Method for detecting and identifying unknown bioagents, including bacteria, viruses and the like, by a combination of nucleic acid amplification and molecular weight determination using primers which hybridize to conserved sequence regions of nucleic acids derived from a bioagent and which bracket variable sequence regions that uniquely identify the bioagent. The result is a âbase composition signatureâ (BCS) which is then matched against a database of base composition signatures, by which the bioagent is identified.
Description[0001] This invention was made with United States Government support under DARPA/SPO contract BAA00-09. The United States Government may have certain rights in the invention.
The present invention relates to methods for rapid detection and identification of bioagents from environmental, clinical or other samples. The methods provide for detection and characterization of a unique base composition signature (BCS) from any bioagent, including bacteria and viruses. The unique BCS is used to rapidly identify the bioagent. [0002]
Rapid and definitive microbial identification is desirable for a variety of industrial, medical, environmental, quality, and research reasons. Traditionally, the microbiology laboratory has functioned to identify the etiologic agents of infectious diseases through direct examination and culture of specimens. Since the mid-1980s, researchers have repeatedly demonstrated the practical utility of molecular biology techniques, many of which form the basis of clinical diagnostic assays. Some of these techniques include nucleic acid hybridization analysis, restriction enzyme analysis, genetic sequence analysis, and separation and purification of nucleic acids (See, e.g., J. Sambrook, E. F. Fritsch, and T. Maniatis, Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989). These procedures, in general, are time-consuming and tedious. Another option is the polymerase chain reaction (PCR) or other amplification procedure which amplifies a specific target DNA sequence based on the flanking primers used. Finally, detection and data analysis convert the hybridization event into an analytical result. [0003]
Other techniques for detection of bioagents include high-resolution mass spectrometry (MS), low-resolution MS, fluorescence, radioiodination, DNA chips and antibody techniques. None of these techniques is entirely satisfactory. [0004]
Mass spectrometry provides detailed information about the molecules being analyzed, including high mass accuracy. It is also a process that can be easily automated. However, high-resolution MS alone fails to perform against unknown or bioengineered agents, or in environments where there is a high background level of bioagents (âclutteredâ background). Low-resolution MS can fail to detect some known agents, if their spectral lines are sufficiently weak or sufficiently close to those from other living organisms in the sample. DNA chips with specific probes can only determine the presence or absence of specifically anticipated organisms. Because there are hundreds of thousands of species of benign bacteria, some very similar in sequence to threat organisms, even arrays with 10,000 probes lack the breadth needed to detect a particular organism. [0005]
Antibodies face more severe diversity limitations than arrays. If antibodies are designed against highly conserved targets to increase diversity, the false alarm problem will dominate, again because threat organisms are very similar to benign ones. Antibodies are only capable of detecting known agents in relatively uncluttered environments. [0006]
Several groups have described detection of PCR products using high resolution electrospray ionization -Fourier transform- ion cyclotron resonance mass spectrometry (ESI-FT-ICR MS). Accurate measurement of exact mass combined with knowledge of the number of at least one nucleotide allowed calculation of the total base composition for PCR duplex products of approximately 100 base pairs. (Aaserud et al., [0007] J. Am. Soc. Mass Spec. 7:1266-1269, 1996; Muddiman et al., Anal. Chem. 69:1543-1549, 1997; Wunschel et al., Anal. Chem. 70:1203-1207, 1998; Muddiman et al., Rev. Anal. Chem. 17:1-68, 1998). Electrospray ionization-Fourier transform-ion cyclotron resistance (ESI-FT-ICR) MS may be used to determine the mass of double-stranded, 500 base-pair PCR products via the average molecular mass (Hurst et al., Rapid Commun. Mass Spec. 10:377-382, 1996). The use of matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry for characterization of PCR products has been described. (Muddiman et al., Rapid Commun. Mass Spec. 13:1201-1204, 1999). However, the degradation of DNAs over about 75 nucleotides observed with MALDI limited the utility of this method.
U.S. Pat. No. 5,849,492 describes a method for retrieval of phylogenetically informative DNA sequences which comprise searching for a highly divergent segment, of genomic DNA surrounded by two highly conserved segments, designing the universal primers for PCR amplification of the highly divergent region, amplifying the genomic DNA by PCR technique using universal primers, and then sequencing the gene to determine the identity of the organism. [0008]
U.S. Pat. No. 5,965,363 discloses methods for screening nucleic acids for polymorphisms by analyzing amplified target nucleic acids using mass spectrometric techniques and to procedures for improving mass resolution and mass accuracy of these methods. [0009]
WO 99/14375 describes methods, PCR primers and kits for use in analyzing preselected DNA tandem nucleotide repeat alleles by mass spectrometry. [0010]
WO 98/12355 discloses methods of determining the mass of a target nucleic acid by mass spectrometric analysis, by cleaving the target nucleic acid to reduce its length, making the target single-stranded and using MS to determine the mass of the single-stranded shortened target. Also disclosed are methods of preparing a double-stranded target nucleic acid for MS analysis comprising amplification of the target nucleic acid, binding one of the strands to a solid support, releasing the second strand and then releasing the first strand which is then analyzed by MS. Kits for target nucleic acid preparation are also provided. [0011]
PCT WO97/33000 discloses methods for detecting mutations in a target nucleic acid by nonrandomly fragmenting the target into a set of single-stranded nonrandom length fragments and determining their masses by MS. [0012]
U.S. Pat. No. 5,605,798 describes a fast and highly accurate mass spectrometer-based process for detecting the presence of a particular nucleic acid in a biological sample for diagnostic purposes. [0013]
WO 98/21066 describes processes for determining the sequence of a particular target nucleic acid by mass spectrometry. Processes for detecting a target nucleic acid present in a biological sample by PCR amplification and mass spectrometry detection are disclosed, as are methods for detecting a target nucleic acid in a sample by amplifying the target with primers that contain restriction sites and tags, extending and cleaving the amplified nucleic acid, and detecting the presence of extended product, wherein the presence of a DNA fragment of a mass different from wild-type is indicative of a mutation. Methods of sequencing a nucleic acid via mass spectrometry methods are also described. [0014]
WO 97/37041, WO 99/31278 and U.S. Pat. No. 5,547,835 describe methods of sequencing nucleic acids using mass spectrometry. U.S. Pat. Nos. 5,622,824, 5,872,003 and 5,691,141 describe methods, systems and kits for exonuclease-mediated mass spectrometric sequencing. [0015]
Thus, there is a need for a method for bioagent detection and identification which is both specific and rapid, and in which no nucleic acid sequencing is required. The present invention addresses this need. [0016]
One embodiment of the present invention is a method of identifying an unknown bioagent comprising (a) contacting nucleic acid from the bioagent with at least one pair of oligonucleotide primers which hybridize to sequences of the nucleic acid and flank a variable nucleic acid sequence; (b) amplifying the variable nucleic acid sequence to produce an amplification product; (c) determining the molecular mass of the amplification product; and (d) comparing the molecular mass to one or more molecular masses of amplification products obtained by performing steps (a)-(c) on a plurality of known organisms, wherein a match identifies the unknown bioagent. In one aspect of this preferred embodiment, the sequences to which the at least one pair of oligonucleotide primers hybridize are highly conserved. Preferably, the amplifying step comprises polymerase chain reaction. Alternatively, the amplifying step comprises ligase chain reaction or strand displacement amplification. In one aspect of this preferred embodiment, the bioagent is a bacterium, virus, cell or spore. Advantageously, the nucleic acid is ribosomal RNA. In another aspect, the nucleic acid encodes RNase P or an RNA-dependent RNA polymerase. Preferably, the amplification product is ionized prior to molecular mass determination. The method may further comprise the step of isolating nucleic acid from the bioagent prior to contacting the nucleic acid with the at least one pair of oligonucleotide primers. The method may further comprise the step of performing steps (a)-(d) using a different oligonucleotide primer pair and comparing the results to one or more molecular mass amplification products obtained by performing steps (a)-(c) on a different plurality of known organisms from those in step (d). Preferably, the one or more molecular mass is contained in a database of molecular masses. In another aspect of this preferred embodiment, the amplification product is ionized by electrospray ionization, matrix-assisted laser desorption or fast atom bombardment. Advantageously, the molecular mass is determined by mass spectrometry. Preferably, the mass spectrometry is Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS), ion trap, quadrupole, magnetic sector, time of flight (TOF), Q-TOF or triple quadrupole. The method may further comprise performing step (b) in the presence of an analog of adenine, thymidine, guanosine or cytidine having a different molecular weight than adenosine, thymidine, guanosine or cytidine. In one aspect, the oligonucleotide primer comprises a base analog or substitute base at [0017] positions 1 and 2 of each triplet within the primer, wherein the base analog or substitute base binds with increased affinity to its complement compared-to the native base. Preferably, the primer comprises a universal base at position 3 of each triplet within the primer. The base analog or substitute base may be 2,6-diaminopurine, propyne T, propyne G, phenoxazines or G-clamp. Preferably, the universal base is inosine, guanidine, uridine, 5-nitroindole, 3-nitropyrrole, dP or dK, or 1-(2-deoxy-β-D-ribofuranosyl)-imidazole-4-carboxamide.
Another embodiment of the present invention is a method of identifying an unknown bioagent comprising (a) contacting nucleic acid from the bioagent with at least one pair of oligonucleotide primers which hybridize to sequences of the nucleic acid and flank a variable nucleic acid sequence; (b) amplifying the variable nucleic acid sequence to produce an amplification product; (c) determining the base composition of the amplification product; and (d) comparing the base composition to one or more base compositions of amplification products obtained by performing steps (a)-(c) on a plurality of known organisms, wherein a match identifies the unknown bioagent. In one aspect of this preferred embodiment, the sequences to which the at least one pair of oligonucleotide primers hybridize are highly conserved. Preferably, the amplifying step comprises polymerase chain reaction. Alternatively, the amplifying step comprises ligase chain reaction or strand displacement amplification. In one aspect of this preferred embodiment, the bioagent is a bacterium, virus, cell or spore. Advantageously, the nucleic acid is ribosomal RNA. In another aspect, the nucleic acid encodes RNase P or an RNA-dependent RNA polymerase. Preferably, the amplification product is ionized prior to molecular mass determination. The method may further comprise the step of isolating nucleic acid from the bioagent prior to contacting the nucleic acid with the at least one pair of oligonucleotide primers. The method may further comprise the step of performing steps (a)-(d) using a different oligonucleotide primer pair and comparing the results to one or more base composition signatures of amplification products obtained by performing steps (a)-(c) on a different plurality of known organisms from those in step (d). Preferably, the one or more base compositions is contained in a database of base compositions. In another aspect of this preferred embodiment, the amplification product is ionized by electrospray ionization, matrix-assisted laser desorption or fast atom bombardment. Advantageously, the molecular mass is determined by mass spectrometry. Preferably, the mass spectrometry is Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS), ion trap, quadrupole, magnetic sector, time of flight (TOF), Q-TOF or triple quadrupole. The method may further comprise performing step (b) in the presence of an analog of adenine, thymidine, guanosine or cytidine having a different molecular weight than adenosine, thymidine, guanosine or cytidine. In one aspect, the oligonucleotide primer comprises a base analog or substitute base at [0018] positions 1 and 2 of each triplet within the primer, wherein the base analog or substitute base, binds with increased affinity to its complement compared to the native base. Preferably, the primer comprises a universal base at position 3 of each triplet within the primer. The base analog or substitute base may be 2,6-diaminopurine, propyne T, propyne G, phenoxazines or G-clamp. Preferably, the universal base is inosine, guanidine, uridine, 5-nitroindole, 3-nitropyrrole, dP or dK, or 1-(2-deoxy-β-D-ribofuranosyl)-imidazole-4-carboxamide.
The present invention also provides a method for detecting a single nucleotide polymorphism in an individual, comprising the steps of (a) isolating nucleic acid from the individual; (b) contacting the nucleic acid with oligonucleotide primers which hybridize to regions of the nucleic acid which flank a region comprising the potential polymorphism; (c) amplifying the region to produce an amplification product; (d) determining the molecular mass of the amplification product; and (e) comparing the molecular mass to the molecular mass of the region in an individual known to have the polymorphism, wherein if the molecular masses are the same then the individual has the polymorphism. [0019]
In one aspect of this preferred embodiment, the primers hybridize to highly conserved sequences. Preferably, the polymorphism is associated with a disease. Alternatively, the polymorphism is a blood group antigen. In one aspect of the preferred embodiment, the amplifying step is polymerase chain reaction. Alternatively, the amplification step is ligase chain reaction or strand displacement amplification. Preferably, the amplification product is ionized prior to mass determination. In one aspect, the amplification product is ionized by electrospray ionization, matrix-assisted laser desorption or fast atom bombardment. Advantageously, the molecular mass is determined by mass spectrometry. Preferably, the mass spectrometry is Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS), ion trap, quadrupole, magnetic sector, time of flight (TOF), Q-TOF or triple quadrupole.[0020]
FIGS. [0021] 1A-1I are consensus diagrams that show examples of conserved regions from 16S rRNA (FIG. 1A-1B), 23S rRNA (3â²-half, FIG. 1C-1D; 5â²-half, FIG. 1E-F), 23S rRNA Domain I (FIG. 1G), 23S rRNA Domain IV (FIG. 1H) and 16S rRNA Domain III (FIG. 1I) which are suitable for use in the present invention. Lines with arrows are examples of regions to which intelligent primer pairs for PCR are designed. The label for each primer pair represents the starting and ending base number of the amplified region on the consensus diagram. Bases in capital letters are greater than 95% conserved; bases in lower case letters are 90-95% conserved, filled circles are 80-90% conserved; and open circles are less than 80% conserved. The label for each primer pair represents the starting and ending base number of the amplified region on the consensus diagram.
FIG. 2 shows a typical primer amplified region from the 16S rRNA Domain III shown in FIG. 1C. [0022]
FIG. 3 is a schematic diagram showing conserved regions in RNase P. Bases in capital letters are greater than 90% conserved; bases in lower case letters are 80-90% conserved; filled circles designate bases which are 70-80% conserved; and open circles designate bases that are less than 70% conserved. [0023]
FIG. 4 is a schematic diagram of base composition signature determination using nucleotide analog âtagsâ to determine base composition signatures. [0024]
FIG. 5 shows the deconvoluted mass spectra of a [0025] Bacillus anthracis region with and without the mass tag phosphorothioate A (A*). The two spectra differ in that the measured molecular weight of the mass tag-containing sequence is greater than the unmodified sequence.
FIG. 6 shows base composition signature (BCS) spectra from PCR products from [0026] Staphylococcus aureus ( S. aureus 16Sâ1337F) and Bacillus anthracus (B. anthr. 16Sâ1337F), amplified using the same primers. The two strands differ by only two (AT-->CG) substitutions and are clearly distinguished on the basis of their BCS.
FIG. 7 shows that a single difference between two sequences (A[0027] 14 in B. anthracis vs. A15 in B. cereus) can be easily detected using ESI-TOF mass spectrometry.
FIG. 8 is an ESI-TOF of [0028] Bacillus anthracis spore coat protein sspE 56 mer plus calibrant. The signals unambiguously identify B. anthracis versus other Bacillus species.
FIG. 9 is an ESI-TOF of a [0029] B. anthracis synthetic 16Sâ1228 duplex (reverse and forward strands). The technique easily distinguishes between the forward and reverse strands.
FIG. 10 is an ESI-FTICR-MS of a [0030] synthetic B. anthracis 16Sâ1337 46 base pair duplex.
FIG. 11 is an ESI-TOF-MS of a 56 mer oligonucleotide (3 scans) from the [0031] B. anthracis saspB gene with an internal mass standard. The internal mass standards are designated by asterisks.
FIG. 12 is an ESI-TOF-MS of an internal standard with 5 mM TBA-TFA buffer showing that charge stripping with tributylammonium trifluoroacetate reduces the most abundant charge state from [Mâ8H[0032] +]8â to [Mâ3H+]3â.
The present invention provides a combination of a non-PCR biomass detection mode, preferably high-resolution MS, with PCR-based BCS technology using âintelligent primersâ which hybridize to conserved sequence regions of nucleic acids derived from a bioagent and which bracket variable sequence regions that uniquely identify the bioagent. The high-resolution MS technique is used to determine the molecular mass and base composition signature (BCS) of the amplified sequence region. This unique âbase composition signatureâ (BCS) is then input to a maximum-likelihood detection algorithm for matching against a database of base composition signatures in the same amplified region. The present method combines PCR-based amplification technology (which provides specificity) and a molecular mass detection mode (which provides speed and does not require nucleic acid sequencing of the amplified target sequence) for bioagent detection and identification. [0033]
The present method allows extremely rapid and accurate detection and identification of bioagents compared to existing methods.- Furthermore, this rapid detection and identification is possible even when sample material is impure. Thus, the method is useful in a wide variety of fields, including, but not limited to, environmental testing (e.g., detection and discrimination of pathogenic vs. non-pathogenic bacteria in water or other samples), germ warfare (allowing immediate identification of the bioagent and appropriate treatment), pharmacogenetic analysis and medical diagnosis (including cancer diagnosis based on mutations and polymorphisms; drug resistance and susceptibility testing; screening for and/or diagnosis of genetic diseases and conditions; and diagnosis of infectious diseases and conditions). The method leverages ongoing biomedical research in virulence, pathogenicity, drug resistance and genome sequencing into a method which provides greatly improved sensitivity, specificity and reliability compared to existing methods, with lower rates of false positives. [0034]
The present method can be used to detect and classify any biological agent, including bacteria, viruses, fungi and toxins. As one example, where the agent is a biological threat, the information obtained is used to determine practical information needed for countermeasures, including toxin genes, pathogenicity islands and antibiotic resistance genes. In addition, the methods can be used to identify natural or deliberate engineering events including chromosome fragment swapping, molecular breeding (gene shuffling) and emerging infectious diseases. [0035]
Bacteria have a common set of absolutely required genes. About 250 genes are present in all bacterial species ([0036] Proc. Natl. Acad. Sci. U.S.A. 93:10268, 1996; Science 270:397, 1995), including tiny genomes like Mycoplasma, Ureaplasma and Rickettsia. These genes encode proteins involved in translation, replication, recombination and repair, transcription, nucleotide metabolism, amino acid metabolism, lipid metabolism, energy generation, uptake, secretion and the like. Examples of these proteins are DNA polymerase III beta, elongation factor TU, heat shock protein groEL, RNA polymerase beta, phosphoglycerate kinase, NADH dehydrogenase, DNA ligase, DNA topoisomerase and elongation factor G. Operons can also be targeted using the present method. One example of an operon is the bfp operon from enteropathogenic E. coli. Multiple core chromosomal genes can be used to classify bacteria at a genus or genus species level to determine if an organism has threat potential. The method can also be used to detect pathogenicity markers (plasmid or chromosomal) and antibiotic resistance genes to confirm the threat potential of an organism and to direct countermeasures.
A theoretically ideal bioagent detector would identify, quantify, and report the complete nucleic acid sequence of every bioagent that reached the sensor. The complete sequence of the nucleic acid component of a pathogen would provide all relevant information about the threat, including its identity and the presence of drug-resistance or pathogenicity markers. This ideal has not yet been achieved. However, the present invention provides a straightforward strategy for obtaining information with the same practical value using base composition signatures (BCS). While the base composition of a gene fragment is not as information-rich as the sequence itself, there is no need to analyze the complete sequence of the gene if the short analyte sequence fragment is properly chosen. A database of reference sequences can be prepared in which each sequence is indexed to a unique base composition signature, so that the presence of the sequence can be inferred with accuracy from the presence of the signature. The advantage of base composition signatures is that they can be quantitatively measured in a massively parallel fashion using multiplex PCR (PCR in which two or more primer pairs amplify target sequences simultaneously) and mass spectrometry. These multiple primer amplified regions uniquely identify most threat and ubiquitous background bacteria and viruses. In addition, cluster-specific primer pairs distinguish important local clusters (e.g., anthracis group). [0037]
In the context of this invention, a âbioagentâ is any organism, living or dead, or a nucleic acid derived from such an organism. Examples of bioagents include but are not limited to cells (including but not limited to human clinical samples, bacterial cells and other pathogens) viruses, toxin genes and bioregulating compounds). Samples may be alive or dead or in a vegetative state (for example, vegetative bacteria or spores) and may be encapsulated or bioengineered. [0038]
As used herein, a
[0039]base composition signatures
(BCS) is the exact base composition from selected fragments of nucleic acid sequences that uniquely identifies the target gene and source organism. BCS can be thought of as unique indexes of specific genes.
