-06-13 2019-10-15 Illumina Cambridge Limited Methods and compositions for preparing sequencing libraries US10829814B2 (en) 2014-06-19 2020-11-10 Illumina, Inc. Methods and compositions for single cell genomics US10017759B2 (en) 2014-06-26 2018-07-10 Illumina, Inc. Library preparation of tagged nucleic acid EP3161154B1 (en) 2014-06-27 2020-04-15 Illumina, Inc. Modified polymerases for improved incorporation of nucleotide analogues CN106661561B (en) 2014-06-30 2020-10-30 亿æè¾¾è¡ä»½æéå ¬å¸ Methods and compositions using unilateral transposition US12297505B2 (en) 2014-07-14 2025-05-13 Veracyte, Inc. Algorithms for disease diagnostics WO2016011377A1 (en) 2014-07-17 2016-01-21 Celmatix Inc. Methods and systems for assessing infertility and related pathologies EP3172321B2 (en) 2014-07-21 2023-01-04 Illumina, Inc. Polynucleotide enrichment using crispr-cas systems US10526641B2 (en) 2014-08-01 2020-01-07 Dovetail Genomics, Llc Tagging nucleic acids for sequence assembly CN107075581B (en) 2014-08-06 2022-03-18 纽äºææ¯å ¬å¸ Digital measurement by targeted sequencing GB201414098D0 (en) 2014-08-08 2014-09-24 Illumina Cambridge Ltd Modified nucleotide linkers CN106796212A (en) 2014-08-12 2017-05-31 æ°ç代åæ©å ¬å¸ System and method for monitoring health based on the body fluid collected US9982250B2 (en) 2014-08-21 2018-05-29 Illumina Cambridge Limited Reversible surface functionalization WO2016036403A1 (en) 2014-09-05 2016-03-10 Population Diagnostics Inc. Methods and compositions for inhibiting and treating neurological conditions US11408024B2 (en) 2014-09-10 2022-08-09 Molecular Loop Biosciences, Inc. Methods for selectively suppressing non-target sequences EP3191606B1 (en) * 2014-09-12 2020-05-27 Illumina, Inc. Methods for detecting the presence of polymer subunits using chemiluminescence JP2017536087A (en) 2014-09-24 2017-12-07 ã°ãã ã¹ã¿ã¼ã ã¸ã§ããã£ã¯ã¹ï¼ ã¤ã³ã³ã¼ãã¬ã¤ããã Process control to increase the robustness of genetic assays EP3201355B1 (en) 2014-09-30 2019-07-31 Illumina, Inc. Modified polymerases for improved incorporation of nucleotide analogues US10494630B2 (en) * 2014-10-14 2019-12-03 Mgi Tech Co., Ltd. Linker element and method of using same to construct sequencing library IL299976B2 (en) 2014-10-17 2024-11-01 Illumina Cambridge Ltd Continuity-preserving transposition EP3209410B1 (en) 2014-10-22 2024-12-25 IntegenX Inc. Electrophoresis cartridge and corresponding method for sample preparation, processing and analysis EP4026913A1 (en) 2014-10-30 2022-07-13 Personalis, Inc. Methods for using mosaicism in nucleic acids sampled distal to their origin EP3632944B1 (en) 2014-10-31 2021-12-01 Illumina Cambridge Limited Polymers and dna copolymer coatings WO2016073768A1 (en) 2014-11-05 2016-05-12 Veracyte, Inc. Systems and methods of diagnosing idiopathic pulmonary fibrosis on transbronchial biopsies using machine learning and high dimensional transcriptional data CN111024936A (en) 2014-11-05 2020-04-17 纳è¿è¾¾æ¯çç©ç§æä¸å¿ Metal composites for enhanced imaging CA2967351A1 (en) 2014-11-11 2016-05-19 Illumina, Inc. Polynucleotide amplification using crispr-cas systems WO2016081712A1 (en) 2014-11-19 2016-05-26 Bigdatabio, Llc Systems and methods for genomic manipulations and analysis KR101750464B1 (en) * 2014-11-28 2017-06-28 ì¼ì´ë§¥ë°ì´ì¤ì¼í°ì£¼ìíì¬ The probe system for real time quantitative and qualitative analyzing of biomaterial, the reaction chamber with said probe system, and the analyzing method thereof EP3234112B1 (en) 2014-12-19 2019-12-11 Vrije Universiteit Brussel In vitro maturation of a mammalian cumulus oocyte complex EP4095261B1 (en) 2015-01-06 2025-05-28 Molecular Loop Biosciences, Inc. Screening for structural variants CN107209120B (en) 2015-02-02 2020-05-15 æ ªå¼ä¼ç¤¾æ¥ç«é«æ°ææ¯ Multicolor fluorescence analysis device WO2016126987A1 (en) 2015-02-04 2016-08-11 Twist Bioscience Corporation Compositions and methods for synthetic gene assembly CA2975852A1 (en) 2015-02-04 2016-08-11 Twist Bioscience Corporation Methods and devices for de novo oligonucleic acid assembly AU2016220135B2 (en) 2015-02-17 2021-07-29 Dovetail Genomics Llc Nucleic acid sequence assembly CA2979850C (en) * 2015-03-24 2020-07-21 Illumina, Inc. Methods, carrier assemblies, and systems for imaging samples for biological or chemical analysis WO2016154540A1 (en) 2015-03-26 2016-09-29 Dovetail Genomics Llc Physical linkage preservation in dna storage DK3901281T3 (en) 2015-04-10 2023-01-23 Spatial Transcriptomics Ab SPATIALLY SEPARATE, MULTIPLEX NUCLEIC ACID ANALYSIS OF BIOLOGICAL SAMPLES US10900030B2 (en) 2015-04-14 2021-01-26 Illumina, Inc. Structured substrates for improving detection of light emissions and methods relating to the same WO2016172377A1 (en) 2015-04-21 2016-10-27 Twist Bioscience Corporation Devices and methods for oligonucleic acid library synthesis US11085073B2 (en) 2015-04-24 2021-08-10 Qiagen Gmbh Method for immobilizing a nucleic acid molecule on a solid support WO2016170179A1 (en) 2015-04-24 2016-10-27 Qiagen Gmbh Method for immobilizing a nucleic acid molecule on solid support DK3760737T3 (en) 2015-05-11 2023-04-11 Illumina Inc Platform for discovery and analysis of therapeutic agents JP6698708B2 (en) 2015-06-09 2020-05-27 ã©ã¤ã ãã¯ããã¸ã¼ãº ã³ã¼ãã¬ã¼ã·ã§ã³ Methods, systems, compositions, kits, devices, and computer-readable media for molecular tagging EP3527672B1 (en) 2015-06-09 2022-10-05 Centrillion Technology Holdings Corporation Oligonucleotide arrays for sequencing nucleic acids WO2017007753A1 (en) 2015-07-07 2017-01-12 Illumina, Inc. Selective surface patterning via nanoimrinting WO2017015018A1 (en) 2015-07-17 2017-01-26 Illumina, Inc. Polymer sheets for sequencing applications WO2017015543A1 (en) 2015-07-23 2017-01-26 Asuragen, Inc. Methods, compositions, kits, and uses for analysis of nucleic acids comprising repeating a/t-rich segments WO2017024138A1 (en) 2015-08-06 2017-02-09 Arc Bio, Llc Systems and methods for genomic analysis WO2017027653A1 (en) 2015-08-11 2017-02-16 The Johns Hopkins University Assaying ovarian cyst fluid WO2017037078A1 (en) 2015-09-02 2017-03-09 Illumina Cambridge Limited Systems and methods of improving droplet operations in fluidic systems EA201890763A1 (en) 2015-09-18 2018-08-31 ТвиÑÑ ÐайоÑÐ°Ð¹ÐµÐ½Ñ ÐоÑпоÑейÑн LIBRARIES OF OPTIONAL OLEG-NUCLEIC ACIDS AND THEIR SYNTHESIS KR102794025B1 (en) 2015-09-22 2025-04-09 í¸ìì¤í¸ ë°ì´ì¤ì¬ì´ì¸ì¤ ì½í¬ë ì´ì Flexible substrates for nucleic acid synthesis AU2016341198B2 (en) 2015-10-19 2023-03-09 Dovetail Genomics, Llc Methods for genome assembly, haplotype phasing, and target independent nucleic acid detection CN115920796A (en) 2015-12-01 2023-04-07 ç¹é¦æ¯ç¹çç©ç§å¦å ¬å¸ Functionalized surfaces and preparation thereof EP3405783B1 (en) 2016-01-22 2022-03-09 Purdue Research Foundation Charged mass