US20060252038A1 - Multiplex genotyping using solid phase capturable dideoxynucleotides and mass spectrometry - Google Patents

Multiplex genotyping using solid phase capturable dideoxynucleotides and mass spectrometry Download PDF

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US20060252038A1
US20060252038A1 US10/521,206 US52120605A US2006252038A1 US 20060252038 A1 US20060252038 A1 US 20060252038A1 US 52120605 A US52120605 A US 52120605A US 2006252038 A1 US2006252038 A1 US 2006252038A1
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linker
dideoxynucleotide
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Jingyue Ju
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Columbia University of New York
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    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813Hybridisation assays
    • C12Q1/6827Hybridisation assays for detection of mutation or polymorphism
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07HSUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07HSUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • C07H21/02Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with ribosyl as saccharide radical
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07HSUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • C07H21/04Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with deoxyribosyl as saccharide radical
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    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6869Methods for sequencing
    • C12Q1/6872Methods for sequencing involving mass spectrometry

Definitions

  • SNPs Single nucleotide polymorphisms
  • MALDI-TOF MS Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry
  • primers designed to anneal immediately adjacent to a polymorphic site are extended by a single dideoxynucleotide that is complementary to the nucleotide at the variable site.
  • a particular SNP can be identified.
  • Current SBE methods to perform multiplex SNP analysis using MS require unambiguous simultaneous detection of a library of primers and their extension products.
  • limitations in resolution and sensitivity of MALDI-TOF MS for longer DNA molecules make it difficult to simultaneously measure DNA fragments over a large mass range (6). The requirement to measure both primers and their extension products in this range limits the scope of multiplexing.
  • a high fidelity DNA sequencing method has been developed which uses solid phase capturable biotinylated dideoxynucleotides (biotin-ddNTPs) by detection with fluorescence (18) or mass spectrometry (19), eliminating false terminations and excess primers.
  • biotin-ddNTPs solid phase capturable biotinylated dideoxynucleotides
  • fluorescence or mass spectrometry (19)
  • Combinatorial fluorescence energy transfer tags and biotin-ddNTPs have also been used to detect SNPs (20).
  • False stops or terminations occur when a deoxynucleotide rather than a dideoxynucleotide terminates a se+quencing fragment. It has been shown that false stops and primers which have dimerized can produce peaks in the mass spectra that can mask the actual results preventing accurate base identification (21).
  • the present application discloses an approach using solid phase capturable biotin-ddNTPs in SBE for multiplex genotyping by MALDI-TOF MS.
  • primers that have different molecular weights and that are specific to the polymorphic sites in the DNA template are extended with biotin-ddNTPs by DNA polymerase to generate 3′-biotinylated DNA extension products.
  • the 3′-biotinylated DNAs are then captured by streptavidin-coated magnetic beads, while the unextended primers and other components in the reaction are washed away.
  • the pure DNA extension products are subsequently released from the magnetic beads, for example by denaturing the biotin-streptavidin interaction with formamide, and analyzed with MALDI-TOF MS.
  • the nucleotide at the polymorphic site is identified by analyzing the mass difference between the primer extension product and an internal mass standard added to the purified DNA products. Since the primer extension products are isolated prior to MS analysis, the resulting mass spectrum is free of non-extended primer peaks and their associated dimers, which increases the accuracy and scope of multiplexing in SNP analysis.
  • the solid phase purification system also facilitates desalting of the captured oligonucleotides. Desalting is critical in sample preparation for MALDI-TOF MS measurement since alkaline and alkaline earth salts can form adducts with DNA fragments that interfere with accurate peak detection (21).
  • This invention is directed to a method for determining the identity of a nucleotide present at a predetermined site in a DNA whose sequence immediately 3′ of such predetermined site is known which comprises:
  • the method further comprises after step (b) the steps of:
  • the method further comprises after step (i) the step of treating the surface to remove primers that have not been extended by a labeled dideoxynucleotide.
  • FIG. 1 Scheme of single base extension for multiplex SNP analysis using biotin-ddNTPs and MALDI-TOF MS.
  • Primers that anneal immediately next to the polymorphic sites in the DNA template are extended by DNA polymerase of a biotin-ddNTP in a sequence-specific manner.
  • MALDI-TOF MS was used to analyze these DNA products to yield a mass spectrum. From the relative mass of each extended primer, compared to the mass of an internal standard, the nucleotide at the polymorphic site is identified.
  • FIG. 2 Multiplex SNP genotyping mass spectra generated using biotin-ddNTPs. Inset is a magnified view of heterozygote peaks. Masses of the extension product in reference to the internal mass standard were listed on each single base extension peak. The mass values in parenthesis indicate the mass difference between the extension products and the corresponding primers.
  • A Detection of six nucleotide variations from synthetic DNA templates mimicking mutations in the p53 gene. Four homozygous (T, G, C and C) and one heterozygous (C/A) genotypes were detected.
  • B Detection of two heterozygotes (A/G and C/G) in the human HFE gene.
  • FIG. 3 Structure of four mass tagged biotinylated ddNTPs. Any of the four ddNTPs (ddATP, ddCTP, ddGTP, ddTTP) can be used with any of the illustrated linkers.
  • FIG. 4 Synthesis scheme for mass tag linkers.
  • the linkers are labeled to correspond to the specific ddNTP with which they are shown coupled in FIGS. 3, 5 , 7 , 8 and 9 .
  • any of the three linkers can be used with any ddNTP.
  • iii) Propargyl amine Propargyl amine.
  • FIG. 5 The synthesis of ddATP-Linker-II-11-Biotin.
  • Linker II tetrakis(triphenylphosphine) palladium(0)
  • POCl 3 tetrakis(triphenylphosphine) palladium(0)
  • POCl 3 tetrakis(triphenylphosphine) palladium(0)
  • POCl 3 tetrakis(triphenylphosphine) palladium(0)
  • POCl 3 Bn 4 N + pyrophosphate
  • NH 4 OH NH 4 OH
  • Sulfo-NHS-LC-Biotin Sulfo-NHS-LC-Biotin.
  • FIG. 6 DNA products are purified by a streptavidin coated porous silica surface. Only the biotinylated fragments are captured. These fragments are then cleaved by light irradiation (hv) to release the captured fragments, leaving the biotin moiety still bound to the streptavidin.
  • hv light irradiation
  • FIG. 7 Mechanism for the cleavage of photocleavable linkers.
  • FIG. 8 The structures of ddNTPs linked to photocleavable (PC) biotin. Any of the four ddNTPs (ddATP, ddCTP, ddGTP, ddTTP) can be used with any of the shown linkers.
  • FIG. 10 Schematic for capturing a DNA fragment terminated with a dideoxynucleoside monophosphate on a surface.
  • the dideoxynucleoside monophosphate (ddNMP) which is on the 3′ end of the DNA fragment is attached via a linker to a chemical moiety “X” which interacts with a compound “Y” on the surface to capture the DNA fragment terminated with the ddNMP.
  • the DNA fragment can be freed from the surface either by disrupting the interaction between chemical moiety X and compound Y (lower scheme) or by cleaving the linker (upper scheme).
  • FIGS. 11 A- 11 C Schematic of a high throughput channel based purification system. Sample solutions can be pushed back and forth between the two plates through glass capillaries and the streptavidin coated channels in the chip. The whole chip can be irradiated to cleave the samples after immobilization.