As used herein,
[0040]intelligent primers
are primers which bind to sequence regions which flank an intervening variable region. In a preferred embodiment, these sequence regions which flank the variable region are highly conserved among different species of bioagent. For example, the sequence regions may be highly conserved among all Bacillus species. By the term
highly conserved
, it is meant that the sequence regions exhibit between about 80-100%, more preferably between about 90-100% and most preferably between about 95-100% identity. Examples of intelligent primers which amplify regions of the 16S and 23S rRNA are shown in Figures
1A-
1I. A typical primer amplified region in 16S rRNA is shown in FIG. 2. The arrows represent primers which bind to highly conserved regions which flank a variable region in 16S rRNA domain III. The amplified region is the stem-loop structure under
1100-1188.
One main advantage of the detection methods of the present invention is that the primers need not be specific for a particular bacterial species, or even genus, such as Bacillus or Streptomyces. Instead, the primers recognize highly conserved regions across hundreds of bacterial species including, but not limited to, the species described herein. Thus, the same primer pair can be used to identify any desired bacterium because it will bind to the conserved regions which flank a variable region specific to a single species, or common to several bacterial species, allowing nucleic acid amplification of the intervening sequence and determination of its molecular weight and base composition. For example, the 16S[0041] â971-1062, 16Sâ1228-1310 and 16Sâ1100-1188 regions are 98-99% conserved in about 900 species of bacteria (16S=16S rRNA, numbers indicate nucleotide position). In one embodiment of the present invention, primers used in the present method bind to one or more of these regions or portions thereof.
The present invention provides a combination of a non-PCR biomass detection mode, preferably high-resolution MS, with nucleic acid amplification-based BCS technology using âintelligent primersâ which hybridize to conserved regions and which bracket variable regions that uniquely identify the bioagent(s). Although the use of PCR is preferred, other nucleic acid amplification techniques may also be used, including ligase chain reaction (LCR) and strand displacement amplification (SDA). The high-resolution MS technique allows separation of bioagent spectral lines from background spectral lines in highly cluttered environments. The resolved spectral lines are then translated to BCS which are input to a maximum-likelihood detection algorithm matched against spectra for one or more known BCS. Preferably, the bioagent BCS spectrum is matched against one or more databases of BCS from vast numbers of bioagents. Preferably, the matching is done using a maximum-likelihood detection algorithm. [0042]
In a preferred embodiment, base composition signatures are quantitatively measured in a massively parallel fashion using the polymerase chain reaction (PCR), preferably multiplex PCR, and mass spectrometric (MS) methods. Sufficient quantities of nucleic acids must be present for detection of bioagents by MS. A wide variety of techniques for preparing large amounts of purified nucleic acids or fragments thereof are well known to those of skill in the art. PCR requires one or more pairs of oligonucleotide primers which bind to regions which flank the target sequence(s) to be amplified. These primers prime synthesis of a different strand of DNA, with synthesis occurring in the direction of one primer towards the other primer. The primers, DNA to be amplified, a thermostable DNA polymerase (e.g. Taq polymerase), the four deoxynucleotide triphosphates, and a buffer are combined to initiate DNA synthesis. The solution is denatured by heating, then cooled to allow annealing of newly added primer, followed by another round of DNA synthesis. This process is typically repeated for about 30 cycles, resulting in amplification of the target sequence. [0043]
The âintelligent primersâ define the target sequence region to be amplified and analyzed. In one embodiment, the target sequence is a ribosomal RNA (rRNA) gene sequence. With the complete sequences of many of the smallest microbial genomes now available, it is possible to identify a set of genes that defines âminimal lifeâ and identify composition signatures that uniquely identify each gene and organism. Genes that encode core life functions such as DNA replication, transcription, ribosome structure, translation, and transport are distributed broadly in the bacterial genome and are preferred regions for BCS analysis. Ribosomal RNA (rRNA) genes comprise regions that provide useful base composition signatures. Like many genes involved in core life functions, rRNA genes contain sequences that are extraordinarily conserved across bacterial domains interspersed with regions of high variability that are more specific to each species. The variable regions can be utilized to build a database of base composition signatures. The strategy.involves creating a structure-based alignment of sequences of the small (16S) and the large (23S) subunits of the rRNA genes. For example, there are currently over 13,000, sequences in the ribosomal RNA database that has been created and maintained by Robin Gutell, University of Texas at Austin, and is publicly available on the Institute for Cellular and Molecular Biology web page (http://www.rna.icmb.utexas.edu/). There is also a publicly available rRNA database created and maintained by the University of Antwerp, Belgium at http://www.rrna.uia.ac.be. [0044]
These databases have been analyzed to determine regions that are useful as base composition signatures. The characteristics of such regions are: a) between about 80 and 100%, preferably>about 95% identity among species of the particular bioagent of interest, of upstream and downstream nucleotide sequences which serve as sequence amplification primer sites; b) an intervening variable region which exhibits no greater than about 5% identity among species; and c) a separation of between about 30 and 1000 nucleotides, preferably no more than about 50-250 nucleotides, and more preferably no more than about 60-100 nucleotides, between the conserved regions. [0045]
Due to their overall conservation, the flanking rRNA primer sequences serve as good âuniversalâ primer binding sites to amplify the region of interest for most, if not all, bacterial species. The intervening region between the sets of primers varies in length and/or composition, and thus provides a unique base composition signature. [0046]
It is advantageous to design the âintelligent primersâ to be as universal as possible to minimize the number of primers which need to be synthesized, and to allow detection of multiple species using a single pair of primers. These primer pairs can be used to amplify variable regions in these species. Because any variation (due to codon wobble in the 3[0047] rd position) in these conserved regions among species is likely to occur in the third position of a DNA triplet, oligonucleotide primers can be designed such that the nucleotide corresponding to this position is a base which can bind to more than one nucleotide, referred to herein as a âuniversal baseâ. For example, under this âwobbleâ pairing, inosine (I) binds to U, C or A; guanine (G) binds to U or C, and uridine (U) binds to U or C. Other examples of universal bases include nitroindoles such as 5-nitroindole or 3-nitropyrrole (Loakes et al., Nucleosides and Nucleotides 14:1001-1003, 1995), the degenerate nucleotides dP or dK (Hill et al.), an acyclic nucleoside analog containing 5-nitroindazole (Van Aerschot et al., Nucleosides and Nucleotides 14:1053-1056, 1995) or the purine analog 1-(2-deoxy-β-D-ribofuranosyl)-imidazole-4-carboxamide (Sala et al., Nucl. Acids Res. 24:3302-3306, 1996).
In another embodiment of the invention, to compensate for the somewhat weaker binding by the âwobbleâ base, the oligonucleotide primers are designed such that the first and second positions of each triplet are occupied by nucleotide analogs which bind with greater affinity than the unmodified nucleotide. Examples of these analogs are 2,6-diaminopurine which binds to thymine, propyne T which binds to adenine and propyne C and phenoxazines, including G-clamp, which binds to G. Propynes are described in U.S. Pat. Nos. 5,645,985, 5,830,653 and 5,484,908, the entire contents of which are incorporated herein by reference. Phenoxazines are described in U.S. Pat. Nos. 5,502,177, 5,763,588, and 6,005,096, the entire contents of which are incorporated herein by reference. G-clamps are described in U.S. Pat. Nos. 6,007,992 and 6,028,183, the entire contents of which are incorporated herein by reference. [0048]
Bacterial biological warfare agents capable of being detected by the present methods include [0049] Bacillus anthracis (anthrax), Yersinia pestis (pneumonic plague), Franciscella tularensis (tularemia), Brucella suis, Brucella abortus, Brucella melitensis (undulant fever), Burkholderia mallei (glanders), Burkholderia pseudomalleii (melioidosis), Salmonella typhi (typhoid fever), Rickettsia typhii (epidemic typhus), Rickettsia prowasekii (endemic typhus) and Coxiella burnetii (Q fever), Rhodobacter capsulatus, Chlamydia pneumoniae, Escherichia coli, Shigella dysenteriae, Shigella flexneri, Bacillus cereus, Clostridium botulinum, Coxiella burnetti, Pseudomonas aeruginosa, Legionella pneumophila, and Vibrio cholerae.
Besides 16S and 23S rRNA, other target regions suitable for use in the present invention for detection of bacteria include 5S rRNA and RNase P (FIG. 3). [0050]
Biological warfare fungus biowarfare agents include coccidioides immitis (Coccidioidomycosis). [0051]
Biological warfare toxin genes capable of being detected by the methods of the present invention include botulism, T-2 mycotoxins, ricin, staph enterotoxin B, shigatoxin, abrin, aflatoxin, [0052] Clostridium perfringens epsilon toxin, conotoxins, diacetoxyscirpenol, tetrodotoxin and saxitoxin.
Biological warfare viral threat agents are mostly RNA viruses (positive-strand and negative-strand), with the exception of smallpox. Every RNA virus is a family of related viruses (quasispecies). These viruses mutate rapidly and the potential for engineered strains (natural or deliberate) is very high. RNA viruses cluster into families that have conserved RNA structural domains on the viral genome (e.g., virion components, accessory proteins) and conserved housekeeping genes that encode core viral proteins including, for single strand positive strand RNA viruses, RNA-dependent RNA polymerase, double stranded RNA helicase, chymotrypsin-like and papain-like proteases and methyltransferases. [0053]
Examples of (â)-strand RNA viruses include arenaviruses (e.g., sabia virus, lassa fever, Machupo, Argentine hemorrhagic fever, flexal virus), bunyaviruses (e.g., hantavirus, nairovirus, phlebovirus, hantaan virus, Congo-crimean hemorrhagic fever, rift valley fever), and mononegavirales (e.g., filovirus, paramyxovirus, ebola virus, Marburg, equine morbillivirus). [0054]
Examples of (+)-strand RNA viruses include picornaviruses (e.g., coxsackievirus, echovirus, human coxsackievirus A, human echovirus, human enterovirus, human poliovirus, hepatitis A virus, human parechovirus, human rhinovirus), astroviruses (e.g., human astrovirus), calciviruses (e.g., chiba virus, chitta virus, human calcivirus, norwalk virus), nidovirales (e.g., human coronavirus, human torovirus), flaviviruses (e.g., dengue virus 1-4, Japanese encephalitis virus, Kyanasur forest disease virus, Murray Valley encephalitis virus, Rocio virus, St. Louis encephalitis virus, West Nile virus, yellow fever virus, hepatitis c virus) and togaviruses (e.g., Chikugunya virus, Eastern equine encephalitis virus, Mayaro virus, O'nyong-nyong virus, Ross River virus, Venezuelan equine encephalitis virus, Rubella virus, hepatitis E virus). The hepatitis C virus has a 5â²-untranslated region of 340 nucleotides, an open reading frame encoding 9 proteins having 3010 amino acids and a 3â²-untranslated region of 240 nucleotides. The 5â²-UTR and 3â²-UTR are 99% conserved in hepatitis C viruses. [0055]
In one embodiment, the target gene is an RNA-dependent RNA polymerase or a helicase encoded by (+)-strand RNA viruses, or RNA polymerase from a (â)-strand RNA virus. (+)-strand RNA viruses are double stranded RNA and replicate by RNA-directed RNA synthesis using RNA-dependent RNA polymerase and the positive strand as a template. Helicase unwinds the RNA duplex to allow replication of the single stranded RNA. These viruses include viruses from the family picornaviridae (e.g., poliovirus, coxsackievirus, echovirus), togaviridae (e.g., alphavirus, flavivirus, rubivirus), arenaviridae (e.g., lymphocytic choriomeningitis virus, lassa fever virus), cononaviridae (e.g., human respiratory virus) and Hepatitis A virus. The genes encoding these proteins comprise variable and highly conserved regions which flank the variable regions. [0056]
In a preferred embodiment, the detection scheme for the PCR products generated from the bioagent(s) incorporates three features. First, the technique simultaneously detects and differentiates multiple (generally about 6-10) PCR products. Second, the technique provides a BCS that uniquely identifies the bioagent from the possible primer sites. Finally, the detection technique is rapid, allowing multiple PCR reactions to be run in parallel. [0057]
In one embodiment, the method can be used to detect the presence of antibiotic resistance and/or toxin genes in a bacterial species. For example, [0058] Bacillus anthracis comprising a tetracycline resistance plasmid and plasmids encoding one or both anthracis toxins (px01 and/or px02) can be detected by using antibiotic resistance primer sets and toxin gene primer sets. If the B. anthracis is positive for tetracycline resistance, then a different antibiotic, for example quinalone, is used.
Mass spectrometry (MS)-based detection of PCR products provides all of these features with additional advantages. MS is intrinsically a parallel detection scheme without the need for radioactive or fluorescent labels, since every amplification product with a unique base composition is identified by its molecular mass. The current state of the art in mass spectrometry is such that less than femtomole quantities of material can be readily analyzed to afford information about the molecular contents of the sample. An accurate assessment of the molecular mass of the material can be quickly obtained, irrespective of whether the molecular weight of the sample is several hundred, or in excess of one hundred thousand atomic mass units (amu) or Daltons. Intact molecular ions can be generated from amplification products using one of a variety of ionization techniques to convert the sample to gas phase. These ionization methods include, but are not limited to, electrospray ionization (ES), matrix-assisted laser desorption ionization (MALDI) and fast atom bombardment (FAB). For example, MALDI of nucleic acids, along with examples of matrices for use in MALDI of nucleic acids, are described in WO 98/54751 (Genetrace, Inc.). [0059]
Upon ionization, several peaks are observed from one sample due to the formation of ions with different charges. Averaging the multiple readings of molecular mass obtained from a single mass spectrum affords an estimate of molecular mass of the bioagent. Electrospray ionization mass spectrometry (ESI-MS) is particularly useful for very high molecular weight polymers such as proteins and nucleic acids having molecular weights greater than 10 kDa, since it yields a distribution of multiply-charged molecules of the sample without causing a significant amount of fragmentation. [0060]
The mass detectors used in the methods of the present invention include, but are not limited to, Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS), ion trap, quadrupole, magnetic sector, time of flight (TOF), Q-TOF, and triple quadrupole. [0061]
In general, the mass spectrometric techniques which can be used in the present invention include, but are not limited to, tandem mass spectrometry, infrared multiphoton dissociation and pyrolytic gas chromatography mass spectrometry (PGC-MS). In one embodiment of the invention, the bioagent detection system operates continually in bioagent detection mode using pyrolytic GC-MS without PCR for rapid detection of increases in biomass (for example, increases in fecal contamination of drinking water or of germ warfare agents). To achieve minimal latency, a continuous sample stream flows directly into the PGC-MS combustion chamber. When an increase in biomass is detected, a PCR process is automatically initiated. Bioagent presence produces elevated levels of large molecular fragments from 100-7,000 Da which are observed in the PGC-MS spectrum. The observed mass spectrum is compared to a threshold level and when levels of biomass are determined to exceed a predetermined threshold, the bioagent classification process described hereinabove(combining PCR and MS, preferably FT-ICR MS) is initiated. Optionally, alarms or other processes (halting ventilation flow, physical isolation) are also initiated by this detected biomass level. [0062]
The accurate measurement of molecular mass for large DNAs is limited by the adduction of cations from the PCR reaction to each strand, resolution of the isotopic peaks from natural abundance [0063] 13C and 15N isotopes, and assignment of the charge state for any ion. The cations are removed by in-line dialysis using a flow-through chip that brings the solution containing the PCR products into contact with a solution containing ammonium acetate in the presence of an electric field gradient orthogonal to the flow. The latter two problems are addressed by operating with a resolving power of >100,000 and by incorporating isotopically depleted nucleotide triphosphates into the DNA. The resolving power of the instrument is also a consideration. At a resolving power of 10,000, the modeled signal from the [Mâ14H+]14â charge state of an 84 mer PCR product is poorly characterized and assignment of the charge state or exact mass is impossible. At a resolving power of 33,000, the peaks from the individual isotopic components are visible. At a resolving power of 100,000, the isotopic peaks are resolved to the baseline and assignment of the charge state for the ion is straightforward. The [13C,15N]-depleted triphosphates are obtained, for example, by growing microorganisms on depleted media and harvesting the nucleotides (Batey et al., Nucl. Acids Res. 20:4515-4523, 1992).
While mass measurements of intact nucleic acid regions are believed to be adequate to determine most bioagents, tandem mass spectrometry (MS[0064] n) techniques may provide more definitive information pertaining to molecular identity or sequence. Tandem MS involves the coupled use of two or more stages of mass analysis where both the separation and detection steps are based on mass spectrometry. The first stage is used to select an ion or component of a sample from which further structural information is to be obtained. The selected ion is then fragmented using, e.g., blackbody irradiation, infrared multiphoton dissociation, or collisional activation. For example, ions generated by electrospray ionization (ESI) can be fragmented using IR multiphoton dissociation. This activation leads to dissociation of glycosidic bonds and the phosphate backbone, producing two series of fragment ions, called the w-series (having an intact 3â² terminus and a 5â² phosphate following internal cleavage) and the a-Base series(having an intact 5â² terminus and a 3â² furan).
The second stage of mass analysis is then used to detect and measure the mass of these resulting fragments of product ions. Such ion selection followed by fragmentation routines can be performed multiple times so as to essentially completely dissect the molecular sequence of a sample. [0065]
If there are two or more targets of similar base composition or mass, or if a single amplification reaction results in a product which has the same mass as two or more bioagent reference standards, they can be distinguished by using mass-modifying âtags.â In this embodiment of the invention, a nucleotide analog or âtagâ is incorporated during amplification (e.g., a 5-(trifluoromethyl) deoxythymidine triphosphate) which has a different molecular weight than the unmodified base so as to improve distinction of masses. Such tags are described in, for example, PCT WO97/33000. This further limits the number of possible base compositions consistent with any mass. For example, 5-(trifluoromethyl)deoxythymidine triphosphate can be used in place of dTTP in a separate nucleic acid amplification reaction. Measurement of the mass shift between a conventional amplification product and the tagged product is used to quantitate the number of thymidine nucleotides in each of the single strands. Because the strands are complementary, the number of adenosine nucleotides in each strand is also determined. [0066]
In another amplification reaction, the number of G and C residues in each strand is determined using, for example, the cytidine analog 5-methylcytosine (5-meC) or propyne C. The combination of the A/T reaction and G/C reaction, followed by molecular weight determination, provides a unique base composition. This method is summarized in FIG. 4 and Table 1.
[0067] TABLE 1 Total Total Total Base Base base base Double Single Îmass info info comp. comp. strand strand this this other Top Bottom Mass tag sequence sequence strand strand strand strand strand T*Îmass T*ACGT*ACGT* T*ACGT*ACGT* 3x 3T 3A 3T 3A (T*âT) = x AT*GCAT*GCA 2A 2T 2C 2G 2G 2C AT*GCAT*GCA 2x 2T 2A C*Îmass TAC*GTAC*GT TAC*GTAC*GT 2x 2C 2G (C*âC) = y ATGC*ATGCA ATGC*ATGC*A 2x 2C 2GThe mass tag phosphorothioate A (A*) was used to distinguish a [0068] Bacillus anthracis cluster. The B. anthracis (A14G9C14T9) had an average MW of 14072.26, and the B. anthracis (A1A*13G9C14T9) had an average molecular weight of 14281.11 and the phosphorothioate A had an average molecular weight of +16.06 as determined by ESI-TOF MS. The deconvoluted spectra are shown in FIG. 5.