labeling system FR3047251A1 (en) * 2016-02-02 2017-08-04 Dna Script INSTALLATION FOR IMPLEMENTING A PROCESS FOR THE ENZYMATIC SYNTHESIS OF NUCLEIC ACIDS JP7441003B2 (en) 2016-02-23 2024-02-29 ãããã¤ã« ã²ããã¯ã¹ ã¨ã«ã¨ã«ã·ã¼ Generation of phased read sets and haplotype phasing for genome assembly KR102531487B1 (en) 2016-03-25 2023-05-10 카리ì°ì¤, ì¸ì½í¬ë ì´í°ë Synthetic nucleic acid spike-ins DK3377226T3 (en) 2016-03-28 2021-04-26 Illumina Inc Micro rows in several levels EP3442706A4 (en) 2016-04-13 2020-02-19 NextGen Jane, Inc. SAMPLE COLLECTION AND PRESERVATION DEVICES, SYSTEMS AND METHODS US11355328B2 (en) 2016-04-13 2022-06-07 Purdue Research Foundation Systems and methods for isolating a target ion in an ion trap using a dual frequency waveform EP3455372B1 (en) 2016-05-11 2019-10-30 Illumina, Inc. Polynucleotide enrichment and amplification using argonaute systems DK3455356T3 (en) 2016-05-13 2021-11-01 Dovetail Genomics Llc RECOVERY OF LONG-TERM BINDING INFORMATION FROM PRESERVED SAMPLES US11299783B2 (en) 2016-05-27 2022-04-12 Personalis, Inc. Methods and systems for genetic analysis CN107619859A (en) * 2016-07-13 2018-01-23 广å·åº·æçåºå å¥åº·ç§ææéå ¬å¸ Gene sequencing reaction device US11147221B2 (en) 2016-08-22 2021-10-19 Biolumic Limited Methods of seed treatment and resulting products CA3034769A1 (en) 2016-08-22 2018-03-01 Twist Bioscience Corporation De novo synthesized nucleic acid libraries WO2018039490A1 (en) 2016-08-24 2018-03-01 Genomedx Biosciences, Inc. Use of genomic signatures to predict responsiveness of patients with prostate cancer to post-operative radiation therapy EP4353830A3 (en) 2016-09-15 2024-07-03 ArcherDX, LLC Methods of nucleic acid sample preparation for analysis of cell-free dna CA3037185A1 (en) 2016-09-15 2018-03-22 ArcherDX, Inc. Methods of nucleic acid sample preparation WO2018057526A2 (en) 2016-09-21 2018-03-29 Twist Bioscience Corporation Nucleic acid based data storage WO2018064311A2 (en) 2016-09-28 2018-04-05 Life Technologies Corporation Methods and systems for reducing phasing errors when sequencing nucleic acids using termination chemistry US10190155B2 (en) 2016-10-14 2019-01-29 Nugen Technologies, Inc. Molecular tag attachment and transfer US11725232B2 (en) 2016-10-31 2023-08-15 The Hong Kong University Of Science And Technology Compositions, methods and kits for detection of genetic variants for alzheimer's disease WO2018112426A1 (en) 2016-12-16 2018-06-21 Twist Bioscience Corporation Variant libraries of the immunological synapse and synthesis thereof WO2018114706A1 (en) * 2016-12-20 2018-06-28 F. Hoffmann-La Roche Ag Single stranded circular dna libraries for circular consensus sequencing EP4575499A2 (en) 2017-01-04 2025-06-25 MGI Tech Co., Ltd. Stepwise sequencing by non-labeled reversible terminators or natural nucleotides GB201704754D0 (en) 2017-01-05 2017-05-10 Illumina Inc Kinetic exclusion amplification of nucleic acid libraries WO2018132916A1 (en) 2017-01-20 2018-07-26 Genomedx Biosciences, Inc. Molecular subtyping, prognosis, and treatment of bladder cancer CA3063364A1 (en) 2017-01-26 2018-08-02 Qiagen Gmbh Method for enriching template nucleic acids US10240205B2 (en) 2017-02-03 2019-03-26 Population Bio, Inc. Methods for assessing risk of developing a viral disease using a genetic test CN118116478A (en) 2017-02-22 2024-05-31 ç¹é¦æ¯ç¹çç©ç§å¦å ¬å¸ Nucleic acid-based data storage EP3593140A4 (en) 2017-03-09 2021-01-06 Decipher Biosciences, Inc. SUBTYPING PROSTATE CANCER TO PREDICT RESPONSE TO HORMONE THERAPY US10894959B2 (en) 2017-03-15 2021-01-19 Twist Bioscience Corporation Variant libraries of the immunological synapse and synthesis thereof WO2018183942A1 (en) 2017-03-31 2018-10-04 Grail, Inc. Improved library preparation and use thereof for sequencing-based error correction and/or variant identification WO2018191563A1 (en) 2017-04-12 2018-10-18 Karius, Inc. Sample preparation methods, systems and compositions WO2018195091A1 (en) 2017-04-18 2018-10-25 Dovetail Genomics, Llc Nucleic acid characteristics as guides for sequence assembly WO2018205035A1 (en) 2017-05-12 2018-11-15 Genomedx Biosciences, Inc Genetic signatures to predict prostate cancer metastasis and identify tumor agressiveness KR20180124789A (en) * 2017-05-12 2018-11-21 ìì¸ëíêµì°ííë ¥ë¨ Method and apparatus for obtaining high purity nucleotides WO2018231864A1 (en) 2017-06-12 2018-12-20 Twist Bioscience Corporation Methods for seamless nucleic acid assembly SG11201912057RA (en) 2017-06-12 2020-01-30 Twist Bioscience Corp Methods for seamless nucleic acid assembly WO2018231818A1 (en) 2017-06-16 2018-12-20 Life Technologies Corporation Control nucleic acids, and compositions, kits, and uses thereof CA3068273A1 (en) 2017-06-21 2018-12-27 Bluedot Llc Systems and methods for identification of nucleic acids in a sample US11217329B1 (en) 2017-06-23 2022-01-04 Veracyte, Inc. Methods and systems for determining biological sample integrity EP3644707A1 (en) 2017-06-29 2020-05-06 Biolumic Limited Method to improve crop yield and/or quality EP4488382A3 (en) 2017-08-01 2025-01-22 Illumina, Inc. Hydrogel beads for nucleotide sequencing EP3665308A1 (en) 2017-08-07 2020-06-17 The Johns Hopkins University Methods and materials for assessing and treating cancer WO2019038594A2 (en) 2017-08-21 2019-02-28 Biolumic Limited High growth and high hardiness transgenic plants SG11202002194UA (en) 2017-09-11 2020-04-29 Twist Bioscience Corp Gpcr binding proteins and synthesis thereof WO2019060716A1 (en) 2017-09-25 2019-03-28 Freenome Holdings, Inc. Methods and systems for sample extraction JP7066840B2 (en) 2017-10-20 2022-05-13 ãã¤ã¹ã ãã¤ãªãµã¤ã¨ã³ã¹ ã³ã¼ãã¬ã¼ã·ã§ã³ Heated nanowells for polynucleotide synthesis US11099202B2 (en) 2017-10-20 2021-08-24 Tecan Genomics, Inc. Reagent delivery system WO2019086531A1 (en) * 2017-11-03 2019-05-09 F. Hoffmann-La Roche Ag Linear consensus sequencing KR102466365B1 (en) 2017-12-26 2022-11-14 ì¼ë£¨ë¯¸ë, ì¸ì½í¬ë ì´í°ë Sensor System KR20250069684A (en) 2018-01-04 2025-05-19 í¸ìì¤í¸ ë°ì´ì¤ì¬ì´ì¸ì¤ ì½í¬ë ì´ì Dna-based digital information storage CA3090102A1 (en) 2018-01-31 2019-08-08 Dovetail Genomics, Llc Sample prep for dna linkage recovery WO2019160820A1 (en) 2018-02-13 2019-08-22 Illumina, Inc. Dna sequencing using hydrogel beads EP3765592A4 (en) 2018-03-16 2021-12-08 Karius Inc. SERIES OF SAMPLES TO DIFFERENTIATE TARGET NUCLEIC ACIDS FROM CONTAMINATING NUCLEIC ACIDS EP3553182A1 (en) 2018-04-11 2019-10-16 Université de Bourgogne Detection method of somatic genetic anomalies, combination of capture probes and kit of detection KR102657606B1 (en) 2018-04-20 2024-04-15 ì¼ë£¨ë¯¸ë, ì¸ì½í¬ë ì´í°ë Methods of encapsulating single cells, the encapsulated cells and uses thereof CN108765247B (en) * 2018-05-15 2023-01-10 è ¾è®¯ç§æï¼æ·±å³ï¼æéå ¬å¸ Image processing method, device, storage medium and equipment CA3100739A1 (en) 2018-05-18 2019-11-21 Twist Bioscience Corporation Polynucleotides, reagents, and methods for nucleic acid hybridization US10801064B2 (en) 2018-05-31 2020-10-13 Personalis, Inc. Compositions, methods and systems for processing or analyzing multi-species nucleic acid samples US11814750B2 (en) 2018-05-31 2023-11-14 Personalis, Inc. Compositions, methods and systems for processing or analyzing multi-species nucleic acid samples JP7145980B2 (en) 2018-06-15 2022-10-03 ã¨ãï¼ãããã³ï¼ã© ãã·ã¥ ã¢ã¼ã²ã¼ System for identifying antigens recognized by T-cell receptors expressed on tumor-infiltrating lymphocytes NL2021377B1 (en) 2018-07-03 2020-01-08 Illumina Inc Interposer with first and second adhesive layers CA3108807A1 (en) 2018-08-08 2020-02-13 Pml Screening, Llc Methods for assessing the risk of developing progressive multifocal leukoencephalopathy caused by john cunningham virus by genetic testing US11519033B2 (en) 2018-08-28 2022-12-06 10X Genomics, Inc. Method for transposase-mediated spatial tagging and analyzing genomic DNA in a biological sample ES3000357T3 (en) 2018-08-28 2025-02-28 10X Genomics Inc Methods for generating spatially barcoded arrays US20210317524A1 (en) 2018-08-28 2021-10-14 10X Genomics, Inc. Resolving spatial arrays BR112021005976A2 (en) 2018-10-26 2021-06-29 Illumina, Inc. modulation of polymer microspheres for DNA processing WO2020092830A1 (en) 2018-10-31 2020-05-07 Illumina, Inc. Polymerases, compositions, and methods of use DK3887540T3 (en) 2018-11-30 2023-09-18 Illumina Inc ANALYSIS OF MULTIPLE ANALYTES USING A SINGLE ASSAY MY204767A (en) 2018-12-05 2024-09-12 Illumina Inc Polymerases, compositions, and methods of use CA3103736A1 (en) 2018-12-05 2020-06-11 Illumina Cambridge Limited Methods and compositions for cluster generation by bridge amplification AU2019392906A1 (en) 2018-12-07 2021-07-22 Octant, Inc. Systems for protein-protein interaction screening EP3894588A1 (en) 2018-12-10 2021-10-20 10X Genomics, Inc. Resolving spatial arrays using deconvolution CN112739830A (en) 2018-12-18 2021-04-30 ä¼å¢ç±³çº³åæ¡¥æéå ¬å¸ Methods and compositions for paired-end sequencing using a single surface primer EP3899037B1 (en) 2018-12-19 2023-10-18 Illumina, Inc. Methods for improving polynucleotide cluster clonality priority US11649485B2 (en) 2019-01-06 2023-05-16 10X Genomics, Inc. Generating capture probes for spatial analysis US11926867B2 (en) 2019-01-06 2024-03-12 10X Genomics, Inc. Generating capture probes for spatial analysis JP2022521551A (en) 2019-02-26 2022-04-08 ãã¤ã¹ã ãã¤ãªãµã¤ã¨ã³ã¹ ã³ã¼ãã¬ã¼ã·ã§ã³ GLP1 receptor mutant nucleic acid library CN113785057A (en) 2019-02-26 2021-12-10 ç¹é¦æ¯ç¹çç©ç§å¦å ¬å¸ Variant nucleic acid libraries for antibody optimization EP3931354A1 (en) 2019-02-28 2022-01-05 10X Genomics, Inc. Profiling of biological analytes with spatially barcoded oligonucleotide arrays WO2020190509A1 (en) 2019-03-15 2020-09-24 10X Genomics, Inc. Methods for using spatial arrays for single cell sequencing WO2020198071A1 (en) 2019-03-22 2020-10-01 10X Genomics, Inc. Three-dimensional spatial analysis MA56037A (en) 2019-05-28 2022-04-06 Octant Inc TRANSCRIPTION RELAY SYSTEM EP3976820A1 (en) 2019-05-30 2022-04-06 10X Genomics, Inc. Methods of detecting spatial heterogeneity of a biological sample WO2020257612A1 (en) 2019-06-21 2020-12-24 Twist Bioscience Corporation Barcode-based nucleic acid sequence assembly AU2020302791A1 (en) 2019-06-27 2022-02-03 Dovetail Genomics, Llc Methods and compositions for proximity ligation SG11202105836XA (en) 2019-07-12 2021-07-29 Illumina Cambridge Ltd Compositions and methods for preparing nucleic acid sequencing libraries using crispr/cas9 immobilized on a solid support AU2020356471A1 (en) 2019-09-23 2022-04-21 Twist Bioscience Corporation Variant nucleic acid libraries for CRTH2 US11287422B2 (en) 2019-09-23 2022-03-29 Element Biosciences, Inc. Multivalent binding composition for nucleic acid analysis AU2020355027A1 (en) 2019-09-23 2022-04-21 Twist Bioscience Corporation Antibodies that bind CD3 Epsilon EP4041310A4 (en) 2019-10-10 2024-05-15 1859, Inc. Methods and systems for microfluidic screening CN113939590A (en) 2019-10-25 2022-01-14 å ç¾çº³åæ¡¥æéå ¬å¸ Method for generating and sequencing asymmetric linkers on the ends of a polynucleotide template comprising hairpin loops EP4055610A4 (en) 2019-11-05 2023-11-29 Personalis, Inc. Estimating tumor purity from single samples US12157124B2 (en) 2019-11-06 2024-12-03 10X Genomics, Inc. Imaging system hardware EP4055185A1 (en) 2019-11-08 2022-09-14 10X Genomics, Inc. Spatially-tagged analyte capture agents for analyte multiplexing GB201919032D0 (en) 2019-12-20 2020-02-05 Cartana Ab Method of detecting an analyte GB201919029D0 (en) 2019-12-20 2020-02-05 Cartana Ab Method of detecting an analyte AU2020412459B2 (en) 2019-12-23 2022-12-08 Singular Genomics Systems, Inc. Methods for long read sequencing WO2021138094A1 (en) 2019-12-31 2021-07-08 Singular Genomics Systems, Inc. Polynucleotide barcodes for long read sequencing US12110548B2 (en) 2020-02-03 2024-10-08 10X Genomics, Inc. Bi-directional in situ analysis US12059674B2 (en) 2020-02-03 2024-08-13 Tecan Genomics, Inc. Reagent storage system CN115485391A (en) 2020-02-17 2022-12-16 10Xåºå ç»å¦æéå ¬å¸ In situ analysis of chromatin interactions JP2023514749A (en) 2020-02-21 2023-04-07 ï¼ï¼ã¨ãã¯ã¹ ã¸ã§ããã¯ã¹ ã¤ã³ã³ã¼ãã¬ã¤ããã Methods and compositions for integrated in situ spatial assays US12188085B2 (en) 2020-03-05 2025-01-07 10X Genomics, Inc. Three-dimensional spatial transcriptomics with sequencing readout US11359238B2 (en) 2020-03-06 2022-06-14 Singular Genomics Systems, Inc. Linked paired strand sequencing WO2021236929A1 (en) 2020-05-22 2021-11-25 10X Genomics, Inc. Simultaneous spatio-temporal measurement of gene expression and cellular activity US12031177B1 (en) 2020-06-04 2024-07-09 10X Genomics, Inc. Methods of enhancing spatial resolution of transcripts EP4162075A4 (en) 2020-06-08 2024-06-26 The Broad Institute, Inc. Single cell combinatorial indexing from amplified nucleic acids CN116171330A (en) 2020-08-06 2023-05-26 Illuminaå ¬å¸ RNA and DNA sequencing libraries using bead-linked transposomes US12297499B2 (en) 2020-08-17 2025-05-13 10X Genomics, Inc. Multicomponent nucleic acid probes for sample analysis WO2022040176A1 (en) 2020-08-18 2022-02-24 Illumina, Inc. Sequence-specific targeted transposition and selection and sorting of nucleic acids US11200446B1 (en) 2020-08-31 2021-12-14 Element Biosciences, Inc. Single-pass primary analysis CA3188197A1 (en) 2020-09-11 2022-03-17 Andrew Slatter Methods of enriching a target sequence from a sequencing library using hairpin adaptors US20230351619A1 (en) 2020-09-18 2023-11-02 10X