  • FIG. 12 The synthesis of streptavidin coated porous surface.
  • FIGS. 13 A- 13 C Simultaneous detection of nucleotide variations in 30 codons of the p53 tumor suppressor gene by MALDI-TOF MS using solid phase capturable biotinylated dideoxynucleotide. Each peak represents a different polymorphism labeled with its nucleotide identity and absolute mass value. The value in parentheses, denoting the mass difference between each DNA extension product and its corresponding primer, is used to determine the nucleotide identity.
  • A A mass spectrum from a Wilms' tumor sample showing 30 wild type p53 sequences.
  • (B) A mass spectrum from a head and neck tumor (primary tumor biopsy) containing a heterozygous genotype G/T (4684/4734 Da) (boxed) in codon 157, corresponding to the wild type and mutant alleles, respectively.
  • (C) A mass spectrum from a colorectal tumor cell line (HT-29) containing a homozygous G to A mutation (boxed) in codon 273 of the p53 gene.
  • the colorectal tumor cell line (SW-480) contained the identical G to A mutation in codon 273.
  • FIGS. 14 A- 14 B (A) A mass spectrum from a head and neck tumor sample showing 30 wild type sequences of the p53 gene. (B) A mass spectrum from a head and neck tumor cell line (SCC-4) containing a homozygous C (5881 Da) to T (5970 Da) mutation (boxed) in codon 151 of the p53 gene. Both spectra were produced using the primers shown in Table 3 with primer 16 replaced by primer 5′-TGTGGGTTGATTCCACA-3′ for detecting the variation in codon 151 (C/TCC).
  • nucleotide bases are used as follows: adenine (A), cytosine (C), guanine (G), thymine (T), and uracil (U).
  • a nucleotide analogue refers to a chemical compound that is structurally and functionally similar to the nucleotide, i.e. the nucleotide analogue can be recognized by polymerase as a substrate. That is, for example, a nucleotide analogue comprising adenine or an analogue of adenine should form hydrogen bonds with thymine, a nucleotide analogue comprising C or an analogue of C should form hydrogen bonds with G, a nucleotide analogue comprising G or an analogue of G should form hydrogen bonds with C, and a nucleotide analogue comprising T or an analogue of T should form hydrogen bonds with A, in a double helix format.
  • This invention is directed to a method for determining the identity of a nucleotide present at a predetermined site in a DNA whose sequence immediately 3′ of such predetermined site is known which comprises:
  • each of the four labeled dideoxynucleotides comprises a chemical moiety attached to the dideoxynucleotide by a different linker which has a molecular weight different from that of each other linker.
  • the method further comprises after step (b) the steps of:
  • the method comprises after step (i) the step of treating the surface to remove primers that have not been extended by a labeled dideoxynucleotide and any non-captured component.
  • step (c) comprises determining the difference in mass between the labeled single base extended primer and an internal mass calibration standard added to the extended primer.
  • the chemical moiety is attached via a different linker to different dideoxynucleotides.
  • the different linkers increase mass separation between different labeled single base extended primers and thereby increase mass spectrometry resolution.
  • the dideoxynucleotide is selected from the group consisting of 2′,3′-dideoxyadenosine 5′-triphosphate (ddATP), 2′,3′-dideoxyguanosine 5′-triphosphate (ddGTP), 2′,3′-dideoxycytidine 5′-triphosphate (ddCTP), and 2′,3′-dideoxythymidine 5′-triphosphate (ddTTP).
  • ddATP 2′,3′-dideoxyadenosine 5′-triphosphate
  • ddGTP 2′,3′-dideoxyguanosine 5′-triphosphate
  • ddCTP 2′,3′-dideoxycytidine 5′-triphosphate
  • ddTTP 2′,3′-dideoxythymidine 5′-triphosphate
  • the interaction between the chemical moiety attached to the dideoxynucleotide by the linker and the compound on the surface comprises a biotin-streptavidin interaction, a phenylboronic acid-salicylhydroxamic acid interaction, or an antigen-antibody interaction.
  • the step of releasing the labeled single base extended primer from the surface comprises disrupting the interaction between the chemical moiety attached by the linker to the dideoxynucleotide and the compound on the surface.
  • the interaction is disrupted by a means selected from the group consisting of one or more of a physical means, a is chemical means, a physical chemical means, heat, and light.
  • the interaction is disrupted by light.
  • the interaction is disrupted by ultraviolet light.
  • the interaction is disrupted by ammonium hydroxide, formamide, or a change in pH (-log H + concentration).
  • the linker can comprise a chain structure, or a structure comprising one or more rings, or a structure comprising a chain and one or more rings.
  • the dideoxynucleotide comprises a cytosine or a thymine with a 5-position, or an adenine or a guanine with a 7-position, and the linker is attached to the dideoxynucleotide at the 5-position of cytosine or thymine or at the 7-position of adenine or guanine.
  • the step of releasing the labeled single base extended primer from the surface comprises cleaving the linker between the chemical moiety and the dideoxynucleotide.
  • the linker is cleaved by a means selected from the group consisting of one or more of a physical means, a chemical means, a physical chemical means, heat, and light.
  • the linker is cleaved by light.
  • the linker is cleaved by ultraviolet light.
  • the linker is cleaved by ammonium hydroxide, formamide, or a change in pH (-log H + concentration);
  • the linker comprises a derivative of 4-aminomethyl benzoic acid. In one embodiment, the linker comprises a 2-nitrobenzyl group or a derivative of a 2-nitrobenzyl group. In one embodiment, the linker comprises one or more fluorine atoms.
  • the linker is selected from the group consisting of:
  • a plurality of different linkers is used to increase mass separation between different labeled single base extended primers and thereby increase mass spectrometry resolution.
  • the chemical moiety comprises biotin
  • the labeled dideoxynucleotide is a biotinylated dideoxynucleotide
  • the labeled single base extended primer is a biotinylated single base extended primer
  • the surface is a streptavidin-coated solid surface.
  • the biotinylated dideoxynucleotide is selected from the group consisting of ddATP-11-biotin, ddCTP-11-biotin, ddGTP-11-biotin, and ddTTP-16-biotin.
  • biotinylated dideoxynucleotide is selected from the group consisting of:
  • ddNTP1, ddNTP2, ddNTF3, and ddNTP4 represent four different dideoxynucleotides, or their analogues.
  • biotinylated dideoxynucleotide is selected from the group consisting of:
  • biotinylated dideoxynucleotide is selected from the group consisting of:
  • ddNTP1, ddNTP2, ddNTP3, and ddNTP4 represent four different dideoxynucleotides or their analogues.
  • biotinylated dideoxynucleotide is selected from the group consisting of:
  • the streptavidin-coated solid surface is a streptavidin-coated magnetic bead or a streptavidin-coated silica glass.
  • steps (a) and (b) are performed in a single container or in a plurality of connected containers.
  • the invention provides methods for determining the identity of nucleotides present at a plurality of predetermined sites, which comprises carrying out any of the methods disclosed herein using a plurality of different primers each having a molecular weight different from that of each other primer, wherein a different primer hybridizes adjacent to a different predetermined site.
  • different linkers each having a molecular weight different from that of each other linker are attached to the different dideoxynucleotides to increase mass separation between different labeled single base extended primers and thereby increase mass spectrometry resolution.