In another example, assume the measured molecular masses of each strand are 30,000.115Da and 31,000.115 Da respectively, and the measured number of dT and dA residues are (30,28) and (28,30). If the molecular mass is accurate to 100 ppm, there are 7 possible combinations of dG+dC possible for each strand. However, if the measured molecular mass is accurate to 10 ppm, there are only 2 combinations of dG+dC, and at 1 ppm accuracy there is only one possible base composition for each strand. [0069]
Signals from the mass spectrometer may be input to a maximum-likelihood detection and classification algorithm such as is widely used in radar signal processing. The detection processing uses matched filtering of BCS observed in mass-basecount space and allows for detection and subtraction of signatures from known, harmless organisms, and for detection of unknown bioagent threats. Comparison of newly observed bioagents to known bioagents is also possible, for estimation of threat level, by comparing their BCS to those of known organisms and to known forms of pathogenicity enhancement, such as insertion of antibiotic resistance genes or toxin genes. [0070]
Processing may end with a Bayesian classifier using log likelihood ratios developed from the observed signals and average background levels. The program emphasizes performance predictions culminating in probability-of-detection versus probability-of-false-alarm plots for conditions involving complex backgrounds of naturally occurring organisms and environmental contaminants. Matched filters consist of a priori expectations of signal values given the set of primers used for each of the bioagents. A genomic sequence database (e.g. GenBank) is used to define the mass basecount matched filters. The database contains known threat agents and benign background organisms. The latter is used to estimate and subtract the signature produced by the background organisms. A maximum likelihood detection of known background organisms is implemented using matched filters and a running-sum estimate of the noise covariance. Background signal strengths are estimated and used along with the matched filters to form signatures which are then subtracted. the maximum likelihood process is applied to this âcleaned upâ data in a similar manner employing matched filters for the organisms and a running-sum estimate of the noise-covariance for the cleaned up data. [0071]
In one embodiment, a strategy to âtriangulateâ each organism by measuring signals from multiple core genes is used to reduce false negative and false positive signals, and enable reconstruction of the origin or hybrid or otherwise engineered bioagents. After identification of multiple core genes, alignments are created from nucleic acid sequence databases. The alignments are then analyzed for regions of conservation and variation, and potential primer binding sites flanking variable regions are identified. Next, amplification target regions for signature analysis are selected which distinguishes organisms based on specific genomic differences (i.e., base composition). For example, detection of signatures for the three part toxin genes typical of [0072] B. anthracis (Bowen, J. E. and C. P. Quinn, J. Appl. Microbiol. 1999, 87, 270-278) in the absence of the expected signatures from the B. anthracis genome would suggest a genetic engineering event.
The present method can also be used to detect single nucleotide polymorphisms (SNPs), or multiple nucleotide polymorphisms, rapidly and accurately. A SNP is defined as a single base pair site in the genome that is different from one individual to another. The difference can be expressed either as a deletion, an insertion or a substitution, and is frequently linked to a disease state. Because they occur every 100-1000 base pairs, SNPs are the most frequently bound type of genetic marker in the human genome. [0073]
For example, sickle cell anemia results from an A-T transition, which encodes a valine rather than a glutamic acid residue. oligonucleotide primers may be designed such that they bind to sequences which flank a SNP site, followed by nucleotide amplification and mass determination of the amplified product. Because the molecular masses of the resulting product from an individual who does not have sickle cell anemia is different from that of the product from an individual who has the disease, the method can be used to distinguish the two individuals. Thus, the method can be used to detect any known SNP in an individual and thus diagnose or determine increased susceptibility to a disease or condition. [0074]
In one embodiment, blood is drawn from an individual and peripheral blood mononuclear cells (PBMC) are isolated and simultaneously tested, preferably in a high-throughput screening method, for one or more SNPs using appropriate primers based on the known sequences which flank the SNP region. The National Center for Biotechnology Information maintains a publicly available database of SNPs (http://www.ncbi.nlm.nih.gov/SNP/). [0075]
The method of the present invention can also be used for blood typing. The gene encoding A, B or O blood type can differ by four single nucleotide polymorphisms. If the gene contains the sequence CGTGGTGACCCTT, antigen A results. If the gene contains the sequence CGTCGTCACCGCTA antigen B results. If the gene contains the sequence CGTGGT-ACCCCTT, blood group O results (âââ indicates a deletion). These sequences can be distinguished by designing a single primer pair which flanks these regions, followed by amplification and mass determination. [0076]
While the present invention has been described with specificity in accordance with certain of its preferred embodiments, the following examples serve only to illustrate the invention and are not intended to limit the same. [0077]
Nucleic Acid Isolation and PCR [0078]
In one embodiment, nucleic acid is isolated from the organisms and amplified by PCR using standard methods prior to BCS determination by mass spectrometry. Nucleic acid is isolated, for example, by detergent lysis of bacterial cells, centrifugation and ethanol precipitation. Nucleic acid isolation methods are described in, for example, [0079] Current Protocols in Molecular Biology (Ausubel et al.) and Molecular Cloning; A Laboratory Manual (Sambrook et al.). The nucleic acid is then amplified using standard methodology, such as PCR, with primers which bind to conserved regions of the nucleic acid which contain an intervening variable sequence as described below.
Mass Spectrometry [0080]
FTICR Instrumentation: The FTICR instrument is based on a 7 tesla actively shielded superconducting magnet and modified Bruker Daltonics Apex II 70e ion optics and vacuum chamber. The spectrometer is interfaced to a LEAP PAL autosampler and a custom fluidics control system for high throughput screening applications. Samples are analyzed directly from 96-well or 384-well microtiter plates at a rate of about 1 sample/minute. The Bruker data-acquisition platform is supplemented with a lab-built ancillary NT datastation which controls the autosampler and contains an arbitrary waveform generator capable of generating complex rf-excite waveforms (frequency sweeps, filtered noise, stored waveform inverse Fourier transform (SWIFT), etc.) for sophisticated tandem MS experiments. For oligonucleotides in the 20-30-mer regime typical performance characteristics include mass resolving power in excess of 100,000 (FWHM), low ppm mass measurement errors, and an operable m/z range between 50 and 5000 m/z. [0081]
Modified ESI Source: In sample-limited analyses, analyte solutions are delivered at 150 nL/minute to a 30 mm i.d. fused-silica ESI emitter mounted on a 3-D micromanipulator. The ESI ion optics consist of a heated metal capillary, an rf-only hexapole, a skimmer cone, and an auxiliary gate electrode. The 6.2 cm rf-only hexapole is comprised of 1 mm diameter rods and is operated at a voltage of 380 Vpp at a frequency of 5 MHz. A lab-built electromechanical shutter can be employed to prevent the electrospray plume from entering the inlet capillary unless triggered to the âopenâ position via a TTL pulse from the data station. When in the âclosedâ position, a stable electrospray plume is maintained between the ESI emitter and the face of the shutter. The back face of the shutter arm contains an elastomeric seal which can be positioned to form a vacuum seal with the inlet capillary. When the seal is removed, a 1 mm gap between the shutter blade and the capillary inlet allows constant pressure in the external ion reservoir regardless of whether the shutter is in the open or closed position. When the shutter is triggered, a âtime sliceâ of ions is allowed to enter the inlet capillary and is subsequently accumulated in the external ion reservoir. The rapid response time of the ion shutter (<25 ms) provides reproducible, user defined intervals during which ions can be injected into and accumulated in the external ion reservoir. [0082]
Apparatus for Infrared Multiphoton Dissociation [0083]
A 25 watt CW CO[0084] 2 laser operating at 10.6 μm has been interfaced to the spectrometer to enable infrared multiphoton dissociation (IRMPD) for oligonucleotide sequencing and other tandem MS applications. An aluminum optical bench is positioned approximately 1.5 m from the actively shielded superconducting magnet such that the laser beam is aligned with the central axis of the magnet. Using standard IR-compatible mirrors and kinematic mirror mounts, the unfocused 3 mm laser beam is aligned to traverse directly through the 3.5 mm holes in the trapping electrodes of the FTICR trapped ion cell and longitudinally traverse the hexapole region of the external ion guide finally impinging on the skimmer cone. This scheme allows IRMPD to be conducted in an m/z selective manner in the trapped ion cell (e.g. following a SWIFT isolation of the species of interest), or in a broadband mode in the high pressure region of the external ion reservoir where collisions with neutral molecules stabilize IRMPD-generated metastable fragment ions resulting in increased fragment ion yield and sequence coverage.
Identification of Bioagents [0085]
Table 1 shows a small cross section of a database of calculated molecular masses for over 9 primer sets and approximately 30 organisms. The primer sets were derived from rRNA alignment. Examples of regions from rRNA consensus alignments are shown in FIGS. [0086] 1A-1C. Lines with arrows are examples of regions to which intelligent primer pairs for PCR are designed. The primer pairs are >95% conserved in the bacterial sequence database (currently over 10,000 organisms). The intervening regions are variable in length and/or composition, thus providing the base composition âsignatureâ (BCS) for each organism. Primer pairs were chosen so the total length of the amplified region is less than about 80-90 nucleotides. The label for each primer pair represents the starting and ending base number of the amplified region on the consensus diagram.
Included in the short bacterial database cross-section in Table 1 are many well known pathogens/biowarfare agents (shown in bold/red typeface) such as
[0087] Bacillus anthracisor
Yersinia pestisas well as some of the bacterial organisms found commonly in the natural environment such as Streptomyces. Even closely related organisms can be distinguished from each other by the appropriate choice of primers. For instance, two low G+C organisms,
Bacillus anthracisand Staph aureus, can be distinguished from each other by using the primer pair defined by
16S â1337 or 23S
â855 (ÎM of 4 Da).
TABLE 2 Cross Section Of A Database Of Calculated Molecular Masses1 Primer Regions âââ> Bug Name 16S_971 16S_1100 16S_1337 16S_1294 16S_1228 23S_1021 23S_855 23S_193 23S_115FIG. 6 shows the use of ESI-FT-ICR MS for measurement of exact mass. The spectra from 46 mer PCR products originating at position 1337 of the 16S rRNA from
[0088] S. aureus(upper) and
B. anthracis(lower) are shown. These data are from the region of the spectrum containing signals from the [Mâ
08H+]
8âcharge states of the respective 5â²-3â² strands. The two strands differ by two (ATâCG) substitutions, and have measured masses of 14206.396 and 14208.373±0.010 Da, respectively. The possible base compositions derived from the masses of the forward and reverse strands for the
B. anthracisproducts are listed in Table 3.
TABLE 3 Possible base composition for B. anthracis products Calc. Mass Error Base Comp. 14208.2935 0.079520 A1 G17 C10 T18 14208.3160 0.056980 A1 G20 C15 T10 14208.3386 0.034440 A1 G23 C20 T2 14208.3074 0.065560 A6 G11 C3 T26 14208.3300 0.043020 A6 G14 C8 T18 14208.3525 0.020480 A6 G17 C13 T10 14208.3751 0.002060 A6 G20 C18 T2 14208.3439 0.029060 A11 G8 C1 T26 14208.3665 0.006520 A11 G11 C6 T18 14208.3890 0.016020 A11 G14 C11 T10 14208.4116 0.038560 A11 G17 C16 T2 14208.4030 0.029980 A16 G8 C4 T18 14208.4255 0.052520 A16 G11 C9 T10 14208.4481 0.075060 A16 G14 C14 T2 14208.4395 0.066480 A21 G5 C2 T18 14208.4620 0.089020 A21 G8 C7 T10 14079.2624 0.080600 A0 G14 C13 T19 14079.2849 0.058060 A0 G17 C18 T11 14079.3075 0.035520 A0 G20 C23 T3 14079.2538 0.089180 A5 G5 C1 T35 14079.2764 0.066640 A5 G8 C6 T27 14079.2989 0.044100 A5 G11 C11 T19 14079.3214 0.021560 A5 G14 C16 T11 14079.3440 0.000980 A5 G17 C21 T3 14079.3129 0.030140 A10 G5 C4 T27 14079.3354 0.007600 A10 G8 C9 T19 14079.3579 0.014940 A10 G11 C14 T11 14079.3805 0.037480 A10 G14 C19 T3 14079.3494 0.006360 A15 G2 C2 T27 14079.3719 0.028900 A15 G5 C7 T19 14079.3944 0.051440 A15 G8 C12 T11 14079.4170 0.073980 A15 G11 C17 T3 14079.4084 0.065400 A20 G2 C5 T19 14079.4309 0.087940 A20 G5 C10 T13Among the 16 compositions for the forward strand and the 18 compositions for the reverse strand that were calculated, only one pair (shown in bold) are complementary, corresponding to the actual base compositions of the [0089] B. anthracis PCR products.
BCS of Region from [0090] Bacillus anthracis and Bacillus cereus
A conserved Bacillus region from [0091] B. anthracis (A14G9C14T9) and B. cereus (A15G9C13T9) having a C to A base change was synthesized and subjected to ESI-TOF MS. The results are shown in FIG. 7 in which the two regions are clearly distinguished using the method of the present invention (MW=14072.26 vs. 14096.29).
Identification of Additional Bioagents [0092]
In other examples of the present invention, the pathogen [0093] Vibrio cholera can be distinguished from Vibrio parahemolyticus with ÎM>600 Da using one of three 16S primer sets shown in Table 2 ( 16S â971, 16S â1228 or 16Sâ1294) as shown in Table 4. The two mycoplasma species in the list (M. genitalium and M. pneumoniae) can also be distinguished from each other, as can the three mycobacteriae. While the direct mass measurements of amplified products can identify and distinguish a large number of organisms, measurement of the base composition signature provides dramatically enhanced the base composition signature provides dramatically enhanced resolving power for closely related organisms. In cases such as Bacillus anthracis and Bacillus cereus that are virtually indistinguishable from each other based solely on mass differences, compositional analysis or fragmentation patterns are used to resolve the differences. The single base difference between the two organisms yields different fragmentation patterns, and despite the presence of the ambiguous/unidentified base N at position 20 in B. anthracis, the two organisms can be identified.
Tables 4a-b show examples of primer pairs from Table 1 which distinguish pathogens from background.
[0094] TABLE 4a Organism name 23S_855 16S_1337 23S_1021 Bacillus anthracis 42650.98 28447.65 30294.98 Staphylococcus aureus 42654.97 28443.67 30297.96Table 4 shows the expected molecular weight and base composition of
[0096] region16S
â1100-1188 in
Mycobacterium aviumand Streptomyces sp.
TABLE 5 Molecular Base Region Organism name Length weight comp. 16S_1100- Mycobacterium 82 25624.1728 A16G32C18T16 1188 avium 16S_1100- Streptomyces sp. 96 29904.871 A17G38C27T14 1188Table 5 shows base composition (single strand) results for 16S
[0097] â1100-1188 primer amplification reactions different species of bacteria. Species which are repeated in the table (e.g.,
Clostridium botulinum) are different strains which have different base compositions in the 16S
â1100-1188 region.
The same organism having different base compositions are different strains. Groups of organisms which are highlighted or in italics have the same base compositions in the amplified region. Some of these organisms can be distinguished using multiple primers. For example,
[0098] Bacillus anthraciscan be distinguished from
Bacillus cereusand
Bacillus thuringiensisusing the
primer16S
â971-1062 (Table 6). Other primer pairs which produce unique base composition signatures are shown in Table 6 (bold). Clusters containing very similar threat and ubiquitous non-threat organisms (e.g.
anthraciscluster) are distinguished at high resolution with focused sets of primer pairs. The known biowarfare agents in Table 6 are
Bacillus anthracis, Yersinia pestis, Francisella tularensisand
Rickettsia prowazekii.
TABLE 7 Organism 16S_971-1062 16S_1228-1310 16S_1100-1188 Aeromonas A21G29C22T20 A22G27C21T13 A23G31C21T15 hydrophila Aeromonas A21G29C22T20 A22G27C21T13 A23G31C21T15 salmonicida Bacillus anthracis A 21 G 27 C 22 T 22 A24G22C19T18 A23G27C20T18 Bacillus cereus A22G27C21T22 A24G22C19T18 A23G27C20T18 Bacillus A22G27C21T22 A24G22C19T18 A23G27C20T18 thuringiensis Chlamydia A 22 G 26 C 20 T 23 A 24 G 23 C 19 T 16 A24G28C21T16 trachomatis Chlamydia A26G23C20T22 A26G22C16T18 A24G28C21T16 pneumoniae AR39 Leptospira A22G26C20T21 A22G25C21T15 A23G26C24T15 borgpetersenii Leptospira A22G26C20T21 A22G25C21T15 A23G26C24T15 interrogans Mycoplasma A28G23C15T22 A 30 G 18 C 15 T 19 A 24 G 19 C 12 T 18 genitalium Mycoplasma A28G23C15T22 A 27 G 19 C 16 T 20 A 23 G 20 C 14 T 16 pneumoniae Escherichia coli A 22 G 28 C 20 T 22 A24G25C21T13 A23G29C22T15 Shigella A 22 G 28 C 21 T 21 A24G25C21T13 A23G29C22T15 dysenteriae Proteus vulgaris A 23 G 26 C 22 T 21 A 26 G 24 C 19 T 14 A24G30C21T15 Yersinia pestis A24G25C21T22 A25G24C20T14 A24G30C21T15 Yersinia A24G25C21T22 A25G24C20T14 A24G30C21T15 pseudo- tuberculosis Francisella A 20 G 25 C 21 T 23 A 23 G 26 C 17 T 17 A 24 G 26 C 19 T 19 tularensis Rickettsia A 21 G 26 C 24 T 25 A 24 G 23 C 16 T 19 A 26 G 28 C 18 T 18 prowazekii Rickettsia A 21 G 26 C 25 T 24 A 24 G 24 C 17 T 17 A 26 G 28 C 20 T 16 rickettsiiThe sequence of
[0099] B. anthracisand
B. cereusin
region16S
â971 is shown below. Shown in bold is the single base difference between the two species which can be detected using the methods of the present invention.
B. anthracishas an ambiguous base at
position 20.
B. anthracis_16S_971 GCGAAGAACCUUACCAGGUNUUGACAUCCUCUGACAACCCUAGAGAUAGGGCUUCUCCUUC (SEQ ID NO: 1) GGGAGCAGAGUGACAGGUGGUGCAUGGUU B. cereus_16S_971 GCGAAGAACCUUACCAGGUCUUGACAUCCUCUGAAACCCUAGAGAUAGGGCUUCUCCUUC (SEQ ID NO: 2) GGGAGCAGAGUGACAGGUGGUGCAUGGUUESI-TOF MS of sspE 56-mer Plus Calibrant [0100]
The mass measurement accuracy that can be obtained using an internal mass standard in the ESI-MS study of PCR products is shown in FIG.8. The mass standard was a 20-mer phosphorothioate oligonucleotide added to a solution containing a 56-mer PCR product from the [0101] B. anthracis spore coat protein sspE. The mass of the expected PCR product distinguishes B. anthracis from other species of Bacillus such as B. thuringiensis and B. cereus.
[0102] B. anthracis ESI- TOF Synthetic 16Sâ1228 Duplex
An ESI-TOF MS spectrum was obtained from an aqueous solution containing 5 μM each of synthetic analogs of the expected forward and reverse PCR products from the nucleotide 1228 region of the [0103] B. anthracis 16S rRNA gene. The results (FIG. 9) show that the molecular weights of the forward and reverse strands can be accurately determined and easily distinguish the two strands. The [Mâ21H+]21â and [Mâ20H+]20â charge states are shown.
ESI-FTICR-MS of [0104] Synthetic B. anthracis 16Sâ1337 46 Base Pair Duplex
An ESI-FTICR-MS spectrum was obtained from an aqueous solution containing 5 μM each of synthetic analogs of the expected forward and reverse PCR products from the nucleotide 1337 region of the [0105] B. anthracis 16S rRNA gene. The results (FIG. 10) show that the molecular weights of the strands can be distinguished by this method. The [Mâ16H+]16â through [Mâ10H+]10â charge states are shown. The insert highlights the resolution that can be realized on the FTICR-MS instrument, which allows the charge state of the ion to be determined from the mass difference between peaks differing by a single 13C substitution.
ESI-TOF MS of 56-mer Oligonucleotide From saspB Gene of [0106] B. anthracis With Internal Mass Standard
ESI-TOF MS spectra were obtained on a synthetic 56-mer oligonucleotide (5 μM )from the saspB gene of [0107] B. anthracis containing an internal mass standard at an ESI of 1.7 μL/min as a function of sample consumption. The results (FIG. 11) show that the signal to noise is improved as more scans are summed, and that the standard and the product are visible after only 100 scans.