Genomics, Inc. Sample handling apparatus and image registration methods US20220186300A1 (en) 2020-12-11 2022-06-16 10X Genomics, Inc. Methods and compositions for multimodal in situ analysis WO2022140570A1 (en) 2020-12-23 2022-06-30 10X Genomics, Inc. Methods and compositions for analyte detection US20220228210A1 (en) 2020-12-30 2022-07-21 10X Genomics, Inc. Molecular array generation using photoresist EP4271509A1 (en) 2020-12-30 2023-11-08 10X Genomics, Inc. Molecular arrays and methods for generating and using the arrays US20220314187A1 (en) 2020-12-30 2022-10-06 10X Genomics, Inc. Methods and compositions for light-controlled surface patterning using a polymer CN116724125A (en) 2021-01-26 2023-09-08 10Xåºå ç»å¦æéå ¬å¸ Nucleic acid analogue probes for in situ analysis ES2993724T3 (en) * 2021-01-29 2025-01-07 10X Genomics Inc Method for transposase mediated spatial tagging and analyzing genomic dna in a biological sample WO2022169972A1 (en) 2021-02-04 2022-08-11 Illumina, Inc. Long indexed-linked read generation on transposome bound beads WO2022174054A1 (en) 2021-02-13 2022-08-18 The General Hospital Corporation Methods and compositions for in situ macromolecule detection and uses thereof WO2022178267A2 (en) 2021-02-19 2022-08-25 10X Genomics, Inc. Modular assay support devices US12275984B2 (en) 2021-03-02 2025-04-15 10X Genomics, Inc. Sequential hybridization and quenching US20220282319A1 (en) 2021-03-03 2022-09-08 10X Genomics, Inc. Analyte detection in situ using nucleic acid origami CA3210451A1 (en) 2021-03-22 2022-09-29 Illumina Cambridge Limited Methods for improving nucleic acid cluster clonality EP4314279A1 (en) 2021-03-29 2024-02-07 Illumina, Inc. Improved methods of library preparation KR20230163434A (en) 2021-03-29 2023-11-30 ì¼ë£¨ë¯¸ë, ì¸ì½í¬ë ì´í°ë Compositions and methods for assessing DNA damage and normalizing amplicon size bias in libraries IL307164A (en) 2021-03-31 2023-11-01 Illumina Inc Methods of preparing directional tagmentation sequencing libraries using transposon-based technology with unique molecular identifiers for error correction WO2022208171A1 (en) 2021-03-31 2022-10-06 UCL Business Ltd. Methods for analyte detection EP4334706A1 (en) 2021-05-05 2024-03-13 Singular Genomics Systems, Inc. Multiomic analysis device and methods of use thereof CN117396613A (en) 2021-06-01 2024-01-12 10Xåºå ç»å¦æéå ¬å¸ Methods and compositions for analyte detection and probe resolution US20230084407A1 (en) 2021-06-02 2023-03-16 10X Genomics, Inc. Sample analysis using asymmetric circularizable probes US11859241B2 (en) 2021-06-17 2024-01-02 Element Biosciences, Inc. Compositions and methods for pairwise sequencing US11427855B1 (en) 2021-06-17 2022-08-30 Element Biosciences, Inc. Compositions and methods for pairwise sequencing CA3223112A1 (en) 2021-06-18 2022-12-22 Mark AMBROSO Engineered polymerases EP4367262A1 (en) 2021-07-09 2024-05-15 10X Genomics, Inc. Methods for detecting analytes using sparse labelling WO2023009842A1 (en) 2021-07-30 2023-02-02 10X Genomics, Inc. Methods and compositions for synchronizing reactions in situ WO2023015192A1 (en) 2021-08-03 2023-02-09 10X Genomics, Inc. Nucleic acid concatemers and methods for stabilizing and/or compacting the same US12077789B2 (en) 2021-08-14 2024-09-03 Illumina, Inc. Polymerases, compositions, and methods of use CN117858958A (en) 2021-08-16 2024-04-09 10Xåºå ç»å¦æéå ¬å¸ Probes comprising segmented barcode regions and methods of use CN118103750A (en) 2021-08-31 2024-05-28 ä¼é²ç±³çº³å ¬å¸ Flow cell with enhanced aperture imaging resolution USD1064308S1 (en) 2021-09-17 2025-02-25 10X Genomics, Inc. Sample handling device EP4413580A1 (en) 2021-10-05 2024-08-14 Personalis, Inc. Customized assays for personalized cancer monitoring US20230167495A1 (en) 2021-11-30 2023-06-01 10X Genomics, Inc. Systems and methods for identifying regions of aneuploidy in a tissue EP4445377A2 (en) 2021-12-10 2024-10-16 10X Genomics, Inc. Multi-resolution in situ decoding WO2023114203A1 (en) 2021-12-13 2023-06-22 Cornell University Genotyping of targeted loci with single-cell chromatin accessibility WO2023122033A1 (en) 2021-12-20 2023-06-29 10X Genomics, Inc. Self-test for pathology/histology slide imaging device CA3222937A1 (en) 2021-12-29 2023-07-06 Jonathan Mark Boutell Methods of nucleic acid sequencing using surface-bound primers US20230279475A1 (en) 2022-01-21 2023-09-07 10X Genomics, Inc. Multiple readout signals for analyzing a sample EP4479933A1 (en) 2022-02-15 2024-12-25 10x Genomics, Inc. Systems and methods for spatial analysis of analytes using fiducial alignment EP4490737A1 (en) 2022-03-08 2025-01-15 10X Genomics, Inc. In situ code design methods for minimizing optical crowding EP4500481A1 (en) 2022-03-29 2025-02-05 10X Genomics, Inc. Spectral unmixing combined with decoding for super-multiplexed in situ analysis EP4253550A1 (en) 2022-04-01 2023-10-04 GenCC GmbH 6 Co. KG Method for the manufacture of a viral system, a vector system or any transport system for cancer-specific crispr complexes EP4504969A1 (en) 2022-04-06 2025-02-12 10X Genomics, Inc. Methods for multiplex cell analysis WO2024006832A1 (en) 2022-06-29 2024-01-04 10X Genomics, Inc. Click chemistry-based dna photo-ligation for manufacturing of high-resolution dna arrays EP4540607A1 (en) 2022-06-17 2025-04-23 10X Genomics, Inc. Catalytic de-crosslinking of samples for in situ analysis WO2024006826A1 (en) 2022-06-29 2024-01-04 10X Genomics, Inc. Compositions and methods for generating molecular arrays using oligonucleotide printing and photolithography ES3005088T3 (en) 2022-06-29 2025-03-13 10X Genomics Inc High definition molecular array feature generation using photoresist US20240167077A1 (en) 2022-06-29 2024-05-23 10X Genomics, Inc. Covalent attachment of splint oligonucleotides for molecular array generation using ligation EP4482617A1 (en) 2022-06-29 2025-01-01 10X Genomics, Inc. Method of generating arrays using microfluidics and photolithography US20240084359A1 (en) 2022-06-29 2024-03-14 10X Genomics, Inc. Methods and compositions for patterned molecular array generation by directed bead delivery EP4376998B1 (en) 2022-06-29 2024-12-04 10X Genomics, Inc. Methods and compositions for refining feature boundaries in molecular arrays EP4547387A1 (en) 2022-06-29 2025-05-07 10X Genomics, Inc. Compositions and methods for oligonucleotide inversion on arrays US20240060127A1 (en) 2022-06-29 2024-02-22 10X Genomics, Inc. Methods and systems for light-controlled surface patterning using photomasks US20240141427A1 (en) 2022-09-30 2024-05-02 Illumina, Inc. Polymerases, compositions, and methods of use WO2024081869A1 (en) 2022-10-14 2024-04-18 10X Genomics, Inc. Methods for analysis of biological samples EP4573210A1 (en) 2022-11-16 2025-06-25 10X