  • the mass spectrometry is matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.
  • Linkers are provided for attaching a chemical moiety to a dideoxynucleotide, wherein the linker comprises a derivative of 4-aminomethyl benzoic acid.
  • the dideoxynucleotide is selected from the group consisting of 2′,3′-dideoxyadenosine 5′-triphosphate (ddATP), 2′,3′-dideoxyguanosine 5′-triphosphate (ddGTP), 2′,3′-dideoxycytidine 5′-triphosphate (ddCTP), and 2′,3′-dideoxythymidine 5′-triphosphate (ddTTP).
  • ddATP 2′,3′-dideoxyadenosine 5′-triphosphate
  • ddGTP 2′,3′-dideoxyguanosine 5′-triphosphate
  • ddCTP 2′,3′-dideoxycytidine 5′-triphosphate
  • ddTTP 2′,3′-dideoxythymidine 5′-triphosphate
  • the linker comprises one or more fluorine atoms.
  • the linker is selected from the group consisting of:
  • the linker can comprise a chain structure, or a structure comprising one or more rings, or a structure comprising a chain and one or more rings.
  • the linker is cleavable by a means selected from the group consisting of one or more of a physical means, a chemical means, a physical chemical means, heat, and light. In one embodiment, the linker is cleavable by ultraviolet light. In different embodiments, the linker is cleavable by ammonium hydroxide, formamide, or a change in pH (-log H + concentration).
  • the chemical moiety comprises biotin, streptavidin or related analogues that have affinity with biotin, phenylboronic acid, salicylhydroxamic acid, an antibody, or an antigen.
  • the dideoxynucleotide comprises a cytosine or a thymine with a 5-position, or an adenine or a guanine with a 7-position, and the linker is attached to the 5-position of cytosine or thymine or to the 7-position of adenine or guanine.
  • the invention provides for the use of any of the linkers described herein in single nucleotide polymorphism detection using mass spectrometry, wherein the linker increases mass separation between different dideoxynucleotides and increases mass spectrometry resolution.
  • Labeled dideoxynucleotides which comprise a chemical moiety attached via a linker to a 5-position of cytosine or thymine or to a 7-position of adenine or guanine.
  • the dideoxynucleotide is selected from the group consisting of 2′,3′-dideoxyadenosine 5′-triphosphate (ddATP), 2′,3′-dideoxyguanosine 5′-triphosphate (ddGTP), 2′,3′-dideoxycytidine 5′-triphosphate (ddCTP), and 2′,3′-dideoxythymidine 5′-triphosphate (ddTTP).
  • ddATP 2′,3′-dideoxyadenosine 5′-triphosphate
  • ddGTP 2′,3′-dideoxyguanosine 5′-triphosphate
  • ddCTP 2′,3′-dideoxycytidine 5′-triphosphate
  • ddTTP 2′,3′-dideoxythymidine 5′-triphosphate
  • the linker can comprise a chain structure, or a structure comprising one or more rings, or a structure comprising a chain and one or more rings.
  • the linker is cleavable by a means selected from the group consisting of one or more of a physical means, a chemical means, a physical chemical means, heat, and light.
  • the linker is cleavable by ultraviolet light.
  • the linker is cleavable by ammonium hydroxide, formamide, or a change in pH -log [H + concentration].
  • the chemical moiety comprises biotin, streptavidin, phenylboronic acid, salicylhydroxamic acid, an antibody, or an antigen.
  • the labeled dideoxynucleotide is selected from the group consisting of:
  • the labeled dideoxynucleotide is selected from the group consisting of:
  • the labeled dideoxynucleotide is selected from the group consisting of: wherein ddNTP1, ddNTP2T ddNTP3, and ddNTP4 represent four different dideoxynucleotides, or their analogues.
  • the labeled dideoxynucleotide is selected from the group consisting of:
  • the labeled dideoxynucleotide has a molecular weight of 844, 977, 1,017, or 1,051. In one embodiment, the labeled dideoxynucleotide has a molecular weight of 1,049, 1,182, 1,222, or 1,257. Other molecular weights with sufficient mass differences to allow resolution in mass spectrometry can also be used.
  • the mass spectrometry is matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.
  • a system for separating a chemical moiety from other components in a sample in solution, which comprises:
  • the interaction between the chemical moiety and the compound coating the surface is a biotin-streptavidin interaction, a phenylboronic acid-salicylhydroxamic acid interaction, or an antigen-antibody interaction.
  • the chemical moiety is a biotinylated moiety and the channel is a streptavidin-coated silica glass channel.
  • the biotinylated moiety is a biotinylated DNA fragment.
  • the chemical moiety can be freed from the surface by disrupting the interaction between the chemical moiety and the compound coating the surface.
  • the interaction can be disrupted by a means selected from the group consisting of one or more of a physical means, a chemical means, a physical chemical means, heat, and light.
  • the interaction can be disrupted by ammonium hydroxide, formamide, or a change in pH -log [H + concentration].
  • the chemical moiety is attached via a linker to another chemical compound.
  • the other chemical compound is a DNA fragment.
  • the linker is cleavable by a means selected from the group consisting of one or more of a physical means, a chemical means, a physical chemical means, heat, and light.
  • the channel is transparent to ultraviolet light and the linker is cleavable by ultraviolet light. Cleaving the linker frees the DNA fragment or other chemical compound from the chemical moiety which remains captured on the surface.
  • Multi-channel systems comprise a plurality of any of the single channel systems disclosed herein.
  • the channels are in a chip.
  • the multi-channel system comprises 96 channels in a chip. Chips can also be used with fewer or greater than 96 channels.
  • the invention provides for the use of any of the separation systems described herein for single nucleotide polymorphism detection.
  • DNA templates containing the polymorphic sites for the human hereditary hemochromatosis gene HFE were amplified from genomic DNA in a total volume of 10 ⁇ l, that contains 20 ng of genomic DNA, 500 pmol each of forward (C282Y; 5′-CTACCCCCAGAACATCACC-3′ (SEQ ID NO: 1), H63D; 5′-GCACTACCTCTTCATGGGTGCC-3′ (SEQ ID NO: 2)) and reverse (C282Y; 5′-CATCAGTCACATACCCCA-3′ (SEQ ID NO: 3), H63D; 5′-CAGTGAACATGTGATCCCACCC-3′ (SEQ ID NO: 4)) primers, 25 ⁇ M dNTPs (Amersham Biosciences, Piscataway, N.J.), 1 U Tag polymerase (Life Technologies, Rockville, Md.), and 1 ⁇ PCR buffer (50 mM KCl, 1.5 mM MgCl 2 , 10 mM Tris-
  • PCR amplification reactions were started at 94° C. for 4 min, followed by 45 cycles of 94° C. for 30 s, 59° C. for 30 s and 72° C. for 10 s, and finished with an additional extension step of 72° C. for 6 min.
  • Excess primers and dNTPs were degraded by adding 2 U shrimp alkaline phosphatase (Roche Diagnostics, Indianapolis, Ind.) and E. Coli exonuclease I (Boehringer Mannheim, Indianapolis, Ind.) in 1 ⁇ shrimp alkaline phosphatase buffer. The reaction mixture was incubated at 37° C. for 45 min followed by enzyme inactivation at 95° C. for 15 min.