ESI-TOF MS of an Internal Standard With Tributylammonium (TBA)-trifluoroacetate (TFA) Buffer [0108]
An ESI-TOF-MS spectrum of a 20-mer phosphorothioate mass standard was obtained following addition of 5 mM TBA-TFA buffer to the solution. This buffer strips charge from the oligonucleotide and shifts the most abundant charge state from [Mâ8H[0109] +]8â to [Mâ3H+]3â(FIG. 12).
1 7 1 90 RNA Bacillus anthracis misc_feature (20)..(20) N = A, U, G or C 1 gcgaagaacc uuaccaggun uugacauccu cugacaaccc uagagauagg gcuucuccuu 60 cgggagcaga gugacaggug gugcaugguu 90 2 90 RNA Bacillus cereus 2 gcgaagaacc uuaccagguc uugacauccu cugaaaaccc uagagauagg gcuucuccuu 60 cgggagcaga gugacaggug gugcaugguu 90 3 1542 RNA Artificial Sequence misc_feature 16S rRNA consensus sequence 3 nnnnnnnaga guuugaucnu ggcucagnnn gaacgcuggc ggnnngcnun anacaugcaa 60 gucgancgnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn agnggcnnac gggugaguaa 120 nncnunnnna nnunccnnnn nnnnnggnan annnnnnnga aannnnnnnu aauaccnnau 180 nnnnnnnnnn nnnnaaagnn nnnnnnnnnn nnnnnnnnnn nnnnnngann nnnnnnngnn 240 nnaunagnun guuggunngg uaanggcnna ccaagncnnn gannnnuagc ngnncugaga 300 ggnngnncng ccacanuggn acugaganac ggnccanacu ccuacgggag gcagcagunn 360 ggaaunuunn ncaauggnng naanncugan nnagcnannc cgcgugnnng anganggnnu 420 nnngnungua aannncunun nnnnnngang annnnnnnnn nnnnnnnnnn nnnnnnnnnu 480 gacnnuannn nnnnannaag nnncggcnaa cuncgugcca gcagccgcgg uaauacgnag 540 gnngcnagcg uunnncggan unanugggcg uaaagngnnn gnaggnggnn nnnnnngunn 600 nnngunaaan nnnnnngcun aacnnnnnnn nnncnnnnnn nacnnnnnnn cungagnnnn 660 nnagnggnnn nnngaauunn nnguguagng gugnaauncg naganaunng nangaanacc 720 nnungcgaag gcnnnnnncu ggnnnnnnac ugacncunan nnncgaaagc nugggnagcn 780 aacaggauua gauacccugg uaguccangc nnuaaacgnu gnnnnnunnn ngnnngnnnn 840 nnnnnnnnnn nnnnnnnnna nnnaacgnnn uaannnnncc gccuggggag uacgnncgca 900 agnnunaaac ucaaangaau ugacggggnc cngcacaagc ngnggagnau guggnuuaau 960 ucgangnnac gcgnanaacc uuaccnnnnn uugacaunnn nnnnnnnnnn nnganannnn 1020 nnnnnnnnnn nnnnnnnnnn nnnacaggug nugcauggnu gucgucagcu cgugnnguga 1080 gnuguugggu uaagucccgn aacgagcgca acccnnnnnn nnnguuncna ncnnnnnnnn 1140 ngngnacucn nnnnnnacug ccnnngnnaa nnnggaggaa ggnggggang acgucaanuc 1200 nucaugnccc uuangnnnng ggcuncacac nuncuacaau ggnnnnnaca nngngnngcn 1260 annnngnnan nnnnagcnaa ncnnnnaaan nnnnucnnag uncggaungn nnncugcaac 1320 ucgnnnncnu gaagnnggan ucgcuaguaa ucgnnnauca gnangnnncg gugaauacgu 1380 ucncgggncu uguacacacc gcccgucann ncangnnagn nnnnnnnncc nnaagnnnnn 1440 nnnnnnncnn nnnngnnnnn nnnnncnang gnnnnnnnnn nganugggnn naagucguaa 1500 caagguancc nuannngaan nugnggnugg aucaccuccu un 1542 4 2904 RNA Artificial Sequence misc_feature 23S rRNA consensus sequence 4 nnnnaagnnn nnaagngnnn nngguggaug ccunggcnnn nnnagncgan gaaggangnn 60 nnnnncnncn nnanncnnng gnnagnngnn nnnnnncnnn nnanccnnng nunuccgaau 120 ggggnaaccc nnnnnnnnnn nnnnnnnnan nnnnnnnnnn nnnnnnnnnn nnnnnnngnn 180 nacnnnnnga anugaaacau cunaguannn nnaggaanag aaannaannn ngauuncnnn 240 nguagnggcg agcgaannng nannagncnn nnnnnnnnnn nnnnnnnnnn nnnannngaa 300 nnnnnuggna agnnnnnnnn nannngguna nannccngua nnnnaaannn nnnnnnnnnn 360 nnnnnnnnnn aguannncnn nncncgngnn annnngunng aannngnnnn gaccannnnn 420 naagncuaaa uacunnnnnn ngaccnauag ngnannagua cngugangga aaggngaaaa 480 gnacccnnnn nangggagug aaanagnncc ugaaaccnnn nncnuanaan nngunnnagn 540 nnnnnnnnnn nnnuganngc gunccuuuug nannaugnnn cngnganuun nnnunnnnng 600 cnagnuuaan nnnnnnnngn agncgnagng aaancgagun nnaanngngc gnnnagunnn 660 nngnnnnaga cncgaancnn ngugancuan nnaugnncag gnugaagnnn nnguaanann 720 nnnuggaggn ccgaacnnnn nnnnguugaa aannnnnngg augannugug nnungnggng 780 aaanncnaan cnaacnnngn nauagcuggu ucucnncgaa annnnuuuag gnnnngcnun 840 nnnnnnnnnn nnnnggnggu agagcacugn nnnnnnnnng gnnnnnnnnn nnnnuacnna 900 nnnnnnnnaa acuncgaaun ccnnnnnnnn nnnnnnnngn agnnanncnn ngngngnuaa 960 nnuncnnngu nnanagggna acancccaga ncnncnnnua aggncccnaa nnnnnnnnua 1020 aguggnaaan gangugnnnn nncnnanaca nnnaggangu uggcuuagaa gcagccancn 1080 uunaaagann gcguaanagc ucacunnucn agnnnnnnng cgcngannau nuancgggnc 1140 uaannnnnnn nccgaannnn nngnnnnnnn nnnnnnnnnn nnnnngguag nngagcgunn 1200 nnnnnnnnnn ngaagnnnnn nngnnannnn nnnuggannn nnnnnnagug ngnaugnngn 1260 naunaguanc gannnnnnnn gugananncn nnnncnccgn annncnaagg nuuccnnnnn 1320 nangnunnuc nnnnnngggu nagucgnnnc cuaagnngag ncnganangn nuagnngaug 1380 gnnannnggu nnauauuccn nnacnnnnnn nnnnnnnnnn nnnnngacgn nnnnngnnnn 1440 nnnnnnnnnn nnnnggnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 1500 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 1560 nnnncnngaa aannnnnnnn nnnnnnnnnn nnnnnnnnnc guaccnnaaa ccgacacagg 1620 ungnnnngnn gagnanncnn aggngnnngn nnnaannnnn nnnaaggaac unngcaaanu 1680 nnnnccguan cuucggnana aggnnnncnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 1740 nnnnnnnnng nnnnannnan nngnnnnnnn cnacuguuua nnaaaaacac agnncnnugc 1800 naanncgnaa gnnganguau anggnnugac nccugcccng ugcnngaagg uuaanngnnn 1860 nnnnnngnnn nngnnnnnnn nnnnannnaa gcccnnguna acggcggnng uaacuauaac 1920 nnuccuaagg uagcgaaauu ccuugucggg uaaguuccga ccngcacgaa nggngnaang 1980 annnnnnnnc ugucucnnnn nnnnncncng ngaanuunna nunnnnguna agaugcnnnn 2040 uncncgcnnn nngacggaaa gaccccnngn ancuuuacun nannnunnna nugnnnnnnn 2100 nnnnnnnnug unnagnauag gunggagncn nngannnnnn nncgnnagnn nnnnnggagn 2160 cnnnnnugnn auacnacncu nnnnnnnnnn nnnnucuaac nnnnnnnnnn nancnnnnnn 2220 nnngacanug nnngnngggn aguuunacug gggcggunnc cuccnaaann guaacggagg 2280 ngnncnaagg unnncunann nnggnnggnn aucnnnnnnn nagunnaann gnanaagnnn 2340 gcnunacugn nagnnnnacn nnncgagcag nnncgaaagn nggnnnuagu gauccggngg 2400 unnnnnnugg aagngccnuc gcucaacgga uaaaagnuac ncnggggaua acaggcunau 2460 nnnncccaag aguncanauc gacggnnnng uuuggcaccu cgaugucggc ucnucncauc 2520 cuggggcugn agnngguccc aagggunngg cuguucgccn nuuaaagngg nacgngagcu 2580 ggguunanaa cgucgugaga caguungguc ccuaucngnn gngngngnnn gannnuugan 2640 nngnnnugnn cnuaguacga gaggaccggn nngnacnnan cncuggugnn ncnguugunn 2700 ngccannngc anngcngnnu agcuannunn ggnnnngaua anngcugaan gcaucuaagn 2760 nngaancnnn cnnnnagann agnnnucncn nnnnnnnnnn nnnnnnnnna gnnncnnnnn 2820 agannannnn gungauaggn nngnnnugna agnnnngnna nnnnunnagn nnacnnnuac 2880 uaaunnnncn nnnnncuunn nnnn 2904 5 13 DNA Artificial Sequence misc_feature Primer 5 cgtggtgacc ctt 13 6 14 DNA Artificial Sequence misc_feature Primer 6 cgtcgtcacc gcta 14 7 13 DNA Artificial Sequence misc_feature Primer 7 cgtggtaccc ctt 13
. A method of identifying an unknown bioagent comprising:
(a) contacting nucleic acid from said bioagent with at least one pair of oligonucleotide primers which hybridize to sequences of said nucleic acid, wherein said sequences flank a variable nucleic acid sequence of the bioagent;
(b) amplifying said variable nucleic acid sequence to produce an amplification product;
(c) determining the molecular mass of said amplification product; and
(d) comparing said molecular mass to one or more molecular masses of amplification products obtained by performing steps (a)-(c) on a plurality of known organisms, wherein a match identifies said unknown bioagent.
2. The method of claim 1 , wherein said sequences to which said at least one pair of oligonucleotide primers hybridize are highly conserved.
3. The method of claim 1 , wherein said amplifying step comprises polymerase chain reaction.
4. The method of claim 1 , wherein said amplifying step comprises ligase chain reaction or strand displacement amplification.
5. The method of claim 1 , wherein said bioagent is a bacterium, virus, cell or spore.
6. The method of claim 1 , wherein said nucleic acid is ribosomal RNA.
7. The method of claim 1 , wherein said nucleic acid encodes RNase P or an RNA-dependent RNA polymerase.
8. The method of claim 1 , wherein said amplification product is ionized prior to molecular mass determination.
9. The method of claim 1 , further comprising the step of isolating nucleic acid from said bioagent prior to contacting said nucleic acid with said at least one pair of oligonucleotide primers.
10. The method of claim 1 , further comprising the step of performing steps (a)-(d) using a different oligonucleotide primer pair and comparing the results to one or more molecular mass amplification product obtained by performing steps (a)-(c) on a different plurality of known organisms from those in step (d).
11. The method of claim 1 , wherein said one or more molecular masses are contained in a database of molecular masses.
12. The method of claim 1 , wherein said amplification product is ionized by electrospray ionization, matrix-assisted laser desorption or fast atom bombardment.
13. The method of claim 1 , wherein said molecular mass is determined by mass spectrometry.
14. The method of claim 11 , wherein said mass spectrometry is selected from the group consisting of Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS), ion trap, quadrupole, magnetic sector, time of flight (TOF), Q-TOF and triple quadrupole.
15. The method of claim 1 , further comprising performing step (b) in the presence of an analog of adenine, thymidine, guanosine or cytidine having a different molecular weight than adenosine, thymidine, guanosine or cytidine.
16. The method of claim 1 , wherein said oligonucleotide primer comprises a base analog at positions 1 and 2 of each triplet within said primer, wherein said base analog binds with increased affinity to its complement compared to the native base.
17. The method of claim 16 , wherein said primer comprises a universal base at position 3 of each triplet within said primer.
18. The method of claim 16 , wherein said base analog is selected from the group consisting of 2,6-diaminopurine, propyne T, propyne G, phenoxazines and G-clamp.
19. The method of claim 16 , wherein said universal base is selected from the group consisting of inosine, guanidine uridine, 5-nitroindole, 3-nitropyrrole, dP, dK, and 1-(2-deoxy-β-D-ribofuranosyl)-imidazole-4-carboxamide.
20. A method of identifying an unknown bioagent comprising:
contacting nucleic acid from said bioagent with at least one pair of oligonucleotide primers which hybridize to sequences of said nucleic acid, wherein said sequences flank a variable nucleic acid sequence;
amplifying said variable nucleic acid sequence to produce an amplification product;
determining the base composition of said amplification product; and
comparing said base composition to one or more base compositions of amplification products obtained by performing steps (a)-(c) on a plurality of known organisms, wherein a match identifies said unknown bioagent.
21. The method of claim 20 , wherein said sequences to which said at least one pair of oligonucleotide primers hybridize are highly conserved.
22. The method of claim 20 , wherein said amplifying step comprises polymerase chain reaction.
23. The method of claim 20 , wherein said amplifying step comprises ligase chain reaction or strand displacement amplification.
24. The method of claim 20 , wherein said bioagent is a bacterium, virus, cell or spore.
25. The method of claim 20 , wherein said nucleic acid is ribosomal RNA.
26. The method of claim 20 , wherein said nucleic acid encodes RNase P or an RNA-dependent RNA polymerase.
27. The method of claim 20 , wherein said amplification product is ionized prior to base composition determination.
28. The method of claim 20 , further comprising the step of isolating nucleic acid from said bioagent prior to contacting said nucleic acid with said at least one pair of oligonucleotide primers.
29. The method of claim 20 , further comprising the step of performing steps (a)-(d) using a different oligonucleotide primer pair and comparing the results to one or more base compositions of amplification product obtained by performing steps (a)-(c) on a different plurality of known organisms from those in step (d).
30. The method of claim 20 , wherein said one or more base composition signatures are contained in a database of base composition signatures.
31. The method of claim 20 , wherein said amplification product is ionized by electrospray ionization, matrix-assisted laser desorption or fast atom bombardment.
32. The method of claim 20 , wherein said base composition signature is determined by mass spectrometry.
33. The method of claim 32 , wherein said mass spectrometry is selected from the group consisting of Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS), ion trap, quadrupole, magnetic sector, time of flight (TOF), q-TOF and triple quadrupole.
34. The method of claim 20 , further comprising performing step (b) in the presence of an analog of adenine, thymidine, guanosine or cytidine having a different molecular weight than adenosine, thymidine, guanosine or cytidine.
35. The method of claim 20 , wherein said oligonucleotide primer comprises a base analog at positions 1 and 2 of each triplet within said primer, wherein said base analog binds with increased affinity to its complement compared to the native base.
36. The method of claim 35 , wherein said primer comprises a universal base at position 3 of each triplet within said primer.
37. The method of claim 35 , wherein said base analog is selected from the group consisting of 2,6-diaminopurine, propyne T, propyne G, phenoxazines and G-clamp.
38. The method of claim 36 , wherein said universal base is selected from the group consisting of inosine, guanidine uridine, 5-nitroindole, 3-nitropyrrole, dP, dK, and 1-(2-deoxy-β-D-ribofuranosyl)-imidazole-4-carboxamide.
39. A method for detecting a single nucleotide polymorphism
in an individual, comprising the steps of:
isolating nucleic acid from said individual;
contacting said nucleic acid with oligonucleotide primers which hybridize to regions of said nucleic acid which flank a region comprising said potential polymorphism;
amplifying said region to produce an amplification product;
determining the molecular mass of said amplification product;
comparing said molecular mass to the molecular mass of said region in an individual known to have said polymorphism, wherein if said molecular masses are the same then said individual has said polymorphism.
40. The method of claim 39 , wherein said polymorphism is associated with a disease.
41. The method of claim 39 , wherein said polymorphism is a blood group antigen.
42. The method of claim 39 , wherein said amplification step is the polymerase chain reaction.
43. The method of claim 39 , wherein said amplification step is ligase chain reaction or strand displacement amplification.
44. The method of claim 39 , wherein said amplification product is ionized prior to mass determination.
45. The method of claim 39 , wherein said amplification product is ionized by electrospray ionization, matrix-assisted laser desorption or fast atom bombardment.
46. The method of claim 39 , wherein said primers hybridize to conserved sequences.
47. The method of claim 39 , wherein said molecular mass is determined by mass spectrometry.
48. The method of claim 47 , wherein said mass spectrometry is selected from the group consisting of Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS), ion trap, quadrupole, magnetic sector, time of flight (TOF), Q-TOF and triple quadrupole.