Genomics, Inc. Methods and compositions for assessing performance of in situ assays WO2024123733A1 (en) 2022-12-05 2024-06-13 Twist Bioscience Corporation Enzymes for library preparation WO2024129672A1 (en) 2022-12-12 2024-06-20 The Broad Institute, Inc. Trafficked rnas for assessment of cell-cell connectivity and neuroanatomy US20240209419A1 (en) 2022-12-14 2024-06-27 Illumina, Inc. Systems and Methods for Capture and Enrichment of Clustered Beads on Flow Cell Substrates US20240218437A1 (en) 2022-12-16 2024-07-04 10X Genomics, Inc. Methods and compositions for assessing performance WO2024220475A1 (en) 2023-04-21 2024-10-24 Twist Bioscience Corporation Polymerase variants WO2024249591A1 (en) 2023-05-31 2024-12-05 Illumina, Inc. Methods for double-stranded sequencing by synthesis WO2025007252A1 (en) * 2023-07-03 2025-01-09 æ·±å³å大æºé ç§æè¡ä»½æéå ¬å¸ Repeated sequencing method US20250012786A1 (en) 2023-07-07 2025-01-09 10X Genomics, Inc. Systems and methods for tissue bounds detection WO2025024672A1 (en) 2023-07-26 2025-01-30 Element Biosciences, Inc. Dna sequencing systems and use thereof WO2025029831A1 (en) 2023-07-31 2025-02-06 10X Genomics, Inc. Methods and systems for targeted rna cleavage and target rna-primed rolling circle amplification US20250061732A1 (en) 2023-08-16 2025-02-20 10X Genomics, Inc. Systems and methods for image segmentation WO2025059212A1 (en) * 2023-09-12 2025-03-20 Cache Dna, Inc. Multiplexed signal amplification for selection of barcoded microparticles WO2025062341A1 (en) 2023-09-20 2025-03-27 Element Biosciences, Inc. Solid-state and configurable optical test targets and flow cell devices WO2025076421A1 (en) 2023-10-06 2025-04-10 10X Genomics, Inc. Feature pyramiding for in situ data visualizations WO2025117738A1 (en) 2023-11-28 2025-06-05 Illumina, Inc. Methods of improving unique molecular index ligation efficiency WO2025122890A1 (en) 2023-12-07 2025-06-12 10X Genomics, Inc. Graphical user interface and method of estimating an instrument run completion time Citations (83) * Cited by examiner, â Cited by third party Publication number Priority date Publication date Assignee Title US4683195A (en) * 1986-01-30 1987-07-28 Cetus Corporation Process for amplifying, detecting, and/or-cloning nucleic acid sequences US4683202A (en) * 1985-03-28 1987-07-28 Cetus Corporation Process for amplifying nucleic acid sequences US4811218A (en) * 1986-06-02 1989-03-07 Applied Biosystems, Inc. Real time scanning electrophoresis apparatus for DNA sequencing US4822746A (en) * 1986-06-25 1989-04-18 Trustees Of Tufts College Radiative and non-radiative energy transfer and absorbance modulated fluorescence detection methods and sensors US4863849A (en) * 1985-07-18 1989-09-05 New York Medical College Automatable process for sequencing nucleotide US4965188A (en) * 1986-08-22 1990-10-23 Cetus Corporation Process for amplifying, detecting, and/or cloning nucleic acid sequences using a thermostable enzyme US4971903A (en) * 1988-03-25 1990-11-20 Edward Hyman Pyrophosphate-based method and apparatus for sequencing nucleic acids US5114984A (en) * 1991-04-26 1992-05-19 Olin Corporation Process for producing an antimicrobially effective polyurethane US5143853A (en) * 1986-06-25 1992-09-01 Trustees Of Tufts College Absorbance modulated fluorescence detection methods and sensors US5171534A (en) * 1984-01-16 1992-12-15 California Institute Of Technology Automated DNA sequencing technique US5244636A (en) * 1991-01-25 1993-09-14 Trustees Of Tufts College Imaging fiber optic array sensors, apparatus, and methods for concurrently detecting multiple analytes of interest in a fluid sample US5250264A (en) * 1991-01-25 1993-10-05 Trustees Of Tufts College Method of making imaging fiber optic sensors to concurrently detect multiple analytes of interest in a fluid sample US5252494A (en) * 1986-06-25 1993-10-12 Trustees Of Tufts College Fiber optic sensors, apparatus, and detection methods using controlled release polymers and reagent formulations held within a polymeric reaction matrix US5254477A (en) * 1986-06-25 1993-10-19 Trustees Of Tufts College Flourescence intramolecular energy transfer conjugate compositions and detection methods US5298741A (en) * 1993-01-13 1994-03-29 Trustees Of Tufts College Thin film fiber optic sensor array and apparatus for concurrent viewing and chemical sensing of a sample US5302509A (en) * 1989-08-14 1994-04-12 Beckman Instruments, Inc. Method for sequencing polynucleotides US5320814A (en) * 1991-01-25 1994-06-14 Trustees Of Tufts College Fiber optic array sensors, apparatus, and methods for concurrently visualizing and chemically detecting multiple analytes of interest in a fluid sample US5405746A (en) * 1988-03-23 1995-04-11 Cemu Bioteknik Ab Method of sequencing DNA US5429807A (en) * 1993-10-28 1995-07-04 Beckman Instruments, Inc. Method and apparatus for creating biopolymer arrays on a solid support surface US5436327A (en) * 1988-09-21 1995-07-25 Isis Innovation Limited Support-bound oligonucleotides US5445971A (en) * 1992-03-20 1995-08-29 Abbott Laboratories Magnetically assisted binding assays using magnetically labeled binding members US5445934A (en) * 1989-06-07 1995-08-29 Affymax Technologies N.V. Array of oligonucleotides on a solid substrate US5512490A (en) * 1994-08-11 1996-04-30 Trustees Of Tufts College Optical sensor, optical sensing apparatus, and methods for detecting an analyte of interest using spectral recognition patterns US5525464A (en) * 1987-04-01 1996-06-11 Hyseq, Inc. Method of sequencing by hybridization of oligonucleotide probes US5534424A (en) * 1992-05-12 1996-07-09 Cemu Bioteknik Ab Chemical method for the analysis of DNA sequences US5604097A (en) * 1994-10-13 1997-02-18 Spectragen, Inc. Methods for sorting polynucleotides using oligonucleotide tags US5633972A (en) * 1995-11-29 1997-05-27 Trustees Of Tufts College Superresolution imaging fiber for subwavelength light energy generation and near-field optical microscopy US5648245A (en) * 1995-05-09 1997-07-15 Carnegie Institution Of Washington Method for constructing an oligonucleotide concatamer library by rolling circle replication US5700637A (en) * 1988-05-03 1997-12-23 Isis Innovation Limited Apparatus and method for analyzing polynucleotide sequences and method of generating oligonucleotide arrays US5714320A (en) * 1993-04-15 1998-02-03 University Of Rochester Rolling circle synthesis of oligonucleotides and amplification of select randomized circular oligonucleotides US5716785A (en) * 1989-09-22 1998-02-10 Board Of Trustees Of Leland Stanford Junior University Processes for genetic manipulations using promoters US5728529A (en) * 1995-06-23 1998-03-17 Baylor College Of Medicine Alternative dye-labeled ribonucleotides, deoxyribonucleotides, and dideoxyribonucleotides