  • SBE reactions contained 20 pmol of primer, 10 pmol of biotin-11-ddATP, 20 pmol of biotin-11-ddGTP, 40 pmol of biotin-11-ddCTP (New England Nuclear Life Science, Boston, Mass.), 80 pmol of biotin-16-ddUTP (Enzo Diagnostics, Inc., Farmingdale, N.Y.), 2 ⁇ l Thermo Sequenase reaction buffer, 1 U Thermo Sequenase in its diluted buffer (Amersham Biosciences) and 20 pmol of either synthetic template or 10 ⁇ l PCR product in a total reaction volume of 20 ⁇ l.
  • the beads were washed twice with modified B/W buffer, twice with 0.2 M triethyl ammonium acetate (TEAA) buffer and twice with deionized water.
  • the primer extension products were released from the magnetic beads by treatment with 8 ⁇ l 98% formamide solution containing 2% 0.2 M TEAA buffer at 94° C. for 5 min.
  • the released primer extension products were precipitated with 100% ethanol at 4° C. for 30 min, and centrifuged at 4° C. and 14000 RPM for 35 min.
  • the purified primer extension is products were dried and re-suspended in 1 ⁇ l deionized water and 2 ⁇ l matrix solution.
  • the matrix solution was made by dissolving 35 mg of 3-hydroxypicolinic acid (3-HPA; Aldrich, Milwaukee, Wis.) and 6 mg of ammonium citrate (Aldrich) in 0.8 ml of 50% acetonitrile. 10 pmol internal mass standard in 1 ⁇ l of 50% acetonitrile was then added to the sample.
  • biotin-ddNTPs Solid phase capturable biotinylated dideoxynucleotides
  • MS mass spectrometry
  • oligonucleotide primers that have different molecular weights and that are specific to the polymorphic sites in the DNA template are extended with biotin-ddNTPs by DNA polymerase to generate 3′-biotinylated DNA extension products ( FIG. 1 ). These products are then captured by streptavidin-coated solid phase magnetic beads, while the unextended primers and other components in the reaction are washed away.
  • the pure extension DNA products are subsequently released from the solid phase and analyzed with matrix-assisted laser desorption/ionization time-of-flight MS.
  • the mass of the extension DNA products is determined using a stable oligonucleotide as a common internal mass standard. Since only the pure extension DNA products are introduced to MS for analysis, the resulting mass spectrum is free of non-extended primer peaks and their associated dimers, which increases the accuracy and scope of multiplexing in single nucleotide polymorphism (SNP) analysis.
  • SNP single nucleotide polymorphism
  • the solid phase purification approach also facilitates desalting of the captured oligonucleotides, which is essential for accurate mass measurement by MS.
  • biotin-ddNTPs with distinct molecular weights were selected to generate extension products that have a two-fold increase in mass difference compared to that with conventional ddNTPs. This increase in mass difference provides improved resolution and accuracy in detecting heterozygotes in the mass spectrum.
  • affinity systems other than biotin-streptavidin can be used.
  • affinity systems include but are not limited to phenylboronic acid-salicylhydroxamic acid (31) and antigen-antibody systems.
  • the multiplex genotyping approach was validated by detecting six nucleotide variations from synthetic DNA templates that mimic mutations in exons 7 and 8 of the p53 gene. Sequences of the templates and the corresponding primers are shown in Table 1 along with the masses of the primers and their extension products. The mass increase of the resulting single base extension products in comparison with the primers is 665 Da for addition of biotin-ddCTP, 688 Da for addition of biotin-ddATP, 704 Da for addition of biotin-ddGTP and 754 Da for addition of biotin-ddUTP. The mass data in Table 1 indicate that the smallest mass difference among any possible extensions of a primer is 16 Da (between biotin-ddATP and biotin-ddGTP).
  • MALDI-TOF MS in comparison to other detection techniques is its ability to simultaneously measure masses of DNA fragments over a certain range.
  • HHC human hereditary hemochromatosis
  • nucleotide analogues (28) and peptide nucleic acid (9) can be used in the construction of the oligonucleotide primers. It has been shown that MALDI-TOF MS could detect DNA fragments up to 100 bp with sufficient resolution (29). The mass difference between each adjacent DNA fragment is approximately 300 Da. Thus, with a mass difference of 100 Da for each primer in designing a multiplex SNP analysis project, at least 300 SNPs can be analyzed in a single spot of the sample plate by MS. It is a routine method now to place 384 spots in each sample plate in MS analysis.
  • each plate can produce over 100,000 SNPs, which is roughly the entire SNPs in all the coding regions of the human genome.
  • This level of multiplexing should be achievable by mass tagging the primers with stable chemical groups in SBE using biotin-ddNTPs.
  • a master database of primers and the resulting masses of all four possible extension products can be constructed.
  • the experimental data from MALDI-TOF MS can then be compared with this database to precisely identify the library of SNPs automatically.
  • This method coupled with future improvements in mass spectrometer detector sensitivity (30) will provide a platform for high-throughput SNP identification unrivaled in speed and accuracy.
  • the ability to distinguish various bases in DNA using mass spectrometry is dependent on the mass differences of the bases in the spectra.
  • the smallest difference in mass between any two nucleotides is 16 daltons (see Table 1).
  • Fei et al. (15) have shown that using dye-labeled ddNTP paired with a regular dNTP to space out the mass difference, an increase in the detection resolution in a single nucleotide extension assay can be achieved.
  • the current application discloses systematic modification of the biotinylated dideoxynucleotides by incorporating mass linkers assembled using 4-aminomethyl benzoic acid derivatives to increase the mass separation of the individual bases.
  • the mass linkers can be modified by incorporating one or two fluorine atoms to further space out the mass differences between the nucleotides.
  • the structures of four biotinylated ddNTPs are shown in FIG. 3 .
  • ddCTP-11-biotin is commercially available (New England Nuclear, Boston).
  • ddTTP-Linker I-11-Biotin, ddATP-Linker II-11-Biotin and ddGTP-Linker III-11-Biotin are synthesized as shown, for example, for ddATP-Linker II-11-Biotin in FIG. 5 .
  • the linkers are attached to the 5-position on the pyrimidine bases (C and T), and to the 7-position on the purines (A and G) for subsequent conjugation with biotin. It has been established that modification of these positions on the bases in the nucleotides, even with bulky energy transfer fluorescent dyes, still allows efficient incorporation of the modified nucleotides into the DNA strand by DNA polymerase (32, 33).
  • the ddNTPs-Linker-11-biotin can be incorporated into the growing strand by the polymerase in DNA sequencing reactions. Larger mass separations will greatly aid in longer read lengths where signal intensity is smaller and resolution is lower.
  • Linker I Three 4-aminomethyl benzoic acid derivatives Linker I, Linker II and Linker III are designed as mass tags as well as linkers-for bridging biotin to the corresponding dideoxynucleotides.
  • the synthesis of Linker II ( FIG. 4 ) is described here to illustrate the synthetic procedure.
  • 3-Fluoro-4-aminomethyl benzoic acid that can be easily prepared via published procedures (41, 42) is first protected with trifluoroacetic anhydride, then converted to N-hydroxysuccinimide (NHS) ester with disuccinimidylcarbonate in the presence of diisopropylethylamine.