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Methods and compositions for the extraction and amplification of nucleic acid from a sample US20080096766A1 (en) * 2006-06-16 2008-04-24 Sequenom, Inc. Methods and compositions for the amplification, detection and quantification of nucleic acid from a sample US20090263809A1 (en) * 2008-03-20 2009-10-22 Zygem Corporation Limited Methods for Identification of Bioagents US7666588B2 (en) 2001-03-02 2010-02-23 Ibis Biosciences, Inc. Methods for rapid forensic analysis of mitochondrial DNA and characterization of mitochondrial DNA heteroplasmy US7666592B2 (en) 2004-02-18 2010-02-23 Ibis Biosciences, Inc. Methods for concurrent identification and quantification of an unknown bioagent US7714275B2 (en) 2004-05-24 2010-05-11 Ibis Biosciences, Inc. Mass spectrometry with selective ion filtration by digital thresholding US7718354B2 (en) 2001-03-02 2010-05-18 Ibis Biosciences, Inc. Methods for rapid identification of pathogens in humans and animals US20100129811A1 (en) * 2003-09-11 2010-05-27 Ibis Biosciences, Inc. Compositions for use in identification of pseudomonas aeruginosa US7741036B2 (en) 2001-03-02 2010-06-22 Ibis Biosciences, Inc. Method for rapid detection and identification of bioagents US7781162B2 (en) 2001-03-02 2010-08-24 Ibis Biosciences, Inc. Methods for rapid identification of pathogens in humans and animals US7811753B2 (en) 2004-07-14 2010-10-12 Ibis Biosciences, Inc. Methods for repairing degraded DNA US20100291544A1 (en) * 2007-05-25 2010-11-18 Ibis Biosciences, Inc. Compositions for use in identification of strains of hepatitis c virus US20110028334A1 (en) * 2009-07-31 2011-02-03 Ibis Biosciences, Inc. Capture primers and capture sequence linked solid supports for molecular diagnostic tests US20110045456A1 (en) * 2007-06-14 2011-02-24 Ibis Biosciences, Inc. Compositions for use in identification of adventitious contaminant viruses US20110065111A1 (en) * 2009-08-31 2011-03-17 Ibis Biosciences, Inc. Compositions For Use In Genotyping Of Klebsiella Pneumoniae WO2011047307A1 (en) 2009-10-15 2011-04-21 Ibis Biosciences, Inc. Multiple displacement amplification US20110097704A1 (en) * 2008-01-29 2011-04-28 Ibis Biosciences, Inc. Compositions for use in identification of picornaviruses US7956175B2 (en) 2003-09-11 2011-06-07 Ibis Biosciences, Inc. Compositions for use in identification of bacteria US20110143358A1 (en) * 2008-05-30 2011-06-16 Ibis Biosciences, Inc. Compositions for use in identification of tick-borne pathogens US7964343B2 (en) 2003-05-13 2011-06-21 Ibis Biosciences, Inc. Method for rapid purification of nucleic acids for subsequent analysis by mass spectrometry by solution capture US20110151437A1 (en) * 2008-06-02 2011-06-23 Ibis Biosciences, Inc. Compositions for use in identification of adventitious viruses US20110166040A1 (en) * 1997-09-05 2011-07-07 Ibis Biosciences, Inc. Compositions for use in identification of strains of e. coli o157:h7 US20110177515A1 (en) * 2008-05-30 2011-07-21 Ibis Biosciences, Inc. Compositions for use in identification of francisella US20110183344A1 (en) * 2008-10-03 2011-07-28 Rangarajan Sampath Compositions for use in identification of clostridium difficile US20110183345A1 (en) * 2008-10-03 2011-07-28 Ibis Biosciences, Inc. Compositions for use in identification of streptococcus pneumoniae US20110183346A1 (en) * 2008-10-03 2011-07-28 Ibis Biosciences, Inc. Compositions for use in identification of neisseria, chlamydia, and/or chlamydophila bacteria US20110183343A1 (en) * 2008-10-03 2011-07-28 Rangarajan Sampath Compositions for use in identification of members of the bacterial class alphaproteobacter US20110190170A1 (en) * 2008-10-03 2011-08-04 Ibis Biosciences, Inc. Compositions for use in identification of antibiotic-resistant bacteria US20110189687A1 (en) * 2008-10-02 2011-08-04 Ibis Bioscience, Inc. Compositions for use in identification of members of the bacterial genus mycoplasma US20110200985A1 (en) * 2008-10-02 2011-08-18 Rangarajan Sampath Compositions for use in identification of herpesviruses WO2011112718A1 (en) 2010-03-10 2011-09-15 Ibis Biosciences, Inc. Production of single-stranded circular nucleic acid US20110223599A1 (en) * 2010-03-14 2011-09-15 Ibis Biosciences, Inc. Parasite detection via endosymbiont detection US8026084B2 (en) 2005-07-21 2011-09-27 Ibis Biosciences, Inc. Methods for rapid identification and quantitation of nucleic acid variants US8046171B2 (en) 2003-04-18 2011-10-25 Ibis Biosciences, Inc. Methods and apparatus for genetic evaluation US8057993B2 (en) 2003-04-26 2011-11-15 Ibis Biosciences, Inc. Methods for identification of coronaviruses US8071309B2 (en) 2002-12-06 2011-12-06 Ibis Biosciences, Inc. Methods for rapid identification of pathogens in humans and animals US8073627B2 (en) 2001-06-26 2011-12-06 Ibis Biosciences, Inc. System for indentification of pathogens US8084207B2 (en) 2005-03-03 2011-12-27 Ibis Bioscience, Inc. Compositions for use in identification of papillomavirus US8088582B2 (en) 2006-04-06 2012-01-03 Ibis Biosciences, Inc. Compositions for the use in identification of fungi US8097416B2 (en) 2003-09-11 2012-01-17 Ibis Biosciences, Inc. Methods for identification of sepsis-causing bacteria US8119336B2 (en) 2004-03-03 2012-02-21 Ibis Biosciences, Inc. Compositions for use in identification of alphaviruses US8148163B2 (en) 2008-09-16 2012-04-03 Ibis Biosciences, Inc. Sample processing units, systems, and related methods US8158354B2 (en) 2003-05-13 2012-04-17 Ibis Biosciences, Inc. Methods for rapid purification of nucleic acids for subsequent analysis by mass spectrometry by solution capture US8158936B2 (en) 2009-02-12 2012-04-17 Ibis Biosciences, Inc. Ionization probe assemblies US8163895B2 (en) 2003-12-05 2012-04-24 Ibis Biosciences, Inc. Compositions for use in identification of orthopoxviruses US8182992B2 (en) 2005-03-03 2012-05-22 Ibis Biosciences, Inc. Compositions for use in identification of adventitious viruses US8298760B2 (en) 2001-06-26 2012-10-30 Ibis Bioscience, Inc. Secondary structure defining database and methods for determining identity and geographic origin of an unknown bioagent thereby WO2013036603A1 (en) 2011-09-06 2013-03-14 Ibis Biosciences, Inc. Sample preparation methods US8407010B2 (en) 2004-05-25 2013-03-26 Ibis Biosciences, Inc. Methods for rapid forensic analysis of mitochondrial DNA US8534447B2 (en) 2008-09-16 2013-09-17 Ibis Biosciences, Inc. Microplate handling systems and related computer program products and methods US8546082B2 (en) 2003-09-11 2013-10-01 Ibis Biosciences, Inc. Methods for identification of sepsis-causing bacteria US8550694B2 (en) 2008-09-16 2013-10-08 Ibis Biosciences, Inc. Mixing cartridges, mixing stations, and related kits, systems, and methods US8563250B2 (en) 2001-03-02 2013-10-22 Ibis Biosciences, Inc. Methods for identifying bioagents US8652780B2 (en) 2007-03-26 2014-02-18 Sequenom, Inc. Restriction endonuclease enhanced polymorphic sequence detection WO2014052590A1 (en) 2012-09-26 2014-04-03 Ibis Biosciences, Inc. Swab interface for a microfluidic device US8722336B2 (en) 2008-03-26 2014-05-13 Sequenom, Inc. Restriction endonuclease enhanced polymorphic sequence detection US8771948B2 (en) 2009-04-03 2014-07-08 Sequenom, Inc. Nucleic acid preparation compositions and methods US8871471B2 (en) 2007-02-23 2014-10-28 Ibis Biosciences, Inc. Methods for rapid forensic DNA analysis US8950604B2 (en) 2009-07-17 2015-02-10 Ibis Biosciences, Inc. Lift and mount apparatus US9051608B2 (en) 2006-12-05 2015-06-09 Agena Bioscience, Inc. Detection and quantification of biomolecules using mass spectrometry US9068017B2 (en) 2010-04-08 2015-06-30 Ibis Biosciences, Inc. Compositions and methods for inhibiting terminal transferase activity US9080209B2 (en) 2009-08-06 2015-07-14 Ibis Biosciences, Inc. Non-mass determined base compositions for nucleic acid detection US9149473B2 (en) 2006-09-14 2015-10-06 Ibis Biosciences, Inc. Targeted whole genome amplification method for identification of pathogens US9194877B2 (en) 2009-07-17 2015-11-24 Ibis Biosciences, Inc. Systems for bioagent indentification US9393564B2 (en) 2009-03-30 2016-07-19 Ibis Biosciences, Inc. Bioagent detection systems, devices, and methods US9404150B2 (en) 2007-08-29 2016-08-02 Sequenom, Inc. Methods and compositions for universal size-specific PCR US9598724B2 (en) 2007-06-01 2017-03-21 Ibis Biosciences, Inc. Methods and compositions for multiple displacement amplification of nucleic acids US9719083B2 (en) 2009-03-08 2017-08-01 Ibis Biosciences, Inc. Bioagent detection methods US9777335B2 (en) 2001-06-04 2017-10-03 Geneohm Sciences Canada Inc. Method for the detection and identification of methicillin-resistant Staphylococcus aureus US9970061B2 (en) 2011-12-27 2018-05-15 Ibis Biosciences, Inc. Bioagent detection oligonucleotides US11834720B2 (en) 2005-10-11 2023-12-05 Geneohm Sciences, Inc. Sequences for detection and identification of methicillin-resistant Staphylococcus aureus (MRSA) of MREJ types xi to xx Families Citing this family (58) * Cited by examiner, â Cited by third party Publication number Priority date Publication date Assignee Title AU2003298030B2 (en) * 2001-03-02 2009-12-10 Ibis Biosciences, Inc. Methods for rapid identification of pathogens in humans and animals WO2004046385A1 (en) * 2002-11-15 2004-06-03 Biotage Ab A method for inter-species differentiation and identification of a gram-positive bacteria CA2541369A1 (en) * 2003-09-05 2005-04-21 The General Hospital Corporation Photodynamic inactivation of bacterial spores US20100035239A1 (en) * 2003-09-11 2010-02-11 Isis Pharmaceuticals, Inc. Compositions for use in identification of bacteria US20080138808A1 (en) * 2003-09-11 2008-06-12 Hall Thomas A Methods for identification of sepsis-causing bacteria US20060240412A1 (en) * 2003-09-11 2006-10-26 Hall Thomas A Compositions for use in identification of adenoviruses US20050142584A1 (en) * 2003-10-01 2005-06-30 Willson Richard C. Microbial identification based on the overall composition of characteristic oligonucleotides US7309410B2 (en) 2003-12-03 2007-12-18 Palo Alto Research Center Incorporated Traveling wave grids and algorithms for biomolecule separation, transport and focusing US7163611B2 (en) * 2003-12-03 2007-01-16 Palo Alto Research Center Incorporated Concentration and focusing of bio-agents and micron-sized particles using traveling wave grids AU2005322640B2 (en) * 2004-02-18 2008-09-18 Ibis Biosciences, Inc. Compositions for use in identification of bacteria US7888011B2 (en) * 2004-10-18 2011-02-15 U.S. Genomics, Inc. Methods for isolation of nucleic acids from prokaryotic spores EP1861704A2 (en) * 2005-02-09 2007-12-05 Correlogic Systems, Inc. Identification of bacteria and spores JP5162447B2 (en) * 2005-04-13 2013-03-13 ã¢ã¤ãã¹ ãã¤ãªãµã¤ã¨ã³ã·ãº ã¤ã³ã³ã¼ãã¬ã¤ãã£ãã Composition for use in adenovirus identification GB0601302D0 (en) * 2006-01-23 2006-03-01 Semikhodskii Andrei Diagnostic methods and apparatus US8455184B2 (en) * 2006-04-18 2013-06-04 The United States Of America As Represented By The Secretary Of The Air Force Differential multiplexing with pattern recognition US8076104B2 (en) * 2007-01-25 2011-12-13 Rogan Peter K Rapid and comprehensive identification of prokaryotic organisms WO2008118809A1 (en) * 2007-03-23 2008-10-02 Ibis Biosciences, Inc. Compositions for use in identification of mixed populations of bioagents US7811766B2 (en) * 2007-03-28 2010-10-12 Thinkvillage, Llc Genetic identification and validation of Echinacea species US8527207B2 (en) * 2007-05-15 2013-09-03 Peter K. Rogan Accurate identification of organisms based on individual information content KR101866766B1 (en) 2007-06-01 2018-06-18 ì¹´ì´ì¤ ì¤ë¸ ì¬ì´ì¸í°í½ ìë ì¸ëì¤í¸ë¦¬ì¼ 리ìì¹ A novel method for simultaneous detection and discrimination of bacterial, fungal, parasitic and viral infections of eye and central nervous system EP2179041A4 (en) * 2007-06-22 2010-12-22 Ibis Biosciences Inc Compositions and methods for identification of subspecies characteristics of mycobacterium tuberculosis US8067730B2 (en) 2007-07-20 2011-11-29 The George Washington University Laser ablation electrospray ionization (LAESI) for atmospheric pressure, In vivo, and imaging mass spectrometry US8901487B2 (en) 2007-07-20 2014-12-02 George Washington University Subcellular analysis by laser ablation electrospray ionization mass spectrometry US7964843B2 (en) 2008-07-18 2011-06-21 The George Washington University Three-dimensional molecular imaging by infrared laser ablation electrospray ionization mass spectrometry CA2741592A1 (en) * 2008-12-19 2010-06-24 Abbott Laboratories Diagnostic test for mutations in codons 12-13 of human k-ras WO2010080616A1 (en) * 2008-12-19 2010-07-15 Abbott Laboratories Molecular assay for diagnosis of malaria GB2510520B (en) * 2009-02-03 2014-11-05 Bruker Daltonik Gmbh Mass spectrometric identification of microorganisms in complex samples DE102009007266B4 (en) * 2009-02-03 2012-04-19 Bruker Daltonik Gmbh Mass spectrometric identification of microorganisms in complex samples US20100279295A1 (en) * 2009-03-18 2010-11-04 Sequenom, Inc. Use of thermostable endonucleases for generating reporter molecules WO2011041695A1 (en) * 2009-10-02 2011-04-07 Ibis Biosciences, Inc. Determination of methylation status of polynucleotides EP2576811A1 (en) 2010-06-02 2013-04-10 Johns Hopkins University System and methods for determining drug resistance in microorganisms WO2012159089A1 (en) 2011-05-19 2012-11-22 Sequenom, Inc. Products and processes for multiplex nucleic acid identification US9273366B2 (en) * 2011-06-15 2016-03-01 Grifols Therapeutics Inc. Methods, compositions, and kits for determining human immunodeficiency virus (HIV) WO2013085572A2 (en) 2011-07-14 2013-06-13 The George Washington University Plume collimation for laser ablation electrospray ionization mass spectrometry EP2753932A4 (en) * 2011-09-07 2015-07-15 Alpha Biotech Ab Determination of bacterial infections of the genus rickettsia and possibly borrelia, in patients exhibiting symptoms of disease and being blood donors CA2847943C (en) * 2011-09-23 2017-08-29 F.Hoffmann-La Roche Ag Use of g-clamp for improved allele-specific pcr US9334491B2 (en) 2011-12-22 2016-05-10 Ibis Biosciences, Inc. Systems and methods for isolating nucleic acids from cellular samples US9803188B2 (en) 2011-12-22 2017-10-31 Ibis Biosciences, Inc. Systems and methods for isolating nucleic acids WO2013101741A1 (en) 2011-12-30 2013-07-04 Abbott Molecular, Inc. Channels with cross-sectional thermal gradients US9732387B2 (en) 2012-04-03 2017-08-15 The Regents Of The University Of Michigan Biomarker associated with irritable bowel syndrome and Crohn's disease CA2905425A1 (en) 2013-03-13 2014-10-02 Abbott Molecular Inc. Methods of isolating nucleic acid using a lysis buffer containing ethanol CN105378107A (en) 2013-03-14 2016-03-02 é å¹ååå ¬å¸ Multiplex methylation-specific amplification systems and methods US9856525B2 (en) 2013-03-15 2018-01-02 Bio-Rad Laboratories, Inc. Digital assays with associated targets WO2014186874A1 (en) 2013-05-23 2014-11-27 Yyz Pharmatech, Inc. Methods and compositions for enzyme linked immuno and hybridization mass spectrometric assay CN103344695B (en) * 2013-06-18 2015-06-10 ä¸å½ç¾ç é¢é²æ§å¶ä¸å¿ä¼ æç é¢é²æ§å¶æ Kit for rapid mass spectrometric detection of leptospira CN103667109B (en) * 2013-10-23 2016-02-03 大è¿å¸æ°´äº§ææ¯æ¨å¹¿æ»ç« A kind of Rhodobacter capsulatus and application thereof JP2017521654A (en) 2014-06-27 2017-08-03 ã¢ãããã»ã©ãã©ããªã¼ãºï¼¡ï½ï½ï½ï½ï½ Ｌï½ï½ï½ï½ï½ï½ï½ï½ï½ï½ ï½ Compositions and methods for detecting human pegivirus 2 (HPgV-2) RU2580412C1 (en) * 2015-02-02 2016-04-10 ФедеÑалÑное гоÑÑдаÑÑÑвенное авÑономное обÑазоваÑелÑное ÑÑÑеждение вÑÑÑего обÑÐ°Ð·Ð¾Ð²Ð°Ð½Ð¸Ñ "ÐаÑионалÑнÑй иÑÑледоваÑелÑÑкий ТомÑкий полиÑÐµÑ Ð½Ð¸ÑеÑкий ÑнивеÑÑиÑеÑ" Method for quantitative determination of t-2 toxin by differential voltammetry CA2983224A1 (en) 2015-04-24 2016-10-27 Agena Bioscience, Inc. Multiplexed method for the identification and quantitation of minor alleles and polymorphisms CN107787371B (en) 2015-04-24 2022-02-01 åºçº³çç©ææ¯æéå ¬å¸ Parallel method for detecting and quantifying minor variants WO2018089978A1 (en) * 2016-11-14 2018-05-17 Wisconsin Alumni Research Foundation Nucleic acid quantification compositions and methods CN107085034A (en) * 2017-04-19 2017-08-22 ä¸å½äººæ°è§£æ¾å第ä¸ãäºå»é¢ Identification of Shigella sonnei by MALDIâTOFâMS CN107988341B (en) * 2018-01-03 2019-04-23 åäº¬æ¯ æ°ååçç©ç§ææéå ¬å¸ Mass spectrometry identification method and product of Vibrio cholerae typing CN108048540B (en) * 2018-01-03 2021-06-08 åäº¬æ¯ æ°ååçç©ç§ææéå ¬å¸ Method for preparing fingerprint library for detection of Vibrio cholerae typing JP7378712B2 (en) * 2019-08-07 2023-11-14 å¦æ ¡æ³äººå¸¸ç¿å¦å Method for testing xenograft material for pathogen infection, method for producing test kit and xenograft material product evaluated for pathogen infection CN111737674B (en) * 2020-08-03 2021-02-09 å¾·è½æ£®æºè½ç§æï¼æé½ï¼æéå ¬å¸ Wisdom garden system based on cloud platform CN113219115A (en) * 2021-05-18 2021-08-06 广ä¸çç§å¦é¢å¾®çç©ç 究æ(广ä¸çå¾®çç©åææ£æµä¸å¿) MALDI-TOF MS-based method for rapidly identifying bacillus cereus and bacillus thuringiensis WO2023077489A1 (en) * 2021-11-06 2023-05-11 æ±æ±å¤§å¦ Mnp marker combination of yersinia pestis, primer pair combination, kit, and application thereof Citations (11) * Cited by examiner, â Cited by third party Publication number Priority date Publication date Assignee Title US5484908A (en) * 1991-11-26 1996-01-16 Gilead Sciences, Inc. Oligonucleotides containing 5-propynyl pyrimidines US5502177A (en) * 1993-09-17 1996-03-26 Gilead Sciences, Inc. Pyrimidine derivatives for labeled binding partners US5547835A (en) * 1993-01-07 1996-08-20 Sequenom, Inc. DNA sequencing by mass spectrometry US5605798A (en) * 1993-01-07 1997-02-25 Sequenom, Inc. DNA diagnostic based on mass spectrometry US5622824A (en) * 1993-03-19 1997-04-22 Sequenom, Inc. DNA sequencing by mass spectrometry via exonuclease degradation US5645985A (en) * 1991-11-26 1997-07-08 Gilead Sciences, Inc. Enhanced triple-helix and double-helix formation with oligomers containing modified pyrimidines US5830653A (en) * 1991-11-26 1998-11-03 Gilead Sciences, Inc. Methods of using oligomers containing modified pyrimidines US5849492A (en) * 