for automated DNA analysis US5744305A (en) * 1989-06-07 1998-04-28 Affymetrix, Inc. Arrays of materials attached to a substrate US5750341A (en) * 1995-04-17 1998-05-12 Lynx Therapeutics, Inc. DNA sequencing by parallel oligonucleotide extensions US5770367A (en) * 1993-07-30 1998-06-23 Oxford Gene Technology Limited Tag reagent and assay method US5780231A (en) * 1995-11-17 1998-07-14 Lynx Therapeutics, Inc. DNA extension and analysis with rolling primers US5795716A (en) * 1994-10-21 1998-08-18 Chee; Mark S. Computer-aided visualization and analysis system for sequence evaluation US5800992A (en) * 1989-06-07 1998-09-01 Fodor; Stephen P.A. Method of detecting nucleic acids US5814524A (en) * 1995-12-14 1998-09-29 Trustees Of Tufts College Optical sensor apparatus for far-field viewing and making optical analytical measurements at remote locations US5821058A (en) * 1984-01-16 1998-10-13 California Institute Of Technology Automated DNA sequencing technique US5830662A (en) * 1993-09-24 1998-11-03 The Trustees Of Columbia University In The City Of New York Method for construction of normalized cDNA libraries US5834252A (en) * 1995-04-18 1998-11-10 Glaxo Group Limited End-complementary polymerase reaction US5846772A (en) * 1995-12-22 1998-12-08 Smithkline Beecham P.L.C. Two component signal transduction system response regulator polynucleotides of Staphylococcus aureus US5846721A (en) * 1996-09-19 1998-12-08 The Trustees Of Columbia University In The City Of New York Efficient and simpler method to construct normalized cDNA libraries with improved representations of full-length cDNAs US5846727A (en) * 1996-06-06 1998-12-08 Board Of Supervisors Of Louisiana State University And Agricultural & Mechanical College Microsystem for rapid DNA sequencing US5854033A (en) * 1995-11-21 1998-12-29 Yale University Rolling circle replication reporter systems US5871697A (en) * 1995-10-24 1999-02-16 Curagen Corporation Method and apparatus for identifying, classifying, or quantifying DNA sequences in a sample without sequencing US5871928A (en) * 1989-06-07 1999-02-16 Fodor; Stephen P. A. Methods for nucleic acid analysis US5882874A (en) * 1998-02-27 1999-03-16 The Trustees Of Columbia University In The City Of New York Reciprocal subtraction differential display US5888819A (en) * 1991-03-05 1999-03-30 Molecular Tool, Inc. Method for determining nucleotide identity through primer extension US5900481A (en) * 1996-11-06 1999-05-04 Sequenom, Inc. Bead linkers for immobilizing nucleic acids to solid supports US5919673A (en) * 1995-03-22 1999-07-06 The Scripps Research Institute One-pot enzymatic sulfation process using 3'-phosphoadenosine-5'-phosphosulfate and recycled phosphorylated adenosine intermediates US5928905A (en) * 1995-04-18 1999-07-27 Glaxo Group Limited End-complementary polymerase reaction US5962228A (en) * 1995-11-17 1999-10-05 Lynx Therapeutics, Inc. DNA extension and analysis with rolling primers US6013445A (en) * 1996-06-06 2000-01-11 Lynx Therapeutics, Inc. Massively parallel signature sequencing by ligation of encoded adaptors US6023540A (en) * 1997-03-14 2000-02-08 Trustees Of Tufts College Fiber optic sensor with encoded microspheres US6036597A (en) * 1998-02-11 2000-03-14 Agco Corporation Combine harvester rotor load control US6040193A (en) * 1991-11-22 2000-03-21 Affymetrix, Inc. Combinatorial strategies for polymer synthesis US6054270A (en) * 1988-05-03 2000-04-25 Oxford Gene Technology Limited Analying polynucleotide sequences US6080585A (en) * 1994-02-01 2000-06-27 Oxford Gene Technology Limited Methods for discovering ligands US6114114A (en) * 1992-07-17 2000-09-05 Incyte Pharmaceuticals, Inc. Comparative gene transcript analysis US6133436A (en) * 1996-11-06 2000-10-17 Sequenom, Inc. Beads bound to a solid support and to nucleic acids US6136543A (en) * 1997-01-31 2000-10-24 Hitachi, Ltd. Method for determining nucleic acids base sequence and apparatus therefor US6146593A (en) * 1995-05-23 2000-11-14 The Regents Of The University Of California High density array fabrication and readout method for a fiber optic biosensor US6150095A (en) * 1995-04-07 2000-11-21 Oxford Gene Technology Limited Method for analyzing a polynucleotide containing a variable sequence US6200737B1 (en) * 1995-08-24 2001-03-13 Trustees Of Tufts College Photodeposition method for fabricating a three-dimensional, patterned polymer microstructure US6210891B1 (en) * 1996-09-27 2001-04-03 Pyrosequencing Ab Method of sequencing DNA US6210910B1 (en) * 1998-03-02 2001-04-03 Trustees Of Tufts College Optical fiber biosensor array comprising cell populations confined to microcavities US6210896B1 (en) * 1998-08-13 2001-04-03 Us Genomics Molecular motors US6221653B1 (en) * 1999-04-27 2001-04-24 Agilent Technologies, Inc. Method of performing array-based hybridization assays using thermal inkjet deposition of sample fluids US6225061B1 (en) * 1999-03-10 2001-05-01 Sequenom, Inc. Systems and methods for performing reactions in an unsealed environment US6255476B1 (en) * 1999-02-22 2001-07-03 Pe Corporation (Ny) Methods and compositions for synthesis of labelled oligonucleotides and analogs on solid-supports US20010006630A1 (en) * 1997-09-02 2001-07-05 Oron Yacoby-Zeevi Introducing a biological material into a patient US6258568B1 (en) * 1996-12-23 2001-07-10 Pyrosequencing Ab Method of sequencing DNA based on the detection of the release of pyrophosphate and enzymatic nucleotide degradation US6263286B1 (en) * 1998-08-13 2001-07-17 U.S. Genomics, Inc. Methods of analyzing polymers using a spatial network of fluorophores and fluorescence resonance energy transfer US20010041335A1 (en) * 1998-09-30 2001-11-15 Affymetrix, Inc. Methods and compositions for amplifying detectable signals in specific binding assays US6333155B1 (en) * 1997-12-19 2001-12-25 Affymetrix, Inc. Exploiting genomics in the search for new drugs US20020009729A1 (en) * 1995-09-18 2002-01-24 Affymetrix, Inc. Methods for testing oligonucleotide arrays US20020022721A1 (en) * 1996-11-14 2002-02-21 Affymetrix, Inc. Methods of array synthesis US6355420B1 (en) * 1997-02-12 2002-03-12 Us Genomics Methods and products for analyzing polymers US6355431B1 (en) * 1999-04-20 2002-03-12 Illumina, Inc. Detection of nucleic acid amplification reactions using bead arrays US20030027126A1 (en) * 1997-03-14 2003-02-06 Walt David R. Methods for detecting target analytes and enzymatic reactions US20030082566A1 (en) * 2001-02-27 2003-05-01 Anna Sylvan Method for determining allele frequencies Family Cites Families (117) * Cited by examiner, â Cited by third party Publication number Priority date Publication date Assignee Title DE3010971A1 (en) 1980-03-21 1981-10-08 Siemens AG, 1000 Berlin und 8000 München METHOD FOR PRODUCING AN OPTICAL 4-DOOR COUPLER US5354825A (en) 1985-04-08 1994-10-11 Klainer Stanley M Surface-bound fluorescent polymers and related methods of synthesis and use US5114864A (en) 1986-06-25 1992-05-19 Trustees Of Tufts College Fiber optic sensors, apparatus, and detection methods using fluid erodible controlled release polymers for delivery of reagent formulations WO1988005533A1 (en) 1987-01-16 1988-07-28 Kelsius, Inc. Amplification of signals from optical fibers GB8810400D0 (en) 1988-05-03 1988-06-08 Southern E Analysing polynucleotide sequences DK175170B1 (en) 1988-11-29 2004-06-21 Sangtec Molecular Diagnostics Method and Reagent Combination to Determine Nucleotide Sequences US6040138A (en) 1995-09-15 2000-03-21 Affymetrix, Inc. Expression monitoring by hybridization to high density oligonucleotide arrays US5244813A (en) 1991-01-25 1993-09-14 Trustees Of Tufts College Fiber optic sensor, apparatus, and methods for detecting an organic analyte in a fluid or vapor sample US5605662A (en) * 1993-11-01 1997-02-25 Nanogen, Inc. Active programmable electronic devices for molecular biological analysis and diagnostics US5587128A (en) * 1992-05-01 1996-12-24 The Trustees Of The University Of Pennsylvania Mesoscale polynucleotide amplification devices US6027880A (en) 1995-08-02 2000-02-22 Affymetrix, Inc. Arrays of nucleic acid probes and methods of using the same for detecting cystic fibrosis US5861242A (en) 1993-06-25 1999-01-19 Affymetrix, Inc. Array of nucleic acid probes on biological chips for diagnosis of HIV and methods of using the same US20030134291A1 (en) 1993-06-25 2003-07-17 Affymetrix, Inc. Polymorphism detection US5837832A (en) 1993-06-25 1998-11-17 Affymetrix, Inc. Arrays of nucleic acid probes on biological chips US6309823B1 (en) 1993-10-26 2001-10-30 Affymetrix, Inc. Arrays of nucleic acid probes for analyzing biotransformation genes and methods of using the same US6156501A (en) 1993-10-26 2000-12-05 Affymetrix, Inc. Arrays of modified nucleic acid probes and methods of use US7375198B2 (en) 1993-10-26 2008-05-20 Affymetrix, Inc. Modified nucleic acid probes EP0730663B1 (en) 1993-10-26 2003-09-24 Affymetrix, Inc. Arrays of nucleic acid probes on biological chips JP2556293B2 (en) * 1994-06-09 1996-11-20 æ¥æ¬é»æ°æ ªå¼ä¼ç¤¾ MOS OTA US20040175718A1 (en) 1995-10-16 2004-09-09 Affymetrix, Inc. Computer-aided visualization and analysis system for sequence evaluation EP0862656B1 (en) 1995-11-21 2001-03-07 Yale University Unimolecular segment amplification and detection EP0880598A4 (en) 1996-01-23 2005-02-23 Affymetrix Inc Nucleic acid analysis techniques JP2002515738A (en) 1996-01-23 2002-05-28 ã¢ãã£ã¡ããªãã¯ã¹ï¼ã¤ã³ã³ã¼ãã¬ã¤ãã£ã Nucleic acid analysis US5837196A (en) 1996-01-26 1998-11-17 The Regents Of The University Of California High density array fabrication and readout method for a fiber optic biosensor US5851772A (en) 1996-01-29 1998-12-22 University Of Chicago Microchip method for the enrichment of specific DNA sequences EP0937159A4 (en) 1996-02-08 2004-10-20 Affymetrix Inc Chip-based speciation and phenotypic characterization of microorganisms US6013440A (en) 1996-03-11 2000-01-11 Affymetrix, Inc. Nucleic acid affinity columns US5712127A (en) 1996-04-29 1998-01-27 Genescape Inc. Subtractive amplification GB9618050D0 (en) 1996-08-29 1996-10-09 Cancer Res Campaign Tech Global amplification of nucleic acids US5856104A (en) 1996-10-28 1999-01-05 Affymetrix, Inc. Polymorphisms in the glucose-6 phosphate dehydrogenase locus JP4963139B2 (en) 1996-11-06 2012-06-27 ã·ã¼ã¯ã¨ãã ã»ã¤ã³ã³ã¼ãã¬ã¼ããã Compositions and methods for immobilizing nucleic acids on solid supports US6519583B1 (en) 1997-05-15 2003-02-11 Incyte Pharmaceuticals, Inc. Graphical viewer for biomolecular sequence data JP2002512511A (en) 1997-03-07 2002-04-23 ã¢ãã£ã¡ããªãã¯ã¹ï¼ã¤ã³ã³ã¼ãã¬ã¤ããã Gene compositions and methods US6327410B1 (en) 1997-03-14 2001-12-04 The Trustees Of Tufts College Target analyte sensors utilizing Microspheres US7144699B2 (en) 1997-03-20 2006-12-05 Affymetrix, Inc. Iterative resequencing US20020055100A1 (en) 1997-04-01 2002-05-09 Kawashima Eric H. Method of nucleic acid sequencing ATE545710T1 (en) 1997-04-01 2012-03-15 Illumina Cambridge Ltd METHOD FOR THE DUPLICATION OF NUCLEIC ACIDS US6406845B1 (en) 1997-05-05 2002-06-18 Trustees Of Tuft College Fiber optic biosensor for selectively detecting oligonucleotide species in a mixed fluid sample US6611828B1 (en) 1997-05-15 2003-08-26 Incyte Genomics, Inc. Graphical viewer for biomolecular sequence data JP4294740B2 (en) 1997-05-23 2009-07-15 ã½ã¬ã¯ãµã»ã¤ã³ã³ã¼ãã¬ã¤ããã System and apparatus for serial processing of analytes ATE319856T1 (en) 1997-06-13 2006-03-15 Affymetrix Inc A Delaware Corp METHOD FOR DETECTING GENE POLYMORPHISMS AND ALLELE EXPRESSION USING PROBE CHIPS NZ502303A (en) 1997-06-18 2002-02-01 Ulrich J Krull Nucleic acid biosensor system & diagnostic use WO1998058529A2 (en) 1997-06-24 1998-12-30 Affymetrix, Inc. Genetic compositions and methods PT1017848E (en) 1997-07-28 2003-02-28 Medical Biosystems Ltd ANALYSIS OF NUCLEIC ACID SEQUENCES US6190868B1 (en) 1997-08-07 2001-02-20 Curagen Corporation Method for identifying a nucleic acid sequence AU9393798A (en) 1997-09-17 1999-04-05 Affymetrix, Inc. Genetic compositions and methods US6399334B1 (en) 1997-09-24 2002-06-04 Invitrogen Corporation Normalized nucleic acid libraries and methods of production thereof US7348181B2 (en) 1997-10-06 2008-03-25 Trustees Of Tufts College Self-encoding sensor with microspheres US7115884B1 (en) 1997-10-06 2006-10-03 Trustees Of Tufts College Self-encoding fiber optic sensor US6013449A (en) 1997-11-26 2000-01-11 The United States Of America As Represented By The Department Of Health And Human Services Probe-based analysis of heterozygous mutations using two-color labelling AU1623899A (en) 1997-12-04 1999-06-16 Packard Bioscience Company Methods of using probes for analyzing polynucleotide sequence NL1007781C2 (en) 1997-12-12 1999-06-15 Packard Instr Bv Microtiter plate. US6232066B1 (en) * 1997-12-19 2001-05-15 Neogen, Inc. High throughput assay system AU2460399A (en) 1998-01-20 1999-08-02 Packard Bioscience Company Gel pad arrays and methods and systems for making them US6050719A (en) 1998-01-30 2000-04-18 Affymetrix, Inc. Rotational mixing method using a cartridge having a narrow interior WO1999039004A1 (en) 1998-02-02 1999-08-05 Affymetrix, Inc. Iterative resequencing WO1999053102A1 (en) 1998-04-16 1999-10-21 Packard Bioscience Company Analysis of polynucleotide sequence US6780981B1 (en) 1998-05-15 2004-08-24 Oxford Gene Technology Ip Limited Libraries of oligomers labeled with different tags GB9811403D0 (en) 1998-05-27 1998-07-22 Isis Innovation Polynucleotide multimers and their use in hybridisation assays GB9813216D0 (en) 1998-06-18 1998-08-19 Pyrosequencing Ab Reaction