  • NHS ester is subsequently coupled with commercially available propargylamine to form the desired compound, Linker II.
  • Linker I and Linker III can be easily constructed.
  • FIG. 5 describes the scheme required to prepare biotinylated ddATP-Linker II-11-Biotin using well-established procedures (34-36).
  • 7-I-ddA is coupled with linker II in the presence of tetrakis(triphenylphosphine) palladium(0) to produce 7-Linker II-ddA, which is phosphorylated with POCl 3 in butylammonium pyrophosphate (37).
  • 7-Linker II-ddATP is produced, which then couples with sulfo-NHS-LC-Biotin (Pierce, Rockford Ill.) to yield the desired ddATP-Linker II-11-Biotin.
  • ddTTP-Linker I-11-Biotin, and ddGTP-Linker III-11-Biotin can be synthesized.
  • FIG. 6 A schematic of capture and cleavage of the photocleavable linker on the streptavidin coated porous surface is shown in FIG. 6 .
  • the reaction mixture consists of excess primers, enzymes, salts, false stops, and the desired DNA fragment.
  • This reaction mixture is passed over a streptavidin-coated surface and allowed to incubate.
  • the biotinylated fragments are captured by the streptavidin surface, while everything else in the mixture is washed away. Then the fragments are released into solution by cleaving the photocleavable linker with near ultraviolet (UV) light, while the biotin remains attached to the streptavidin that is covalently bound to the surface.
  • UV near ultraviolet
  • the pure DNA fragments can then be crystallized in matrix solution and analyzed by mass spectrometry. It is advantageous to cleave the biotin moiety since it contains sulfur which has several relatively abundant isotopes.
  • the rest of the DNA fragments and linkers contain only carbon, nitrogen, hydrogen, oxygen, fluorine and phosphorous, whose dominant isotopes are found with a relative abundance of 99% to 100%. This allows high resolution mass spectra to be obtained.
  • the photocleavage mechanism (38, 39) is shown in FIG. 7 . Upon irradiation with ultraviolet light at 300-350 nm, the light sensitive o-nitroaromatic carbonamide functionality on DNA fragment 1 is cleaved, producing DNA fragment 2, PC-biotin and carbon dioxide. The partial chemical linker remaining on DNA fragment 2 is stable for detection by mass spectrometry.
  • ddCTP-PC-Biotin Four new biotinylated ddNTPs disclosed here, ddCTP-PC-Biotin, ddTTP-Linker I-PC-Biotin, ddATP-Linker II-PC-Biotin and ddGTP-Linker III-PC-Biotin are shown in FIG. 8 . These compounds are synthesized by a similar chemistry as shown for the synthesis of ddATP-Linker II-11-Biotin in FIG. 6 . The only difference is that in the final coupling step NHS-PC-LC-Biotin (Pierce, Rockford Ill.) is used, as shown in FIG. 9 .
  • the photocleavable linkers disclosed here allow the use of solid phase capturable terminators and mass spectrometry to be turned into a high throughput technique for DNA analysis.
  • the DNA fragment is terminated with a dideoxynucleoside monophosphate (ddNMP).
  • the ddNMP is attached via a linker to a chemical moiety (“X” in FIG. 10 ).
  • the DNA fragment terminated with ddNMP is captured on the surface through interaction between chemical moiety “X” and a compound on or attached to the surface (“Y” in FIG. 10 ).
  • the present application discloses two methods for freeing the captured DNA fragment terminated with ddNMP. In the situation illustrated in the lower part of FIG. 10 , the DNA fragment terminated with ddNMP is freed from the surface by disrupting or breaking the interaction between chemical moiety “X” and compound “Y”. In the upper part of FIG. 10 , the DNA fragment terminated with ddNMP is attached to chemical moiety “X” via a cleavable linker which can be cleaved to free the DNA fragment terminated with ddNMP.
  • X-“Y” affinity system which include but are not limited to, biotin-streptavidin, phenylboronic acid-salicylhydroxamic acid (31), and antigen-antibody systems.
  • the cleavable linker can be cleaved and the “X”-“Y” interaction can be disrupted by a means selected from the group consisting of one or more of a physical means, a chemical means, a physical chemical means, heat, and light.
  • a means selected from the group consisting of one or more of a physical means, a chemical means, a physical chemical means, heat, and light.
  • ultraviolet light can be used to cleave the cleavable linker.
  • Chemical means include, but are not limited to, ammonium hydroxide (40), formamide, or a change in pH (-log H + concentration) of the solution.
  • Streptavidin coated magnetic beads are not ideal for using the photocleavable biotin capture and release process for DNA fragments, since they are not transparent to UV light. Therefore, the photocleavage reaction is not efficient.
  • a high-density surface coated with streptavidin is essential. It is known that the commercially available 96-well streptavidin coated plates cannot provide a sufficient surface area for efficient capture of the biotinylated DNA fragments. Disclosed in this application is a porous silica channel system designed to-overcome this limitation.
  • porous channels are coated with a high density of streptavidin.
  • ninety-six (96) porous silica glass channels can be etched into a silica chip ( FIG. 11 ).
  • the surfaces of the channels are modified to contain streptavidin as shown in FIG. 12 .
  • the channel is first treated with 0.5 M NaOH, washed with water, and then briefly pre-etched with dilute hydrogen fluoride. Upon cleaning with water, the capillary channel is coated with high density 3-aminopropyltrimethoxysilane in aqueous ethanol (43).
  • FIG. 11 A 96-well plate that can be used with biotinylated terminators for DNA analysis is shown in FIG. 11 .
  • each end of a channel is connected to a single well.
  • the end of a channel could be connected to a plurality of wells.
  • Pressure is applied to drive the samples through a glass capillary into the channels on the chip. Inside the channels the biotin is captured by the covalently bound streptavidin. After passing through the channel, the sample enters into a clean plate in the other end of the chip. Pressure applied in reverse drives the sample through the channel multiple times and ensures a highly efficient solid phase capture. Water is similarly added to drive out the reaction mixture and thoroughly wash the captured fragments.
  • the chip After washing, the chip is irradiated with ultraviolet light to cleave the photosensitive linker and release the DNA fragments. The fragment solution is then driven out of the channel and into a collection plate. After matrix solution is added, the samples are spotted on a chip and allowed to crystallize for detection by MALDI-TOF mass spectrometry.
  • the purification cassette is cleaned by chemically cleaving the biotin-streptavidin linkage, and is then washed and reused.
  • the following experiments show the simultaneous genotyping of 30 nucleotide variations in the p53 gene from human tumors in one tube, by using solid phase capturable dideoxynucleotides to generate single base extension products which are detected by mass spectrometry. Both homozygous and heterozygous genotypes are accurately determined with digital resolution. This is the highest level of SNP multiplexing reported thus far using mass spectrometry, indicating the approach will have wide applications in screening a repertoire of genotypes in candidate genes as potential markers for cancer and other diseases.
  • Multiplex PCR and single base extension reactions Multiplex PCR was performed to amplify 3 regions in exons 5, 7 and 8 of the p53 gene.
  • the primers for each region were 5′-TATCTGTTCACTTGTGCCC-3′ (exon 5, forward), 5′-CAGAGGCCTGGGGA-CCCTG-3′(exon 5, reverse), 5′-CTGCTTGCCACAGGTCTC-3′(exon 7, forward), 5′-CACAGCAG-GCCAGTGTGC-3′ (exon 7, reverse), 5 1 -GGACCTGATTTCCTTAC-TG-3′ (exon 8, forward), and 5′-TGAATCTGAGGCATAACTG-3′ (exon 8, reverse).