1994-02-28 1998-12-15 Phylogenetix Laboratories, Inc. Method for rapid identification of prokaryotic and eukaryotic organisms US5965363A (en) * 1996-09-19 1999-10-12 Genetrace Systems Inc. Methods of preparing nucleic acids for mass spectrometric analysis US6007992A (en) * 1997-11-10 1999-12-28 Gilead Sciences, Inc. Pyrimidine derivatives for labeled binding partners US6028183A (en) * 1997-11-07 2000-02-22 Gilead Sciences, Inc. Pyrimidine derivatives and oligonucleotides containing same Family Cites Families (416) * Cited by examiner, â Cited by third party Publication number Priority date Publication date Assignee Title US4075475A (en) 1976-05-03 1978-02-21 Chemetron Corporation Programmed thermal degradation-mass spectrometry analysis method facilitating identification of a biological specimen US5288611A (en) 1983-01-10 1994-02-22 Gen-Probe Incorporated Method for detecting, identifying, and quantitating organisms and viruses CA1215904A (en) * 1983-01-10 1986-12-30 David E. Kohne Method for detection identification and quantitation of non-viral organisms US5641632A (en) 1983-01-10 1997-06-24 Gen-Probe Incorporated Method for preparing rRNA for hybridization with a probe US4965188A (en) 1986-08-22 1990-10-23 Cetus Corporation Process for amplifying, detecting, and/or cloning nucleic acid sequences using a thermostable enzyme 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 ATE163680T1 (en) 1986-11-24 1998-03-15 Gen Probe Inc NUCLEIC ACID PROBE FOR DETECTION AND/OR QUANTIFICATION OF NON-VIRAL ORGANISMS WO1988006633A1 (en) 1987-03-02 1988-09-07 Arnold Lyle John Jr Polycationic supports for nucleic acid purification, separation and hybridization US5188963A (en) 1989-11-17 1993-02-23 Gene Tec Corporation Device for processing biological specimens for analysis of nucleic acids US5270030A (en) 1988-12-29 1993-12-14 Bio-Technology General Corp. Fibrin binding domain polypeptide and method of producing US5198543A (en) 1989-03-24 1993-03-30 Consejo Superior Investigaciones Cientificas PHI29 DNA polymerase IE81145B1 (en) * 1989-04-20 2000-05-03 Thomas Gerard Barry Generation of specific probes for target nucleotide sequences WO1990015157A1 (en) 1989-05-31 1990-12-13 Gene-Trak Systems Universal eubacteria nucleic acid probes and methods US5219727A (en) 1989-08-21 1993-06-15 Hoffmann-Laroche Inc. Quantitation of nucleic acids using the polymerase chain reaction US5192659A (en) 1989-08-25 1993-03-09 Genetype Ag Intron sequence analysis method for detection of adjacent and remote locus alleles as haplotypes US5213961A (en) 1989-08-31 1993-05-25 Brigham And Women's Hospital Accurate quantitation of RNA and DNA by competetitive polymerase chain reaction AU7060591A (en) 1990-01-12 1991-08-05 Hsc Research Development Corporation Introns and exons of the cystic fibrosis gene and mutations at various positions of the gene US5770029A (en) 1996-07-30 1998-06-23 Soane Biosciences Integrated electrophoretic microdevices US5143905A (en) 1990-05-03 1992-09-01 The Regents Of The University Of California Method and means for extending the host range of insecticidal proteins US5015845A (en) 1990-06-01 1991-05-14 Vestec Corporation Electrospray method for mass spectrometry US5712125A (en) 1990-07-24 1998-01-27 Cemv Bioteknik Ab Competitive PCR for quantitation of DNA DE4030262A1 (en) 1990-09-25 1992-03-26 Suedzucker Ag METHOD FOR PRODUCING RHAMNOSE FROM RHAMNOLIPID NL9002259A (en) * 1990-10-17 1992-05-18 Eurodiagnostics B V METHOD FOR DETERMINING A GENOTYPE BY COMPARING THE NUCLEOTID SEQUENCE OF MEM FAMILY MEMBERS AND KIT FOR DETECTING GENETIC VARIATIONS. WO1992009703A1 (en) 1990-11-26 1992-06-11 Cbr Laboratories, Inc. Testing for spirochetal nucleic acid sequences in samples US5072115A (en) 1990-12-14 1991-12-10 Finnigan Corporation Interpretation of mass spectra of multiply charged ions of mixtures WO1992013629A1 (en) 1991-01-31 1992-08-20 Wayne State University A method for analyzing an organic sample US5866429A (en) 1991-04-03 1999-02-02 Bloch; Will Precision and accuracy of anion-exchange separation of nucleic acids US5472843A (en) 1991-04-25 1995-12-05 Gen-Probe Incorporated Nucleic acid probes to Haemophilus influenzae US5213796A (en) 1991-05-06 1993-05-25 Dana Farber Cancer Institute Assay for polyomavirus in humans and uses thereof US6055487A (en) 1991-07-30 2000-04-25 Margery; Keith S. Interactive remote sample analysis system ATE172500T1 (en) 1991-07-31 1998-11-15 Hoffmann La Roche METHOD AND REAGENTS FOR DETERMINING BACTERIA IN CEREBROSPINAL FLUID ES2214472T3 (en) 1991-08-02 2004-09-16 Biomerieux B.V. QUANTIFICATION OF NUCLEIC ACIDS. PT787807E (en) 1991-08-27 2003-08-29 Hoffmann La Roche INITIATORS AND PROBES FOR HEPATITIS C DETECTION AU2580892A (en) * 1991-09-05 1993-04-05 Isis Pharmaceuticals, Inc. Determination of oligonucleotides for therapeutics, diagnostics and research reagents AU2931692A (en) 1991-10-23 1993-05-21 Baylor College Of Medicine Fingerprinting bacterial strains using repetitive dna sequence amplification FR2683827B1 (en) 1991-11-15 1994-03-04 Institut Nal Sante Recherc Medic METHOD FOR DETERMINING THE QUANTITY OF A FRAGMENT OF DNA OF INTEREST BY AN ENZYMATIC AMPLIFICATION METHOD. US6235887B1 (en) 1991-11-26 2001-05-22 Isis Pharmaceuticals, Inc. Enhanced triple-helix and double-helix formation directed by oligonucleotides containing modified pyrimidines IL103935A0 (en) 1991-12-04 1993-05-13 Du Pont Method for the identification of microorganisms by the utilization of directed and arbitrary dna amplification JPH07504749A (en) 1992-03-13 1995-05-25 ãµã¼ã¢ãã¤ã¯ãã¹ã³ã¼ãã¹ãã³ã¼ãã¬ã¼ã·ã§ã³ scanning probe electron microscope US5981176A (en) * 1992-06-17 1999-11-09 City Of Hope Method of detecting and discriminating between nucleic acid sequences US6303297B1 (en) 1992-07-17 2001-10-16 Incyte Pharmaceuticals, Inc. Database for storage and analysis of full-length sequences FR2694754B1 (en) 1992-08-12 1994-09-16 Bio Merieux Mycobacteria DNA fragments, amplification primers, hybridization probes, reagents and method for detecting detection of mycobacteria. AU5128593A (en) 1992-09-16 1994-04-12 University Of Tennessee Research Corporation, The Antigen of hybrid m protein and carrier for group a streptococcal vaccine EP0672187A4 (en) 1992-10-08 1999-11-17 Univ California PCR ASSAYS FOR DETERMINING THE PRESENCE AND CONCENTRATION OF A TARGET. US5503980A (en) * 1992-11-06 1996-04-02 Trustees Of Boston University Positional sequencing by hybridization US6436635B1 (en) * 1992-11-06 2002-08-20 Boston University Solid phase sequencing of double-stranded nucleic acids US6372424B1 (en) * 1995-08-30 2002-04-16 Third Wave Technologies, Inc Rapid detection and identification of pathogens US6194144B1 (en) * 1993-01-07 2001-02-27 Sequenom, Inc. DNA sequencing by mass spectrometry FR2701961B1 (en) 1993-02-24 1995-04-21 Bio Merieux Method for destabilizing an intracatenary secondary structure of a single-stranded polynucleotide, and for capturing said nucleotide. US6074823A (en) * 1993-03-19 2000-06-13 Sequenom, Inc. DNA sequencing by mass spectrometry via exonuclease degradation US5639606A (en) 1993-04-06 1997-06-17 The University Of Rochester Method for quantitative measurement of gene expression using multiplex competitive reverse transcriptase-polymerase chain reaction US6323041B1 (en) 1993-06-11 2001-11-27 Pfizer Inc. Screening novel human phosphodiesterase IV isozymes for compounds which modify their enzymatic activity JPH0775585A (en) 1993-06-14 1995-03-20 Immuno Japan:Kk Hepatitis c virus-related oligonucleotide and method for judging virus gene type US5830853A (en) 1994-06-23 1998-11-03 Astra Aktiebolag Systemic administration of a therapeutic preparation US6001584A (en) 1993-07-19 1999-12-14 The Regents Of The University Of California Oncoprotein protein kinase AU7551594A (en) 1993-07-29 1995-02-28 MURASHIGE, Kate H. Method for recognition of the nucleotide sequence of a purified dna segment GB9315847D0 (en) * 1993-07-30 1993-09-15 Isis Innovation Tag reagent and assay method US5527675A (en) * 1993-08-20 1996-06-18 Millipore Corporation Method for degradation and sequencing of polymers which sequentially eliminate terminal residues US6376178B1 (en) * 1993-09-03 2002-04-23 Duke University Method of nucleic acid sequencing US5683869A (en) 1993-09-03 1997-11-04 Duke University Method of nucleic acid sequencing US5484909A (en) * 1993-09-10 1996-01-16 Amoco Corporation Nucleic acid probes for the detection of bacteria of the genera Pediococcus and Lactobacillus and methods for the detection of the bacterial agents causing spoilage of beer WO1995011996A1 (en) 1993-10-27 1995-05-04 Cornell Research Foundation, Inc. Detection assay for listeria and erwinia microorganisms US5504327A (en) 1993-11-04 1996-04-02 Hv Ops, Inc. (H-Nu) Electrospray ionization source and method for mass spectrometric analysis DE4338119A1 (en) 1993-11-08 1995-05-11 Bayer Ag Specific gene probes and methods for the quantitative detection of methicillin-resistant staphylococci NL9301957A (en) 1993-11-11 1995-06-01 U Gene Research Bv Method for identifying microorganisms, and useful tools. US5928905A (en) 1995-04-18 1999-07-27 Glaxo Group Limited End-complementary polymerase reaction JP3396942B2 (en) 1994-02-21 2003-04-14 ä¸è±åå¦æ ªå¼ä¼ç¤¾ Method for producing aromatic polycarbonate US5608217A (en) 1994-03-10 1997-03-04 Bruker-Franzen Analytik Gmbh Electrospraying method for mass spectrometric analysis DE4444229C2 (en) 1994-03-10 1996-07-25 Bruker Franzen Analytik Gmbh Methods and devices for electrospray ionization for storage mass spectrometers US5976798A (en) 1994-03-30 1999-11-02 Mitokor Methods for detecting mitochondrial mutations diagnostic for Alzheimer's disease and methods for determining heteroplasmy of mitochondrial nucleic acid AU7242994A (en) 1994-05-20 1995-12-18 United States Of America, As Represented By The Secretary Of The Army, The Model for testing immunogenicity of peptides US5814442A (en) 1994-06-10 1998-09-29 Georgetown University Internally controlled virion nucleic acid amplification reaction for quantitation of virion and virion nucleic acid DE4421901A1 (en) 1994-06-23 1996-01-04 Bayer Ag A rapid DNA test for the detection of quinolone-resistant Staphylococcus aureus pathogens in clinical specimens US5541405A (en) 1994-07-27 1996-07-30 Parker-Hannifin Corporation Method and device for continuous pattern sensing using fiber optics GB9417211D0 (en) 1994-08-25 1994-10-12 Solicitor For The Affairs Of H Nucleotide sequencing method US6001564A (en) 1994-09-12 1999-12-14 Infectio Diagnostic, Inc. Species specific and universal DNA probes and amplification primers to rapidly detect and identify common bacterial pathogens and associated antibiotic resistance genes from clinical specimens for routine diagnosis in microbiology laboratories US20020055101A1 (en) 1995-09-11 2002-05-09 Michel G. Bergeron Specific and universal probes and amplification primers to rapidly detect and identify common bacterial pathogens and antibiotic resistance genes from clinical specimens for routine diagnosis in microbiology laboratories CA2118048C (en) 1994-09-30 2003-04-08 James W. Schumm Multiplex amplification of short tandem repeat loci US5753489A (en) 1994-11-10 1998-05-19 Immuno Ag Method for producing viruses and vaccines in serum-free culture US5654141A (en) 1994-11-18 1997-08-05 Thomas Jefferson University Amplification based detection of bacterial infection KR100399813B1 (en) 1994-12-14 2004-06-09 ê°ë¶ìí¤ê°ì´ì¤ ëì½ Exposure apparatus US6180339B1 (en) 1995-01-13 2001-01-30 Bayer Corporation Nucleic acid probes for the detection and identification of fungi US5763169A (en) 1995-01-13 1998-06-09 Chiron Diagnostics Corporation Nucleic acid probes for the detection and identification of fungi US5707802A (en) 1995-01-13 1998-01-13 Ciba Corning Diagnostics Corp. Nucleic acid probes for the detection and identification of fungi US5702895A (en) 1995-01-19 1997-12-30 Wakunaga Seiyaku Kabushiki Kaisha Method and kit for detecting methicillin-resistant Staphylococcus aureus US5484808A (en) 1995-02-09 1996-01-16 Eli Lilly And Company Methods of inhibiting cell-cell adhesion GB9504598D0 (en) * 1995-03-03 1995-04-26 Imp Cancer Res Tech Method of nucleic acid analysis US6428955B1 (en) * 1995-03-17 2002-08-06 Sequenom, Inc. DNA diagnostics based on mass spectrometry AU5544696A (en) 1995-04-11 1996-10-30 Trustees Of Boston University Solid phase sequencing of biopolymers US5932220A (en) 1995-05-08 1999-08-03 Board Of Regents University Of Texas System Diagnostic tests for a new spirochete, Borrelia lonestari sp. nov. US5625184A (en) * 1995-05-19 1997-04-29 Perseptive Biosystems, Inc. Time-of-flight mass spectrometry analysis of biomolecules US5830655A (en) * 1995-05-22 1998-11-03 Sri International Oligonucleotide sizing using cleavable primers US5700642A (en) 1995-05-22 1997-12-23 Sri International Oligonucleotide sizing using immobilized cleavable primers US5630653A (en) * 1995-06-02 1997-05-20 Polka; John G. Wheel cover mounting bracket CA2222793A1 (en) 1995-06-07 1996-12-19 Commonwealth Scientific And Industrial Research Organisation Optimized minizymes and miniribozymes and uses thereof US5856174A (en) 1995-06-29 1999-01-05 Affymetrix, Inc. Integrated nucleic acid diagnostic device US6218529B1 (en) 1995-07-31 2001-04-17 Urocor, Inc. Biomarkers and targets for diagnosis, prognosis and management of prostate, breast and bladder cancer US6146854A (en) 1995-08-31 2000-11-14 Sequenom, Inc. Filtration processes, kits and devices for isolating plasmids US5994066A (en) 1995-09-11 1999-11-30 Infectio Diagnostic, Inc. Species-specific and universal DNA probes and amplification primers to rapidly detect and identify common bacterial pathogens and associated antibiotic resistance genes from clinical specimens for routine diagnosis in microbiology laboratories US5869242A (en) * 1995-09-18 1999-02-09 Myriad Genetics, Inc. Mass spectrometry to assess DNA sequence polymorphisms US5727202A (en) 1995-10-18 1998-03-10 Palm Computing, Inc. Method and apparatus for synchronizing information on two different computer systems US5972693A (en) 1995-10-24 1999-10-26 Curagen Corporation Apparatus for identifying, classifying, or quantifying DNA sequences in a sample without sequencing 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 US5716825A (en) * 1995-11-01 1998-02-10 Hewlett Packard Company Integrated nucleic acid analysis system for MALDI-TOF MS US6312893B1 (en) * 1996-01-23 2001-11-06 Qiagen Genomics, Inc. Methods and compositions for determining the sequence of nucleic acid molecules GB9602028D0 (en) 1996-02-01 1996-04-03 Amersham Int Plc Nucleoside analogues US6852487B1 (en) 1996-02-09 2005-02-08 Cornell Research Foundation, Inc. Detection of nucleic acid sequence differences using the ligase detection reaction with addressable arrays US6051378A (en) * 1996-03-04 2000-04-18 Genetrace Systems Inc. Methods of screening nucleic acids using mass spectrometry WO1997037041A2 (en) 1996-03-18 1997-10-09 Sequenom, Inc. Dna sequencing by mass spectrometry ES2188926T3 (en) 1996-03-20 2003-07-01 Bio Merieux ISOLATION OF NUCLEIC ACID. JP3365198B2 (en) 1996-03-21 2003-01-08 ããã«ã¿æ ªå¼ä¼ç¤¾ Image forming device US5745751A (en) 1996-04-12 1998-04-28 Nelson; Robert W. Civil site information system US6214555B1 (en) 1996-05-01 2001-04-10 Visible Genetics Inc. Method compositions and kit for detection US5928906A (en) * 1996-05-09 1999-07-27 Sequenom, Inc. Process for direct sequencing during template amplification JP2000512497A (en) * 1996-06-10 2000-09-26 ã¦ããã¼ã·ãã£ã»ãªãã»ã¦ã¿ã»ãªãµã¼ãã»ãã¡ã¦ã³ãã¤ã·ã§ã³ Rapid and accurate identification of mutant DNA sequences by electrospray mass spectrometry WO1998003684A1 (en) 1996-07-19 1998-01-29 Hybridon, Inc. Method for sequencing nucleic acids using matrix-assisted laser desorption ionization time-of-flight mass spectrometry US6563025B1 (en) 1996-07-26 2003-05-13 Board Of Trustees Of The University Of Illinois Nucleotide sequences encoding anthranilate synthase US5831046A (en) 1996-08-05 1998-11-03 Prolinx, Incorporated Boronic acid-contaning nucleic acid monomers DE19633436A1 (en) * 1996-08-20 1998-02-26 Boehringer Mannheim Gmbh Method for the detection of nucleic acids by determining the mass CA2301875A1 (en) 1996-09-19 1998-03-26 Genetrace Systems Methods of preparing nucleic acids for mass spectrometric analysis US5777324A (en) * 1996-09-19 1998-07-07 Sequenom, Inc. Method and apparatus for maldi analysis US6361940B1 (en) * 1996-09-24 2002-03-26 Qiagen Genomics, Inc. Compositions and methods for enhancing hybridization and priming specificity US5864137A (en) * 1996-10-01 1999-01-26 Genetrace Systems, Inc. Mass spectrometer GB9620769D0 (en) 1996-10-04 1996-11-20 Brax Genomics Ltd Nucleic acid sequencing US5885775A (en) 1996-10-04 1999-03-23 Perseptive Biosystems, Inc. Methods for determining sequences information in polynucleotides using mass spectrometry US6110710A (en) 1996-10-15 2000-08-29 The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Sequence modification of oligonucleotide primers to manipulate non-templated nucleotide addition US5900481A (en) 1996-11-06 1999-05-04 Sequenom, Inc. Bead linkers for immobilizing nucleic acids to solid supports US6024925A (en) 1997-01-23 2000-02-15 Sequenom, Inc. Systems and methods for preparing low volume analyte array elements US6140053A (en) * 1996-11-06 2000-10-31 Sequenom, Inc. DNA sequencing by mass spectrometry via exonuclease degradation DE69735112T2 (en) 1996-11-06 2006-09-07 Sequenom, Inc., San Diego Method of analysis and device US7285422B1 (en) 1997-01-23 2007-10-23 Sequenom, Inc. Systems and methods for preparing and analyzing low volume analyte array elements US6133436A (en) 1996-11-06 2000-10-17 Sequenom, Inc. Beads bound to a solid support and to nucleic acids ATE375403T1 (en) * 1996-11-06 2007-10-15 Sequenom Inc DNA DIAGNOSTICS USING MASS SPECTROMETRY WO1998021066A1 (en) 1996-11-12 1998-05-22 Hp-Chemie Pelzer Research And Development Ltd. Reusable shaped floor carpets US6060246A (en) 1996-11-15 2000-05-09 Avi Biopharma, Inc. Reagent and method for isolation and detection of selected nucleic acid sequences DE69726309T2 (en) 1996-11-28 2004-09-16 New Japan Chemical Co., Ltd. SUGAR DERIVATIVES, GELLING AGENTS, GELING AGENT PREPARATIONS, METHOD FOR THEIR PRODUCTION AND GEL PREPARATIONS US5822824A (en) 1996-12-03 