monitoring systems EP2045334A1 (en) 1998-06-24 2009-04-08 Illumina, Inc. Decoding of array sensors with microspheres CA2339121A1 (en) 1998-07-30 2000-02-10 Shankar Balasubramanian Arrayed biomolecules and their use in sequencing US6306643B1 (en) 1998-08-24 2001-10-23 Affymetrix, Inc. Methods of using an array of pooled probes in genetic analysis AU6131299A (en) 1998-08-26 2000-03-21 Trustees Of Tufts College Combinatorial polymer synthesis of sensors for polymer-based sensor arrays US20020012913A1 (en) 1998-09-15 2002-01-31 Kevin L. Gunderson Nucleic acid analysis using complete n-mer arrays AU758630B2 (en) 1998-11-06 2003-03-27 Solexa Ltd. A method for reproducing molecular arrays US6285807B1 (en) 1998-11-16 2001-09-04 Trustees Of Tufts College Fiber optic sensor for long-term analyte measurements in fluids US6429027B1 (en) 1998-12-28 2002-08-06 Illumina, Inc. Composite arrays utilizing microspheres US7510841B2 (en) 1998-12-28 2009-03-31 Illumina, Inc. Methods of making and using composite arrays for the detection of a plurality of target analytes GB9901475D0 (en) 1999-01-22 1999-03-17 Pyrosequencing Ab A method of DNA sequencing CA2361223A1 (en) 1999-01-29 2000-08-03 Illumina, Inc. Centrifuging apparatus and method for separation of liquid phases and organic synthesis US20020150909A1 (en) 1999-02-09 2002-10-17 Stuelpnagel John R. Automated information processing in randomly ordered arrays WO2000047996A2 (en) 1999-02-09 2000-08-17 Illumina, Inc. Arrays comprising a fiducial and automated information processing in randomly ordered arrays WO2000048000A1 (en) 1999-02-09 2000-08-17 Illumina, Inc. Intrabead screening methods and compositions GB9906477D0 (en) 1999-03-19 1999-05-12 Pyrosequencing Ab Liquid dispensing apparatus AU3567900A (en) 1999-03-30 2000-10-16 Solexa Ltd. Polynucleotide sequencing GB9907813D0 (en) 1999-04-06 1999-06-02 Medical Biosystems Ltd Synthesis GB9907812D0 (en) 1999-04-06 1999-06-02 Medical Biosystems Ltd Sequencing US20030215821A1 (en) 1999-04-20 2003-11-20 Kevin Gunderson Detection of nucleic acid reactions on bead arrays US20030108867A1 (en) * 1999-04-20 2003-06-12 Chee Mark S Nucleic acid sequencing using microsphere arrays EP1923472B1 (en) 1999-04-20 2012-04-11 Illumina, Inc. Detection of nucleic acid reactions on bead arrays US6620584B1 (en) 1999-05-20 2003-09-16 Illumina Combinatorial decoding of random nucleic acid arrays US6544732B1 (en) 1999-05-20 2003-04-08 Illumina, Inc. Encoding and decoding of array sensors utilizing nanocrystals CA2374596A1 (en) 1999-05-20 2000-11-30 Illumina, Inc. Method and apparatus for retaining and presenting at least one microsphere array to solutions and/or to optical imaging systems US20020051971A1 (en) 1999-05-21 2002-05-02 John R. Stuelpnagel Use of microfluidic systems in the detection of target analytes using microsphere arrays CA2382157C (en) 1999-08-18 2012-04-03 Illumina, Inc. Compositions and methods for preparing oligonucleotide solutions AU2246601A (en) 1999-08-30 2001-04-10 Illumina, Inc. Methods for improving signal detection from an array US7244559B2 (en) * 1999-09-16 2007-07-17 454 Life Sciences Corporation Method of sequencing a nucleic acid US6274320B1 (en) * 1999-09-16 2001-08-14 Curagen Corporation Method of sequencing a nucleic acid GB9923324D0 (en) 1999-10-01 1999-12-08 Pyrosequencing Ab Separation apparatus and method GB9923644D0 (en) 1999-10-06 1999-12-08 Medical Biosystems Ltd DNA sequencing GB9929381D0 (en) 1999-12-10 2000-02-09 Pyrosequencing Ab A method of assessing the amount of nucleic acid in a sample US7390459B2 (en) 1999-12-13 2008-06-24 Illumina, Inc. Oligonucleotide synthesizer ATE527053T1 (en) 1999-12-13 2011-10-15 Illumina Inc SYNTHESIS DEVICE FOR OLIGONUCLEOTIDES USING CENTRIFLY FORCE AU3436601A (en) 1999-12-23 2001-07-03 Illumina, Inc. Decoding of array sensors with microspheres WO2001057268A2 (en) 2000-02-07 2001-08-09 Illumina, Inc. Nucleic acid detection methods using universal priming US7955794B2 (en) 2000-09-21 2011-06-07 Illumina, Inc. Multiplex nucleic acid reactions US7361488B2 (en) 2000-02-07 2008-04-22 Illumina, Inc. Nucleic acid detection methods using universal priming AU2001238068A1 (en) 2000-02-07 2001-08-14 Illumina, Inc. Nucleic acid detection methods using universal priming US20040121364A1 (en) 2000-02-07 2004-06-24 Mark Chee Multiplex nucleic acid reactions US7611869B2 (en) 2000-02-07 2009-11-03 Illumina, Inc. Multiplexed methylation detection methods AU3976001A (en) * 2000-02-10 2001-08-20 Illumina Inc Alternative substrates and formats for bead-based array of arrays<sup>TM</sup> US6770441B2 (en) 2000-02-10 2004-08-03 Illumina, Inc. Array compositions and methods of making same DE60136335D1 (en) 2000-02-16 2008-12-11 Illumina Inc PARALLEL GENOTYPING OF SEVERAL PATIENT SAMPLES AU2001247417A1 (en) 2000-03-14 2001-09-24 Trustees Of Tufts College Cross-reactive sensors SE0001768D0 (en) 2000-05-12 2000-05-12 Helen Andersson Microfluidic flow cell for manipulation of particles EP1307283A2 (en) 2000-06-28 2003-05-07 Illumina, Inc. Composite arrays utilizing microspheres with a hybridization chamber CA2419058A1 (en) 2000-08-09 2002-02-14 Illumina, Inc. Automated information processing in randomly ordered arrays AU2001288393A1 (en) 2000-08-25 2002-03-04 Illumina, Inc. Probes and decoder oligonucleotides US20030096239A1 (en) 2000-08-25 2003-05-22 Kevin Gunderson Probes and decoder oligonucleotides US20050158702A1 (en) 2000-09-05 2005-07-21 Stuelpnagel John R. Cellular arrays comprising encoded cells GB0021977D0 (en) 2000-09-07 2000-10-25 Pyrosequencing Ab Method of sequencing DNA GB0022069D0 (en) 2000-09-08 2000-10-25 Pyrosequencing Ab Method WO2002028530A2 (en) 2000-10-06 2002-04-11 Trustees Of Tufts College Self-encoding sensor arrays with microspheres US6720007B2 (en) 2000-10-25 2004-04-13 Tufts University Polymeric microspheres US20040018491A1 (en) 2000-10-26 2004-01-29 Kevin Gunderson Detection of nucleic acid reactions on bead arrays WO2002099982A2 (en) 2001-03-01 2002-12-12 Illumina, Inc. Methods for improving signal detection from an arrayOwner name: 454 LIFE SCIENCES CORPORATION, CONNECTICUT
Free format text: CHANGE OF NAME;ASSIGNOR:454 CORPORATION;REEL/FRAME:019042/0017
Effective date: 20030917
Owner name: 454 CORPORATION, CONNECTICUT
Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:ROTHBERG, JONATHAN M.;BADER, JOEL S.;DEWELL, SCOTT B.;AND OTHERS;REEL/FRAME:019041/0912;SIGNING DATES FROM 20020717 TO 20020807
2007-10-15 STCB Information on status: application discontinuationFree format text: ABANDONED -- FAILURE TO RESPOND TO AN OFFICE ACTION
RetroSearch is an open source project built by @garambo | Open a GitHub Issue
Search and Browse the WWW like it's 1997 | Search results from DuckDuckGo
HTML:
3.2
| Encoding:
UTF-8
| Version:
0.7.4