  • the 45 1 PCR reaction consisted of 180 ng genomic DNA, 1.5 nmol dNTP, 4.5 1 10 ⁇ PCR buffer, 15 mM MgCl 2 , 4 pmol of forward and reverse primers for exons 5 and 7, 6 pmol of forward and reverse primers for exon 8, and 1.0 U of JumpStart RedAccuTaq DNA Polymerase.
  • the touchdown PCR program was performed with 10 cycles of 96° C. (30 sec), 67° C. to 57° C. ( ⁇ 1.0° C. per cycle, 30 sec) and 72° C. (30 sec), an additional 30 cycles of 96° C. (30 sec), 57° C. (30 sec) and 72° C.
  • the 30 SBE primers (Table 3) were designed to yield extension products with a sufficient mass difference and to be extended simultaneously in a single tube. Primer sequences were designed to avoid any overlap in mass, and the formation of secondary structures. To evenly separate the masses of such a large number of primers for SBE, some primers were synthesized using methyl-dC and dU phosphoramidites (Glen Research) to replace dC and dT respectively. Substitution of dC by methyl-dC increased the primer mass by 14 Da whereas a change from dT to dU decreased the mass by 14 Da. Primers were synthesized using an Applied Biosystems DNA synthesizer.
  • the SPC-SBE genotyping approach was used to analyze nucleotide variations in 30 codons of 3 exons of the p53 gene from 30 Wilms' tumors, 19 head and neck squamous carcinomas and 3 colorectal carcinomas. Primer sequences are shown in Table 3 along with the masses of the primers and their extension products. Extension products of all 30 primers were resolved in the mass spectrum, free from any unextended primers, yielding digital data to unambiguously determine each nucleotide variation ( FIGS. 13A-13C ). Unextended primers occupy the mass range in the mass spectrum decreasing the scope of multiplexing, and excess primers can dimerize to form false peaks in the mass spectrum (21).
  • the primer sequence and modification is specified and the modified nucleotides are shown in bold.
  • the mass of each primer is indicated along with the mass of all four possible SBE products.

Abstract

This invention provides methods for detecting single nucleotide polymorphisms and multiplex genotyping using dideoxynucleotides and mass spectrometry.

Description

  • This application is a continuation-in-part and claims priority of U.S. Ser. No. 10/194,882, filed Jul. 12, 2002, the contents of which are hereby incorporated by reference into this application.
  • BACKGROUND OF THE INVENTION
  • Throughout this application, various publications are referenced in parentheses. Citations for these references may be found at the end of the specification immediately preceding the claims. The disclosures of these publications in their entireties are hereby incorporated by reference into this application to more fully describe the state of the art to which this invention pertains.
  • Single nucleotide polymorphisms (SNPs), the most common genetic variations in the human genome, are important markers for identifying disease genes and for pharmacogenetic studies (1, 2). SNPs appear in the human genome with an average density of once every 1000-base pairs (3). To perform large-scale SNP genotyping, a rapid, precise and cost-effective method is required. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) (4) allows rapid and accurate sample measurements (5-7) and has been used in a variety of SNP detection methods including hybridization (8-10), invasive cleavage (11, 12) and single base extension (SBE) (5, 13-17). SBE is widely used for multiplex SNP analysis. In this method, primers designed to anneal immediately adjacent to a polymorphic site are extended by a single dideoxynucleotide that is complementary to the nucleotide at the variable site. By measuring the mass of the resulting extension product, a particular SNP can be identified. Current SBE methods to perform multiplex SNP analysis using MS require unambiguous simultaneous detection of a library of primers and their extension products. However, limitations in resolution and sensitivity of MALDI-TOF MS for longer DNA molecules make it difficult to simultaneously measure DNA fragments over a large mass range (6). The requirement to measure both primers and their extension products in this range limits the scope of multiplexing.
  • A high fidelity DNA sequencing method has been developed which uses solid phase capturable biotinylated dideoxynucleotides (biotin-ddNTPs) by detection with fluorescence (18) or mass spectrometry (19), eliminating false terminations and excess primers. Combinatorial fluorescence energy transfer tags and biotin-ddNTPs have also been used to detect SNPs (20).
  • False stops or terminations occur when a deoxynucleotide rather than a dideoxynucleotide terminates a se+quencing fragment. It has been shown that false stops and primers which have dimerized can produce peaks in the mass spectra that can mask the actual results preventing accurate base identification (21).
  • The present application discloses an approach using solid phase capturable biotin-ddNTPs in SBE for multiplex genotyping by MALDI-TOF MS. In this method primers that have different molecular weights and that are specific to the polymorphic sites in the DNA template are extended with biotin-ddNTPs by DNA polymerase to generate 3′-biotinylated DNA extension products. The 3′-biotinylated DNAs are then captured by streptavidin-coated magnetic beads, while the unextended primers and other components in the reaction are washed away. The pure DNA extension products are subsequently released from the magnetic beads, for example by denaturing the biotin-streptavidin interaction with formamide, and analyzed with MALDI-TOF MS. The nucleotide at the polymorphic site is identified by analyzing the mass difference between the primer extension product and an internal mass standard added to the purified DNA products. Since the primer extension products are isolated prior to MS analysis, the resulting mass spectrum is free of non-extended primer peaks and their associated dimers, which increases the accuracy and scope of multiplexing in SNP analysis. The solid phase purification system also facilitates desalting of the captured oligonucleotides. Desalting is critical in sample preparation for MALDI-TOF MS measurement since alkaline and alkaline earth salts can form adducts with DNA fragments that interfere with accurate peak detection (21).
  • SUMMARY OF THE INVENTION
  • This invention is directed to a method for determining the identity of a nucleotide present at a predetermined site in a DNA whose sequence immediately 3′ of such predetermined site is known which comprises:
      • (a) treating the DNA with an oligonucleotide primer whose sequence is complementary to such known sequence so that the oligonucleotide primer hybridizes to the DNA and forms a complex in which the 3′ end of the oligonucleotide primer is located immediately adjacent to the predetermined site in the DNA;
      • (b) simultaneously contacting the complex from step (a) with four different labeled dideoxynucleotides, in the presence of a polymerase under conditions permitting a labeled dideoxynucleotide to be added to the 3′ end of the primer so as to generate a labeled single base extended primer, wherein each of the four different labeled dideoxynucleotides (i) is complementary to one of the four nucleotides present in the DNA and (ii) has a molecular weight which can be distinguished from the molecular weight of the other three labeled dideoxynucleotides using mass spectrometry; and
      • (c) determining the difference in molecular weight between the labeled single base extended primer and the oligonucleotide primer so as to identify the dideoxynucleotide incorporated into the single base extended primer and thereby determine the identity of the nucleotide present at the predetermined site in the DNA.
  • In one embodiment, the method further comprises after step (b) the steps of:
      • (i) contacting the labeled single base extended primer with a surface coated with a compound that specifically interacts with a chemical moiety attached to the dideoxynucleotide by a linker so as to thereby capture the extended primer on the surface; and
      • (ii) treating the labeled single base extended primer so as to release it from the surface.