1998-10-20 Dion; William D. Mountable washing device JP2001524808A (en) 1996-12-10 2001-12-04 ã¸ã¼ã³ãã¬ã¤ã¹ã»ã·ã¹ãã ãºã»ã¤ã³ã³ã¼ãã¬ã¤ããã Releasable non-volatile mass labeling molecules US5981190A (en) 1997-01-08 1999-11-09 Ontogeny, Inc. Analysis of gene expression, methods and reagents therefor US5876936A (en) * 1997-01-15 1999-03-02 Incyte Pharmaceuticals, Inc. Nucleic acid sequencing with solid phase capturable terminators US6046005A (en) * 1997-01-15 2000-04-04 Incyte Pharmaceuticals, Inc. Nucleic acid sequencing with solid phase capturable terminators comprising a cleavable linking group DE69838519T2 (en) 1997-01-15 2008-07-03 Xzillion Gmbh & Co. Kg Mass-labeled hybridization probes WO1998035057A1 (en) 1997-02-06 1998-08-13 The National University Of Singapore Diagnosis of plasmodium infection by analysis of extrachromosomal genetic material US5858062A (en) * 1997-02-10 1999-01-12 Litton Systems, Inc. Oxygen concentrator US6727061B2 (en) 1997-02-20 2004-04-27 Cabtec, Inc. Methods for identifying species or Shigella and E. coli using operon sequence analysis US5828062A (en) 1997-03-03 1998-10-27 Waters Investments Limited Ionization electrospray apparatus for mass spectrometry US6553317B1 (en) 1997-03-05 2003-04-22 Incyte Pharmaceuticals, Inc. Relational database and system for storing information relating to biomolecular sequences and reagents DE19710166C1 (en) 1997-03-12 1998-12-10 Bruker Franzen Analytik Gmbh Two-step method of DNA amplification for MALDI-TOF measurements AU6553498A (en) 1997-03-14 1998-09-29 Hybridon, Inc. Method for sequencing of modified nucleic acids using electrospray ionization-fourier transform mass spectrometry US5849497A (en) 1997-04-03 1998-12-15 The Research Foundation Of State University Of New York Specific inhibition of the polymerase chain reaction using a non-extendable oligonucleotide blocker US6018713A (en) 1997-04-09 2000-01-25 Coli; Robert D. Integrated system and method for ordering and cumulative results reporting of medical tests DE19717085C2 (en) 1997-04-23 1999-06-17 Bruker Daltonik Gmbh Processes and devices for extremely fast DNA multiplication using polymerase chain reactions (PCR) US20010039263A1 (en) 1997-05-02 2001-11-08 Max-Delbruck-Centrum Fur Molekulare Medizin Chimeric oligonucleotides and the use thereof US6054278A (en) * 1997-05-05 2000-04-25 The Perkin-Elmer Corporation Ribosomal RNA gene polymorphism based microorganism identification US6159681A (en) 1997-05-28 2000-12-12 Syntrix Biochip, Inc. Light-mediated method and apparatus for the regional analysis of biologic material US6994960B1 (en) 1997-05-28 2006-02-07 The Walter And Eliza Hall Institute Of Medical Research Nucleic acid diagnostics based on mass spectrometry or mass separation and base specific cleavage EP0998749A1 (en) 1997-05-30 2000-05-10 Genetrace Systems, Inc. Volatile matrices for matrix-assisted laser desorption/ionization mass spectrometry US6061686A (en) 1997-06-26 2000-05-09 Digital Equipment Corporation Updating a copy of a remote document stored in a local computer system WO1999005319A2 (en) 1997-07-22 1999-02-04 Rapigene, Inc. Methods and compounds for analyzing nucleic acids by mass spectrometry DE19732086C2 (en) 1997-07-25 2002-11-21 Univ Leipzig Method for the quantitative determination of eubacteria US6207370B1 (en) 1997-09-02 2001-03-27 Sequenom, Inc. Diagnostics based on mass spectrometric detection of translated target polypeptides GB9719044D0 (en) 1997-09-08 1997-11-12 Inst Of Ophthalmology Assay WO1999014375A2 (en) 1997-09-19 1999-03-25 Genetrace Systems, Inc. Dna typing by mass spectrometry with polymorphic dna repeat markers US6063031A (en) 1997-10-14 2000-05-16 Assurance Medical, Inc. Diagnosis and treatment of tissue with instruments US6111096A (en) 1997-10-31 2000-08-29 Bbi Bioseq, Inc. Nucleic acid isolation and purification JP3423597B2 (en) 1997-11-05 2003-07-07 ä¸äºè¾²ææ ªå¼ä¼ç¤¾ Bacterial identification method ATE217028T1 (en) 1997-12-05 2002-05-15 Max Planck Gesellschaft METHOD FOR IDENTIFYING NUCLEIC ACIDS BY MATRIX-ASSISTED LASER DESORPTION/IONIZATION MASS SPECTROMETRY US6914137B2 (en) 1997-12-06 2005-07-05 Dna Research Innovations Limited Isolation of nucleic acids US6268131B1 (en) * 1997-12-15 2001-07-31 Sequenom, Inc. Mass spectrometric methods for sequencing nucleic acids US6458533B1 (en) * 1997-12-19 2002-10-01 High Throughput Genomics, Inc. High throughput assay system for monitoring ESTs GB9815166D0 (en) * 1998-07-13 1998-09-09 Brax Genomics Ltd Compounds for mass spectrometry US20030096232A1 (en) 1997-12-19 2003-05-22 Kris Richard M. High throughput assay system JP3793342B2 (en) 1997-12-26 2006-07-05 ã½ãã¼ã±ãã«ã«æ ªå¼ä¼ç¤¾ Acrylic emulsion and adhesive tape DE19802905C2 (en) 1998-01-27 2001-11-08 Bruker Daltonik Gmbh Process for the preferred production of only one strand of selected genetic material for mass spectrometric measurements US6428956B1 (en) 1998-03-02 2002-08-06 Isis Pharmaceuticals, Inc. Mass spectrometric methods for biomolecular screening WO1999046047A2 (en) * 1998-03-10 1999-09-16 Large Scale Proteomics Corporation Detection and characterization of microorganisms US6268146B1 (en) * 1998-03-13 2001-07-31 Promega Corporation Analytical methods and materials for nucleic acid detection US6270974B1 (en) * 1998-03-13 2001-08-07 Promega Corporation Exogenous nucleic acid detection US6312902B1 (en) * 1998-03-13 2001-11-06 Promega Corporation Nucleic acid detection US6391551B1 (en) * 1998-03-13 2002-05-21 Promega Corporation Detection of nucleic acid hybrids US6235480B1 (en) * 1998-03-13 2001-05-22 Promega Corporation Detection of nucleic acid hybrids US6270973B1 (en) * 1998-03-13 2001-08-07 Promega Corporation Multiplex method for nucleic acid detection US6277578B1 (en) * 1998-03-13 2001-08-21 Promega Corporation Deploymerization method for nucleic acid detection of an amplified nucleic acid target US6261769B1 (en) 1998-03-31 2001-07-17 The United States Of America As Represented By The Secretary Of Agriculture Intergenic spacer target sequence for detecting and distinguishing Chlamydial species or strains US20030228597A1 (en) 1998-04-13 2003-12-11 Cowsert Lex M. Identification of genetic targets for modulation by oligonucleotides and generation of oligonucleotides for gene modulation US7321828B2 (en) 1998-04-13 2008-01-22 Isis Pharmaceuticals, Inc. System of components for preparing oligonucleotides US6223186B1 (en) 1998-05-04 2001-04-24 Incyte Pharmaceuticals, Inc. System and method for a precompiled database for biomolecular sequence information US6723564B2 (en) * 1998-05-07 2004-04-20 Sequenom, Inc. IR MALDI mass spectrometry of nucleic acids using liquid matrices DE19822108A1 (en) 1998-05-12 2000-02-03 Schering Ag Method for the detection of microorganisms in products, in particular in medicines and cosmetics US6221587B1 (en) 1998-05-12 2001-04-24 Isis Pharmceuticals, Inc. Identification of molecular interaction sites in RNA for novel drug discovery US6468743B1 (en) 1998-05-18 2002-10-22 Conagra Grocery Products Company PCR techniques for detecting microbial contaminants in foodstuffs DE19922161A1 (en) 1998-05-18 1999-12-09 Fraunhofer Ges Forschung Anti-adhesion coating for e.g. soldering/welding tools and electric contacts US6104028A (en) * 1998-05-29 2000-08-15 Genetrace Systems Inc. Volatile matrices for matrix-assisted laser desorption/ionization mass spectrometry DE19824280B4 (en) 1998-05-29 2004-08-19 Bruker Daltonik Gmbh Mutation analysis using mass spectrometry GB2339905A (en) 1998-06-24 2000-02-09 Bruker Daltonik Gmbh Use of mass-specrometry for detection of mutations CA2333253C (en) 1998-07-02 2010-09-07 Gen-Probe Incorporated Molecular torches US6074831A (en) 1998-07-09 2000-06-13 Agilent Technologies, Inc. Partitioning of polymorphic DNAs US6218118B1 (en) * 1998-07-09 2001-04-17 Agilent Technologies, Inc. Method and mixture reagents for analyzing the nucleotide sequence of nucleic acids by mass spectrometry US6432651B1 (en) * 1998-07-10 2002-08-13 Cetek Corporation Method to detect and analyze tight-binding ligands in complex biological samples using capillary electrophoresis and mass spectrometry US6605433B1 (en) 1998-08-20 2003-08-12 The Johns Hopkins University Mitochondrial dosimeter US6146144A (en) * 1998-09-29 2000-11-14 Fowler; Ernest R. Rug hooking kit and method for handicapped US6610492B1 (en) 1998-10-01 2003-08-26 Variagenics, Inc. Base-modified nucleotides and cleavage of polynucleotides incorporating them US6238927B1 (en) * 1998-10-05 2001-05-29 Mosaic Technologies, Incorporated Reverse displacement assay for detection of nucleic acid sequences IT1303330B1 (en) 1998-11-05 2000-11-06 Ilpea Ind Spa GASKET FOR FROGORIFERI WITH SHAPED COUNTERPORT. DE19852167C2 (en) 1998-11-12 2000-12-14 Bruker Saxonia Analytik Gmbh Simple SNP analysis using mass spectrometry EP1144594A1 (en) 1998-11-24 2001-10-17 Regents Of The University Of Minnesota Transgenic circulating endothelial cells US6994962B1 (en) 1998-12-09 2006-02-07 Massachusetts Institute Of Technology Methods of identifying point mutations in a genome DE19859723A1 (en) 1998-12-23 2000-06-29 Henkel Kgaa Preparations for coloring keratinous fibers US6503718B2 (en) 1999-01-10 2003-01-07 Exact Sciences Corporation Methods for detecting mutations using primer extension for detecting disease US6638714B1 (en) 1999-02-03 2003-10-28 Ortho-Clinical Diagnostics, Inc. Oligonucleotide primers for efficient detection of hepatitis C virus (HCV) and methods of use thereof US6153389A (en) * 1999-02-22 2000-11-28 Haarer; Brian K. DNA additives as a mechanism for unambiguously marking biological samples EP1035219A1 (en) 1999-02-25 2000-09-13 Universiteit Gent Gastric helicobacter 16 S rDNA sequences from cattle and pigs and their use for detection and typing of Helicobacter strains DE19910592A1 (en) 1999-03-10 2000-09-14 Volkswagen Ag Restraint US6436640B1 (en) * 1999-03-18 2002-08-20 Exiqon A/S Use of LNA in mass spectrometry US6613509B1 (en) * 1999-03-22 2003-09-02 Regents Of The University Of California Determination of base (nucleotide) composition in DNA oligomers by mass spectrometry JP2002541839A (en) 1999-04-21 2002-12-10 ã¢ããã¹ï¼ ã¤ã³ã³ã¼ãã¬ã¤ããã Magnetic DNA extraction kit for plants US6649351B2 (en) * 1999-04-30 2003-11-18 Aclara Biosciences, Inc. Methods for detecting a plurality of analytes by mass spectrometry US6140067A (en) 1999-04-30 2000-10-31 Mitokor Indicators of altered mitochondrial function in predictive methods for determining risk of type 2 diabetes mellitus ATE437242T1 (en) 1999-05-03 2009-08-15 Gen Probe Inc POLYNUCLEOTIDE MATRIX-BASED METHOD FOR IDENTIFYING MICROORGANISMS US20020086289A1 (en) 1999-06-15 2002-07-04 Don Straus Genomic profiling: a rapid method for testing a complex biological sample for the presence of many types of organisms WO2001000828A2 (en) 1999-06-30 2001-01-04 Corixa Corporation Compositions and methods for the therapy and diagnosis of lung cancer EP1198597A1 (en) 1999-07-22 2002-04-24 Artus Gesellschaft Für Molekularbiologische Diagnostik und Entwicklung MbH. Method for the species-specific detection of organisms US6723505B1 (en) 1999-08-13 2004-04-20 Nye Colifast As Method for identification of the indicators of contamination in liquid samples US6266144B1 (en) 1999-08-26 2001-07-24 Taiwan Semiconductor Manufacturing Company Stepper and scanner new exposure sequence with intra-field correction DE19943374A1 (en) 1999-09-10 2001-03-29 Max Planck Gesellschaft Method for binding nucleic acids to a solid phase US7005274B1 (en) 1999-09-15 2006-02-28 Migenix Corp. Methods and compositions for diagnosing and treating arthritic disorders and regulating bone mass WO2001023608A2 (en) 1999-09-27 2001-04-05 Merck Sharp & Dohme De Espana, S.A.E. Hybridization probes which specifically detect strains of the genera microbispora, microtetraspora, nonomuria and planobispora EP2660335B1 (en) 1999-09-28 2018-11-21 Geneohm Sciences Canada, Inc. Highly conserved genes and their use to generate species-specific, genus-specific, family-specific, group-specific and universal nucleic acid probes and amplification primers to rapidly detect and identify algal, archaeal, bacterial, fungal and parasitical microorgamisms from clinical specimens for diagnosis US6296188B1 (en) 1999-10-01 2001-10-02 Perfect Plastic Printing Corporation Transparent/translucent financial transaction card including an infrared light filter CA2387035A1 (en) 1999-10-13 2001-04-19 Sequenom, Inc. Methods for generating databases and databases for identifying polymorphic genetic markers US6787302B2 (en) 1999-10-25 2004-09-07 Genprime, Inc. Method and apparatus for prokaryotic and eukaryotic cell quantitation CA2388528A1 (en) 1999-11-04 2001-05-10 California Institute Of Technology Methods and apparatus for analyzing polynucleotide sequences US6286146B1 (en) 1999-11-15 2001-09-11 Debra Rocker Method of wearing weighted training vest while listening to audio equipment US6856914B1 (en) 1999-11-19 2005-02-15 The University Of British Columbia Method, apparatus, media and signals for identifying associated cell signaling proteins EP1268764A2 (en) 1999-11-29 2003-01-02 Aventis Pharma S.A. Method for obtaining nucleic acids from an environment sample US6608190B1 (en) * 1999-12-16 2003-08-19 E. I. Du Pont De Nemours And Company Nucleic acid fragments for the identification of bacteria in industrial wastewater bioreactors US6936414B2 (en) 1999-12-22 2005-08-30 Abbott Laboratories Nucleic acid isolation method and kit DE60025850T2 (en) * 1999-12-29 2006-08-03 Keygene N.V. PROCESS FOR THE PRODUCTION OF OLIGONUCLEOTIDES, IN PARTICULAR FOR THE DETECTION OF AMPLIFIED RESTRICTION FRAGMENTS OBTAINED BY AFLP SE0000061D0 (en) 2000-01-10 2000-01-10 Bjoern Herrmann A method for detection of pathogenic organisms WO2001051661A2 (en) 2000-01-13 2001-07-19 Amsterdam Support Diagnostics B.V. A universal nucleic acid amplification system for nucleic acids in a sample US20010034250A1 (en) 2000-01-24 2001-10-25 Sanjay Chadha Hand-held personal computing device with microdisplay CA2298181C (en) 2000-02-02 2006-09-19 Dayan Burke Goodnough Non-targeted complex sample analysis US20020009727A1 (en) 2000-02-02 2002-01-24 Schultz Gary A. Detection of single nucleotide polymorphisms US6453244B1 (en) 2000-02-10 2002-09-17 Stanford University Detection of polymorphisms by denaturing high-performance liquid chromatography WO2001061616A2 (en) 2000-02-14 2001-08-23 First Opinion Corporation Automated diagnostic system and method US6393367B1 (en) 2000-02-19 2002-05-21 Proteometrics, Llc Method for evaluating the quality of comparisons between experimental and theoretical mass data DE10015797B4 (en) 2000-03-26 2006-02-02 Bruker Daltonik Gmbh Multiplex analysis of DNA mixtures using photolytically readable DNA chips DE10015262A1 (en) 2000-03-28 2001-10-04 Basf Ag Paper coating composition useful for off set printing, contains a binding agent prepared by radical polymerization of ethylenically unsaturated compounds US6713258B2 (en) 2000-03-29 2004-03-30 Center For The Application Of Molecular Biology To International Agriculture (Cambia) Methods for genotyping by hybridization analysis DK2278029T3 (en) 2000-04-10 2016-10-03 Taxon Biosciences Inc Methods of study and genetic analysis of populations US6475736B1 (en) * 2000-05-23 2002-11-05 Variagenics, Inc. Methods for genetic analysis of DNA using biased amplification of polymorphic sites US6716634B1 (en) 2000-05-31 2004-04-06 Agilent Technologies, Inc. Increasing ionization efficiency in mass spectrometry US6507837B1 (en) 2000-06-08 2003-01-14 Hyperphrase Technologies, Llc Tiered and content based database searching AU2001268259A1 (en) 2000-06-09 2001-12-24 Corixa Corporation Compositions and methods for the therapy and diagnosis of colon cancer DE60137722D1 (en) * 2000-06-13 2009-04-02 Univ Boston USE OF MASS-MATCHED NUCLEOTIDES IN THE ANALYSIS OF OLIGONUCLEOTIDE MIXTURES AND IN THE HIGH-MULTIPLEXIC NUCLEIC ACID SEQUENCING EP1170379A1 (en) * 2000-06-30 2002-01-09 Centre National de Genotypage Sample generation for genotyping by mass spectrometry FR2811321A1 (en) 2000-07-04 2002-01-11 Bio Merieux New oligonucleotide primers, useful for identifying bacteria, particularly in cases of septicemia, provide amplification of bacterial 16S ribosomal nucleic acid US6504021B2 (en) 2000-07-05 2003-01-07 Edge Biosystems, Inc. Ion exchange method for DNA purification CA2412946A1 (en) 2000-07-06 2002-01-10 Bio Merieux Method for controlling the microbiological quality of an aqueous medium and kit therefor US6783939B2 (en) 2000-07-07 2004-08-31 Alphavax, Inc. Alphavirus vectors and virosomes with modified HIV genes for use in vaccines US6811977B2 (en) * 2000-07-27 2004-11-02 California Institute Of Technology Rapid, quantitative method for the mass spectrometric analysis of nucleic acids for gene expression and genotyping AUPQ909000A0 (en) 2000-07-28 2000-08-24 University Of Sydney, The A method of detecting microorganisms US20030190635A1 (en) 2002-02-20 2003-10-09 Mcswiggen James A. RNA interference mediated treatment of Alzheimer's disease using short interfering RNA GB0021286D0 (en) 2000-08-30 2000-10-18 Gemini Genomics Ab Identification of drug metabolic capacity US20040005555A1 (en) * 2000-08-31 2004-01-08 Rothman Richard E. Molecular diagnosis of bactermia US7349808B1 (en) 2000-09-06 2008-03-25 Egenomics, Inc. System and method for tracking and controlling infections US6813615B1 (en) 2000-09-06 2004-11-02 Cellomics, Inc. Method and system for interpreting and validating experimental data with automated reasoning US20020120408A1 (en) 2000-09-06 2002-08-29 Kreiswirth Barry N. System and method for tracking and controlling infections WO2002021108A2 (en) 2000-09-08 2002-03-14 Large Scale Proteomics Corporation Method for detecting molecules or chemical reactions by determining variation of conductance SE0003286D0 (en) 2000-09-15 2000-09-15 Ulf Gyllensten Method and kit for human identification WO2002024876A2 (en) 2000-09-25 2002-03-28 Polymun Scientific Immunbiologische Forschung Gmbh Live influenza vaccine and method of manufacture US20030148281A1 (en) 2000-10-05 2003-08-07 Glucksmann Maria A. 65499 and 58875, novel seven transmembrane receptors and uses thereof US6996472B2 (en) 2000-10-10 2006-02-07 The United States Of America As Represented By The Department Of Health And Human Services Drift compensation