  • In one embodiment, the method further comprises after step (i) the step of treating the surface to remove primers that have not been extended by a labeled dideoxynucleotide.
  • BRIEF DESCRIPTION OF THE FIGURES
  • FIG. 1: Scheme of single base extension for multiplex SNP analysis using biotin-ddNTPs and MALDI-TOF MS. Primers that anneal immediately next to the polymorphic sites in the DNA template are extended by DNA polymerase of a biotin-ddNTP in a sequence-specific manner. After solid phase capture and isolation of the 3′-biotinylated DNA extension fragments, MALDI-TOF MS was used to analyze these DNA products to yield a mass spectrum. From the relative mass of each extended primer, compared to the mass of an internal standard, the nucleotide at the polymorphic site is identified.
  • FIG. 2. Multiplex SNP genotyping mass spectra generated using biotin-ddNTPs. Inset is a magnified view of heterozygote peaks. Masses of the extension product in reference to the internal mass standard were listed on each single base extension peak. The mass values in parenthesis indicate the mass difference between the extension products and the corresponding primers. (A) Detection of six nucleotide variations from synthetic DNA templates mimicking mutations in the p53 gene. Four homozygous (T, G, C and C) and one heterozygous (C/A) genotypes were detected. (B) Detection of two heterozygotes (A/G and C/G) in the human HFE gene.
  • FIG. 3: Structure of four mass tagged biotinylated ddNTPs. Any of the four ddNTPs (ddATP, ddCTP, ddGTP, ddTTP) can be used with any of the illustrated linkers.
  • FIG. 4: Synthesis scheme for mass tag linkers. For illustrative purposes, the linkers are labeled to correspond to the specific ddNTP with which they are shown coupled in FIGS. 3, 5, 7, 8 and 9. However, any of the three linkers can be used with any ddNTP. (i) (CF3CO)2O; (ii) Disuccinimidylcarbonate/diisopropylethylamine; (iii) Propargyl amine.
  • FIG. 5: The synthesis of ddATP-Linker-II-11-Biotin. (i) Linker II, tetrakis(triphenylphosphine) palladium(0); (ii) POCl3, Bn4N+ pyrophosphate; (iii) NH4OH; (iv) Sulfo-NHS-LC-Biotin.
  • FIG. 6: DNA products are purified by a streptavidin coated porous silica surface. Only the biotinylated fragments are captured. These fragments are then cleaved by light irradiation (hv) to release the captured fragments, leaving the biotin moiety still bound to the streptavidin.
  • FIG. 7: Mechanism for the cleavage of photocleavable linkers.
  • FIG. 8: The structures of ddNTPs linked to photocleavable (PC) biotin. Any of the four ddNTPs (ddATP, ddCTP, ddGTP, ddTTP) can be used with any of the shown linkers.
  • FIG. 9: The synthesis of ddATP-Linker-II-PC-Biotin. PC=photocleavable.
  • FIG. 10: Schematic for capturing a DNA fragment terminated with a dideoxynucleoside monophosphate on a surface. The dideoxynucleoside monophosphate (ddNMP) which is on the 3′ end of the DNA fragment is attached via a linker to a chemical moiety “X” which interacts with a compound “Y” on the surface to capture the DNA fragment terminated with the ddNMP. The DNA fragment can be freed from the surface either by disrupting the interaction between chemical moiety X and compound Y (lower scheme) or by cleaving the linker (upper scheme).
  • FIGS. 11A-11C: Schematic of a high throughput channel based purification system. Sample solutions can be pushed back and forth between the two plates through glass capillaries and the streptavidin coated channels in the chip. The whole chip can be irradiated to cleave the samples after immobilization.
  • FIG. 12: The synthesis of streptavidin coated porous surface.
  • FIGS. 13A-13C: Simultaneous detection of nucleotide variations in 30 codons of the p53 tumor suppressor gene by MALDI-TOF MS using solid phase capturable biotinylated dideoxynucleotide. Each peak represents a different polymorphism labeled with its nucleotide identity and absolute mass value. The value in parentheses, denoting the mass difference between each DNA extension product and its corresponding primer, is used to determine the nucleotide identity. (A) A mass spectrum from a Wilms' tumor sample showing 30 wild type p53 sequences. (B) A mass spectrum from a head and neck tumor (primary tumor biopsy) containing a heterozygous genotype G/T (4684/4734 Da) (boxed) in codon 157, corresponding to the wild type and mutant alleles, respectively. (C) A mass spectrum from a colorectal tumor cell line (HT-29) containing a homozygous G to A mutation (boxed) in codon 273 of the p53 gene. The colorectal tumor cell line (SW-480) contained the identical G to A mutation in codon 273.
  • FIGS. 14A-14B: (A) A mass spectrum from a head and neck tumor sample showing 30 wild type sequences of the p53 gene. (B) A mass spectrum from a head and neck tumor cell line (SCC-4) containing a homozygous C (5881 Da) to T (5970 Da) mutation (boxed) in codon 151 of the p53 gene. Both spectra were produced using the primers shown in Table 3 with primer 16 replaced by primer 5′-TGTGGGTTGATTCCACA-3′ for detecting the variation in codon 151 (C/TCC).
  • DETAILED DESCRIPTION OF THE INVENTION
  • The following definitions are presented as an aid in understanding this invention.
  • The standard abbreviations for nucleotide bases are used as follows: adenine (A), cytosine (C), guanine (G), thymine (T), and uracil (U).
  • A nucleotide analogue refers to a chemical compound that is structurally and functionally similar to the nucleotide, i.e. the nucleotide analogue can be recognized by polymerase as a substrate. That is, for example, a nucleotide analogue comprising adenine or an analogue of adenine should form hydrogen bonds with thymine, a nucleotide analogue comprising C or an analogue of C should form hydrogen bonds with G, a nucleotide analogue comprising G or an analogue of G should form hydrogen bonds with C, and a nucleotide analogue comprising T or an analogue of T should form hydrogen bonds with A, in a double helix format.
  • This invention is directed to a method for determining the identity of a nucleotide present at a predetermined site in a DNA whose sequence immediately 3′ of such predetermined site is known which comprises:
      • (a) treating the DNA with an oligonucleotide primer whose sequence is complementary to such known sequence so that the oligonucleotide primer hybridizes to the DNA and forms a complex in which the 3′ end of the oligonucleotide primer is located immediately adjacent to the predetermined site in the DNA;
      • (b) simultaneously contacting the complex from step (a) with four different labeled dideoxynucleotides, in the presence of a polymerase under conditions permitting a labeled dideoxynucleotide to be added to the 3′ end of the primer so as to generate a labeled single base extended primer, wherein each of the four different labeled dideoxynucleotides (i) is complementary to one of the four nucleotides present in the DNA and (ii) has a molecular weight which can be distinguished from the molecular weight of the other three labeled dideoxynucleotides using mass spectrometry; and
      • (c) determining the difference in molecular weight between the labeled single base extended primer and the oligonucleotide primer so as to identify the dideoxynucleotide incorporated into the single base extended primer and thereby determine the identity of the nucleotide present at the predetermined site in the DNA.
  • In one embodiment, each of the four labeled dideoxynucleotides comprises a chemical moiety attached to the dideoxynucleotide by a different linker which has a molecular weight different from that of each other linker.