method for fingerprint spectra JP2004511788A (en) 2000-10-13 2004-04-15 ã¢ã¤ã¢ã¼ã«ã¨ã ãã¨ã«ã¨ã«ã·ã¼ High throughput processing system and method of use AU2002246612B2 (en) 2000-10-24 2007-11-01 The Board Of Trustees Of The Leland Stanford Junior University Direct multiplex characterization of genomic DNA US6906316B2 (en) 2000-10-27 2005-06-14 Fuji Electric Co., Ltd. Semiconductor device module US6682889B1 (en) * 2000-11-08 2004-01-27 Becton, Dickinson And Company Amplification and detection of organisms of the Chlamydiaceae family AU2002222614A1 (en) 2000-12-12 2002-07-01 Chugai Seiyaku Kabushiki Kaisha Method of detecting polymorphism in dna by using mass spectroscopy US6800289B2 (en) 2000-12-21 2004-10-05 Her Majesty The Queen In Right Of Canada, As Represented By The Minister Of National Defence Strain of the western equine encephalitis virus US6586584B2 (en) 2001-01-29 2003-07-01 Becton, Dickinson And Company Sequences and methods for detection of Hepatitis C virus DE10108453B4 (en) 2001-02-22 2005-10-20 Bruker Daltonik Gmbh Mass spectrometric mutation analysis with photolytically cleavable primers CA2439504A1 (en) 2001-02-28 2002-09-12 Chondrogene Inc. Compositions and methods relating to osteoarthritis US6699670B2 (en) 2001-03-01 2004-03-02 The Johns Hopkins University Quantitative assay for the simultaneous detection and speciation of bacterial infections US20040121314A1 (en) 2002-12-06 2004-06-24 Ecker David J. Methods for rapid detection and identification of bioagents in containers US7718354B2 (en) 2001-03-02 2010-05-18 Ibis Biosciences, Inc. Methods for rapid identification of pathogens in humans and animals US7666588B2 (en) 2001-03-02 2010-02-23 Ibis Biosciences, Inc. Methods for rapid forensic analysis of mitochondrial DNA and characterization of mitochondrial DNA heteroplasmy US20030027135A1 (en) 2001-03-02 2003-02-06 Ecker David J. Method for rapid detection and identification of bioagents US20040121335A1 (en) 2002-12-06 2004-06-24 Ecker David J. Methods for rapid detection and identification of bioagents associated with host versus graft and graft versus host rejections AU2003298030B2 (en) 2001-03-02 2009-12-10 Ibis Biosciences, Inc. Methods for rapid identification of pathogens in humans and animals WO2004060278A2 (en) 2002-12-06 2004-07-22 Isis Pharmaceuticals, Inc. Methods for rapid identification of pathogens in humans and animals US20040121310A1 (en) 2002-12-18 2004-06-24 Ecker David J. Methods for rapid detection and identification of bioagents in forensic studies US7226739B2 (en) 2001-03-02 2007-06-05 Isis Pharmaceuticals, Inc Methods for rapid detection and identification of bioagents in epidemiological and forensic investigations US20040038206A1 (en) * 2001-03-14 2004-02-26 Jia Zhang Method for high throughput assay of genetic analysis US20030104410A1 (en) 2001-03-16 2003-06-05 Affymetrix, Inc. Human microarray EP1832567A3 (en) 2001-03-19 2009-08-26 President And Fellows Of Harvard College Evolving new molecular function CN1316034C (en) 2001-03-28 2007-05-16 ç§å¦ä¸å·¥ä¸ç ç©¶ä¼ Universal primers for wildlife identification US7630836B2 (en) 2001-05-30 2009-12-08 The Kitasato Institute Polynucleotides CA2348042A1 (en) 2001-06-04 2002-12-04 Ann Huletsky Sequences for detection and identification of methicillin-resistant staphylococcus aureus CA2449885A1 (en) 2001-06-06 2002-12-12 Roche Vitamins Ag Improved isoprenoid production US20020187477A1 (en) * 2001-06-06 2002-12-12 Hong Xue Method for detecting single nucleotide polymorphisms (SNPs) and point mutations GB0113907D0 (en) 2001-06-07 2001-08-01 Univ London Virus detection using degenerate PCR primers US20020187490A1 (en) 2001-06-07 2002-12-12 Michigan State University Microbial identification chip based on DNA-DNA hybridization GB0113908D0 (en) 2001-06-07 2001-08-01 Univ London Designing degenerate PCR primers US7217510B2 (en) 2001-06-26 2007-05-15 Isis Pharmaceuticals, Inc. Methods for providing bacterial bioagent characterizing information US8073627B2 (en) 2001-06-26 2011-12-06 Ibis Biosciences, Inc. System for indentification of pathogens DE10132147B4 (en) 2001-07-03 2004-04-15 Universität Leipzig Method for the rapid quantitative determination of Eu bacteria GB0117054D0 (en) 2001-07-12 2001-09-05 Plant Bioscience Ltd Methods and means for modification of plant characteristics EP1407051B1 (en) 2001-07-19 2006-04-12 Infectio Diagnostic (I.D.I.) INC. Universal method and composition for the rapid lysis of cells for the release of nucleic acids and their detection US20040191769A1 (en) 2001-07-24 2004-09-30 Transgenomic, Inc. Methods, compositions, and kits for mutation detection in mitochondrial DNA AU2002325197A1 (en) 2001-07-30 2003-02-17 Den Kgl. Veterinaer- Og Landbohojskole Bacterial strains belonging to lactobacillus species and their use in food and feed industry US7115385B2 (en) 2001-08-02 2006-10-03 North Carolina State University Media and methods for cultivation of microorganisms AT411174B (en) 2001-08-09 2003-10-27 Lambda Labor Fuer Molekularbio METHOD AND CHIP FOR ANALYZING NUCLEIC ACIDS US20030039976A1 (en) * 2001-08-14 2003-02-27 Haff Lawrence A. Methods for base counting WO2003016546A1 (en) 2001-08-21 2003-02-27 Flinders Technologies Pty Ltd. Method and device for simultaneously molecularly cloning and polylocus profiling of genomes or genome mixtures US7105296B2 (en) 2001-08-29 2006-09-12 E. I. Du Pont De Nemours And Company Genes encoding Baeyer-Villiger monooxygenases US7049286B2 (en) 2001-08-30 2006-05-23 Diatos, S.A. Insulin conjugates and methods of use thereof US7060809B2 (en) 2001-09-04 2006-06-13 Exiqon A/S LNA compositions and uses thereof US20040101809A1 (en) 2001-09-21 2004-05-27 Weiss Ervin I Device, method and materials for mobilizing substances into dentinal tubules in root canal treatment DE10150121B4 (en) 2001-10-11 2005-12-01 Bernhard-Nocht-Institut für Tropenmedizin Real-time detection of DNA amplification products US6977148B2 (en) 2001-10-15 2005-12-20 Qiagen Gmbh Multiple displacement amplification US7297485B2 (en) 2001-10-15 2007-11-20 Qiagen Gmbh Method for nucleic acid amplification that results in low amplification bias US20040029129A1 (en) 2001-10-25 2004-02-12 Liangsu Wang Identification of essential genes in microorganisms DE10152821B4 (en) 2001-10-25 2006-11-16 Bruker Daltonik Gmbh Mass spectra without electronic noise EP1308506A1 (en) 2001-11-06 2003-05-07 Eidgenössische Technische Hochschule Zürich Mixtures of Propionibacterium jensenii and Lactobacillus sp. with antimicrobial activities for use as a natural preservation system BR122016004546B8 (en) 2001-11-13 2021-07-27 Univ Pennsylvania recombinant adeno-associated virus, method of generating said virus and composition comprising said virus AU2002365229A1 (en) * 2001-11-15 2003-09-02 Whatman, Inc. Methods and materials for detecting genetic material US20040023209A1 (en) 2001-11-28 2004-02-05 Jon Jonasson Method for identifying microorganisms based on sequencing gene fragments JP3692067B2 (en) 2001-11-30 2005-09-07 æ ªå¼ä¼ç¤¾æ±è Polishing slurry for copper CMP and method of manufacturing semiconductor device using the same US20030148284A1 (en) * 2001-12-17 2003-08-07 Vision Todd J. Solid phase detection of nucleic acid molecules TW509116U (en) 2001-12-18 2002-11-01 Ind Tech Res Inst Device for clipping and tightening spindle of honing and milling machine US20030175709A1 (en) 2001-12-20 2003-09-18 Murphy George L. Method and system for depleting rRNA populations US7468185B2 (en) 2001-12-21 2008-12-23 Pfizer Inc. Vaccine for periodontal disease KR100444230B1 (en) 2001-12-27 2004-08-16 ì¼ì±ì 기주ìíì¬ Nonreducible dielectric ceramic composition US20060088826A1 (en) 2001-12-28 2006-04-27 Van Eijk Michael Josephus Ther Discrimination and detection of target nucleotide sequences using mass spectrometry ATE358185T1 (en) 2002-02-01 2007-04-15 Bruker Daltonik Gmbh MUTATION ANALYSIS USING PCR AND MASS SPECTROMETRY CN1202204C (en) 2002-02-27 2005-05-18 è´¢å¢æ³äººå·¥ä¸ææ¯ç ç©¶é¢ Red-light organic electroluminescent compound and components and devices made of it KR100600988B1 (en) 2002-03-13 2006-07-13 주ìíì¬ ìì§ìëª ê³¼í Increasing immune response by co-administering the influenza NP gene DNA in DNA immunization US7024370B2 (en) 2002-03-26 2006-04-04 P) Cis, Inc. Methods and apparatus for early detection of health-related events in a population US6897027B2 (en) 2002-03-27 2005-05-24 Decode Genetics Ehf. Method for desalting nucleic acids WO2003100035A2 (en) 2002-04-01 2003-12-04 Isis Pharmaceuticals, Inc. Method for rapid detection and identification of viral bioagents US7539579B2 (en) 2002-04-09 2009-05-26 Beattie Kenneth L Oligonucleotide probes for genosensor chips JP2004000200A (en) 2002-04-19 2004-01-08 Menicon Co Ltd Method for detecting microorganism FR2838740A1 (en) 2002-04-22 2003-10-24 Centre Nat Rech Scient Detecting primate T cell lymphoma/leukemia viruses, useful e.g. for diagnosis and drug screening, using degenerate oligonucleotides based on conserved regions of envelope protein GB0209812D0 (en) 2002-04-30 2002-06-05 Renovo Ltd Genetic testing DE10222632B4 (en) 2002-05-17 2006-03-09 Con Cipio Gmbh Microsatellite markers for genetic analysis and for the differentiation of roses US6906319B2 (en) 2002-05-17 2005-06-14 Micromass Uk Limited Mass spectrometer EP1365031A1 (en) 2002-05-21 2003-11-26 MTM Laboratories AG Method for detection of somatic mutations using mass spectometry AU2003237249A1 (en) 2002-05-24 2003-12-12 Isis Pharmaceuticals, Inc. Oligonucleotides having modified nucleoside units US20030220844A1 (en) * 2002-05-24 2003-11-27 Marnellos Georgios E. Method and system for purchasing genetic data AU2003240150B2 (en) 2002-05-29 2008-10-30 Aresa Biodetection Aps Reporter system for plants GB0212666D0 (en) 2002-05-31 2002-07-10 Secr Defence Immunogenic sequences AU2003238930A1 (en) 2002-06-07 2003-12-22 Incyte Corporation Enzymes WO2003106635A2 (en) 2002-06-13 2003-12-24 Regulome Corp Functional sites KR100484144B1 (en) 2002-06-20 2005-04-18 ì¼ì±ì ì주ìíì¬ Remote management server and the method thereof WO2004003511A2 (en) 2002-07-01 2004-01-08 Wayne State University Methods and compositions for the identification of antibiotics that are not susceptible to antibiotic resistance AU2003254093A1 (en) 2002-07-19 2004-02-09 Isis Pharmaceuticals, Inc. Methods for mass spectrometry analysis utilizing an integrated microfluidics sample platform US6916483B2 (en) * 2002-07-22 2005-07-12 Biodynamics, Llc Bioabsorbable plugs containing drugs GB0217434D0 (en) 2002-07-27 2002-09-04 Royal Vetinary College Biological material US20040022764A1 (en) 2002-07-31 2004-02-05 Hanan Polansky Inhibition of microcompetition with a foreign polynucleotide as treatment of chronic disease US7172868B2 (en) 2002-08-01 2007-02-06 The Regents Of The University Of California Nucleotide sequences specific to Francisella tularensis and methods for the detection of Francisella tularensis US20040038385A1 (en) * 2002-08-26 2004-02-26 Langlois Richard G. System for autonomous monitoring of bioagents JP4683922B2 (en) 2002-09-06 2011-05-18 ãã©ã¹ãã£ã¼ãº ãªã ãã¹ãã³ ã¦ããã¼ã·ã㣠Gene expression quantification method US20040110138A1 (en) 2002-11-01 2004-06-10 University Of Ottawa Method for the detection of multiple genetic targets JP2006505275A (en) 2002-11-12 2006-02-16 ã¸ã§ãã©ã¤ã One-step real-time RT-PCR kit for universal detection of microorganisms in industrial products US7250496B2 (en) 2002-11-14 2007-07-31 Rosetta Genomics Ltd. Bioinformatically detectable group of novel regulatory genes and uses thereof DE60331587D1 (en) 2002-11-15 2010-04-15 Gen Probe Inc TEST AND COMPOSITIONS FOR DETECTION OF BACILLUS ANTHRACIS NUCLEIC ACID US6680476B1 (en) 2002-11-22 2004-01-20 Agilent Technologies, Inc. Summed time-of-flight mass spectrometry utilizing thresholding to reduce noise JP2004201679A (en) 2002-12-10 2004-07-22 Kao Corp Primer for detecting Fusobacterium nucleatum by PCR and method for detecting the same US20040117354A1 (en) 2002-12-16 2004-06-17 Azzaro Steven Hector Process for tagging and measuring quality US20040121312A1 (en) 2002-12-18 2004-06-24 Ecker David J. Methods for rapid detection and identification of the absence of bioagents US20040121315A1 (en) 2002-12-18 2004-06-24 Ecker David J. Secondary structure defining database and methods for determining identity and geographic origin of an unknown bioagent in containers thereby US20040121329A1 (en) 2002-12-18 2004-06-24 Ecker David J. Secondary structure defining database and methods for determining identity and geographic origin of an unknown bioagent in blood, bodily fluids, and bodily tissues thereby US20040122598A1 (en) 2002-12-18 2004-06-24 Ecker David J. Secondary structure defining database and methods for determining identity and geographic origin of an unknown bioagent in food products and cosmetics thereby US20040121340A1 (en) 2002-12-18 2004-06-24 Ecker David J. Secondary structure defining database and methods for determining identity and geographic origin of an unknown bioagent associated with host versus graft and graft versus host rejections thereby US20040122857A1 (en) 2002-12-18 2004-06-24 Ecker David J. Secondary structure defining database and methods for determining identity and geographic origin of an unknown bioagent in forensic studies thereby US9487823B2 (en) 2002-12-20 2016-11-08 Qiagen Gmbh Nucleic acid amplification JP2004201641A (en) 2002-12-26 2004-07-22 Mitsubishi Kagaku Bio-Clinical Laboratories Inc Fungus detection method US20040170954A1 (en) 2003-02-10 2004-09-02 Mckenney Keith Pathogen inactivation assay US20040170981A1 (en) 2003-02-10 2004-09-02 Mckenney Keith Real-time polymerase chain reaction using large target amplicons US20040185438A1 (en) 2003-03-10 2004-09-23 Ecker David J. Methods of detection and notification of bioagent contamination US20050065813A1 (en) 2003-03-11 2005-03-24 Mishelevich David J. Online medical evaluation system US8046171B2 (en) 2003-04-18 2011-10-25 Ibis Biosciences, Inc. Methods and apparatus for genetic evaluation WO2004097369A2 (en) 2003-04-25 2004-11-11 Sequenom, Inc. Fragmentation-based methods and systems for de novo sequencing US8057993B2 (en) 2003-04-26 2011-11-15 Ibis Biosciences, Inc. Methods for identification of coronaviruses WO2005009202A2 (en) 2003-05-12 2005-02-03 Isis Pharmaceuticals, Inc. Automatic identification of bioagents US8158354B2 (en) 2003-05-13 2012-04-17 Ibis Biosciences, Inc. Methods for rapid purification of nucleic acids for subsequent analysis by mass spectrometry by solution capture US7964343B2 (en) 2003-05-13 2011-06-21 Ibis Biosciences, Inc. Method for rapid purification of nucleic acids for subsequent analysis by mass spectrometry by solution capture EP1644520B1 (en) 2003-07-03 2007-09-19 Danmarks og Gronlands Geologiske Undersogelse Method for selective detection of a target nucleic acid AU2003257013A1 (en) 2003-07-29 2005-03-07 Sigma Aldrich Co. Methods and compositions for amplification of dna JP4304292B2 (en) 2003-07-30 2009-07-29 æ¥æ¬é»æ°æ ªå¼ä¼ç¤¾ Mobile communication system, mobile communication terminal, power control method used therefor, and program thereof KR100632429B1 (en) 2003-08-01 2006-10-09 íë¡í ì¨ ì£¼ìíì¬ Screening system of reassortant influenza viruses using primer dependent multiplex RT-PCR US7956175B2 (en) 2003-09-11 2011-06-07 Ibis Biosciences, Inc. Compositions for use in identification of bacteria US20060240412A1 (en) 2003-09-11 2006-10-26 Hall Thomas A Compositions for use in identification of adenoviruses US20050142584A1 (en) 2003-10-01 2005-06-30 Willson Richard C. Microbial identification based on the overall composition of characteristic oligonucleotides WO2005036369A2 (en) 2003-10-09 2005-04-21 Isis Pharmaceuticals, Inc. Database for microbial investigations FR2861743B1 (en) 2003-11-04 2007-10-19 Univ Aix Marseille Ii MOLECULAR IDENTIFICATION OF BACTERIA OF THE GENUS CORYNEBACTERIUM JP3861871B2 (en) 2003-11-26 2006-12-27 ãµã³ã±ã³é»æ°æ ªå¼ä¼ç¤¾ Switching power supply US20050250125A1 (en) 2003-12-19 2005-11-10 Novakoff James L Method for conducting pharmacogenomics-based studies JP4619368B2 (en) 2004-02-10 2011-01-26 ã¨ãï¼ãããã³âã© ãã·ã¥ ã¢ã¼ã²ã¼ Novel primers and probes for detection of parvovirus B19 AU2005322640B2 (en) 2004-02-18 2008-09-18 Ibis Biosciences, Inc. Compositions for use in identification of bacteria US7666592B2 (en) 2004-02-18 2010-02-23 Ibis Biosciences, Inc. Methods for concurrent identification and quantification of an unknown bioagent US8119336B2 (en) 2004-03-03 2012-02-21 Ibis Biosciences, Inc. Compositions for use in identification of alphaviruses US7312036B2 (en) 2004-03-22 2007-12-25 Isis Pharmaceuticals, Inc. Compositions for use in identification of viral hemorrhagic fever viruses US20050266411A1 (en) 2004-05-25 2005-12-01 Hofstadler Steven A Methods for rapid forensic analysis of mitochondrial DNA WO2006135400A2 (en) 2004-08-24 2006-12-21 Isis Pharmaceuticals, Inc. Methods for rapid identification of recombinant organisms US7627437B2 (en) 2005-01-14 2009-12-01 Idaho Research Foundation Categorization of microbial communities DE102005008583B4 (en) 2005-02-24 2007-10-25 Johannes-Gutenberg-Universität Mainz A method of typing an individual by short tandem repeat (STR) loci of the genomic DNA WO2006094238A2 (en) 2005-03-03 2006-09-08 Isis Pharmaceuticals, Inc. Compositions for use in identification of adventitious viruses JP5162447B2 (en) 2005-04-13 2013-03-13 ã¢ã¤ãã¹ ãã¤ãªãµã¤ã¨ã³ã·ãº ã¤ã³ã³ã¼ãã¬ã¤ãã£ãã Composition for use in adenovirus identification AU2006239993A1 (en) 2005-04-21 2006-11-02 Ibis Biosciences, Inc. Compositions for use in identification of bacteria AU2006272776B2 (en) 2005-07-21 2012-01-19 Ibis Biosciences, Inc. Methods for rapid identification and quantitation of nucleic acid variants US20070026435A1 (en) 2005-07-28 2007-02-01 Polysciences, Inc. Hydroxysilane functionalized magnetic particles and nucleic acid separation method WO2008104002A2 (en) 2007-02-23 2008-08-28 Ibis Biosciences, Inc. Methods for rapid forensic dna analysis WO2008118809A1 (en) 2007-03-23 2008-10-02 Ibis Biosciences, Inc. Compositions for use in identification of mixed populations of bioagents JP5067239B2 (en) 2008-03-28 2012-11-07 å¯å£«éæ ªå¼ä¼ç¤¾ Cable management device and electronic device deviceOwner name: ISIS PHARMACEUTICALS, INC., CALIFORNIA
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