  • In one embodiment, the method further comprises after step (b) the steps of:
      • (i) contacting the labeled single base extended primer with a surface coated with a compound that specifically interacts with a chemical moiety attached to the dideoxynucleotide by a linker so as to thereby capture the extended primer on the surface; and
      • (ii) treating the labeled single base extended primer so as to release it from the surface.
  • In a further embodiment, the method comprises after step (i) the step of treating the surface to remove primers that have not been extended by a labeled dideoxynucleotide and any non-captured component.
  • In one embodiment of the method step (c) comprises determining the difference in mass between the labeled single base extended primer and an internal mass calibration standard added to the extended primer. In one embodiment, the internal mass standard is 5′-TTTTTCTTTTTCT-3′ (SEQ ID NO: 5) (MW=3855 Da).
  • In one embodiment, the chemical moiety is attached via a different linker to different dideoxynucleotides. In one embodiment, the different linkers increase mass separation between different labeled single base extended primers and thereby increase mass spectrometry resolution.
  • In one embodiment, the dideoxynucleotide is selected from the group consisting of 2′,3′-dideoxyadenosine 5′-triphosphate (ddATP), 2′,3′-dideoxyguanosine 5′-triphosphate (ddGTP), 2′,3′-dideoxycytidine 5′-triphosphate (ddCTP), and 2′,3′-dideoxythymidine 5′-triphosphate (ddTTP).
  • In different embodiments of the methods described herein, the interaction between the chemical moiety attached to the dideoxynucleotide by the linker and the compound on the surface comprises a biotin-streptavidin interaction, a phenylboronic acid-salicylhydroxamic acid interaction, or an antigen-antibody interaction.
  • In one embodiment, the step of releasing the labeled single base extended primer from the surface comprises disrupting the interaction between the chemical moiety attached by the linker to the dideoxynucleotide and the compound on the surface. In different embodiments, the interaction is disrupted by a means selected from the group consisting of one or more of a physical means, a is chemical means, a physical chemical means, heat, and light. In one embodiment, the interaction is disrupted by light. In one embodiment, the interaction is disrupted by ultraviolet light. In different embodiments, the interaction is disrupted by ammonium hydroxide, formamide, or a change in pH (-log H+ concentration).
  • In different embodiments, the linker can comprise a chain structure, or a structure comprising one or more rings, or a structure comprising a chain and one or more rings. In different embodiments, the dideoxynucleotide comprises a cytosine or a thymine with a 5-position, or an adenine or a guanine with a 7-position, and the linker is attached to the dideoxynucleotide at the 5-position of cytosine or thymine or at the 7-position of adenine or guanine.
  • In different embodiments, the step of releasing the labeled single base extended primer from the surface comprises cleaving the linker between the chemical moiety and the dideoxynucleotide. In different embodiments, the linker is cleaved by a means selected from the group consisting of one or more of a physical means, a chemical means, a physical chemical means, heat, and light. In one embodiment, the linker is cleaved by light. In one embodiment, the linker is cleaved by ultraviolet light. In different embodiments, the linker is cleaved by ammonium hydroxide, formamide, or a change in pH (-log H+ concentration);
  • In one embodiment, the linker comprises a derivative of 4-aminomethyl benzoic acid. In one embodiment, the linker comprises a 2-nitrobenzyl group or a derivative of a 2-nitrobenzyl group. In one embodiment, the linker comprises one or more fluorine atoms.
  • In one embodiment, the linker is selected from the group consisting of:
    Figure US20060252038A1-20061109-C00001
  • In one embodiment, a plurality of different linkers is used to increase mass separation between different labeled single base extended primers and thereby increase mass spectrometry resolution.
  • In one embodiment, the chemical moiety comprises biotin, the labeled dideoxynucleotide is a biotinylated dideoxynucleotide, the labeled single base extended primer is a biotinylated single base extended primer, and the surface is a streptavidin-coated solid surface. In one embodiment, the biotinylated dideoxynucleotide is selected from the group consisting of ddATP-11-biotin, ddCTP-11-biotin, ddGTP-11-biotin, and ddTTP-16-biotin.
  • In one embodiment, the biotinylated dideoxynucleotide is selected from the group consisting of:
    Figure US20060252038A1-20061109-C00002
  • wherein ddNTP1, ddNTP2, ddNTF3, and ddNTP4 represent four different dideoxynucleotides, or their analogues.
  • In one embodiment, the biotinylated dideoxynucleotide is selected from the group consisting of:
    Figure US20060252038A1-20061109-C00003
  • In one embodiment, the biotinylated dideoxynucleotide is selected from the group consisting of:
    Figure US20060252038A1-20061109-C00004
  • wherein ddNTP1, ddNTP2, ddNTP3, and ddNTP4 represent four different dideoxynucleotides or their analogues.
  • In one embodiment, the biotinylated dideoxynucleotide is selected from the group consisting of:
    Figure US20060252038A1-20061109-C00005
  • In one embodiment, the streptavidin-coated solid surface is a streptavidin-coated magnetic bead or a streptavidin-coated silica glass.
  • In one embodiment of the method, steps (a) and (b) are performed in a single container or in a plurality of connected containers.
  • The invention provides methods for determining the identity of nucleotides present at a plurality of predetermined sites, which comprises carrying out any of the methods disclosed herein using a plurality of different primers each having a molecular weight different from that of each other primer, wherein a different primer hybridizes adjacent to a different predetermined site. In one embodiment, different linkers each having a molecular weight different from that of each other linker are attached to the different dideoxynucleotides to increase mass separation between different labeled single base extended primers and thereby increase mass spectrometry resolution.
  • In one embodiment, the mass spectrometry is matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.
  • Linkers are provided for attaching a chemical moiety to a dideoxynucleotide, wherein the linker comprises a derivative of 4-aminomethyl benzoic acid.
  • In one embodiment, the dideoxynucleotide is selected from the group consisting of 2′,3′-dideoxyadenosine 5′-triphosphate (ddATP), 2′,3′-dideoxyguanosine 5′-triphosphate (ddGTP), 2′,3′-dideoxycytidine 5′-triphosphate (ddCTP), and 2′,3′-dideoxythymidine 5′-triphosphate (ddTTP).
  • In one embodiment, the linker comprises one or more fluorine atoms.
  • In one embodiment, the linker is selected from the group consisting of:
    Figure US20060252038A1-20061109-C00006
  • In different embodiments, the linker can comprise a chain structure, or a structure comprising one or more rings, or a structure comprising a chain and one or more rings.
  • In different embodiments, the linker is cleavable by a means selected from the group consisting of one or more of a physical means, a chemical means, a physical chemical means, heat, and light. In one embodiment, the linker is cleavable by ultraviolet light. In different embodiments, the linker is cleavable by ammonium hydroxide, formamide, or a change in pH (-log H+ concentration).
  • In different embodiments of the linker, the chemical moiety comprises biotin, streptavidin or related analogues that have affinity with biotin, phenylboronic acid, salicylhydroxamic acid, an antibody, or an antigen.
  • In different embodiments, the dideoxynucleotide comprises a cytosine or a thymine with a 5-position, or an adenine or a guanine with a 7-position, and the linker is attached to the 5-position of cytosine or thymine or to the 7-position of adenine or guanine.
  • The invention provides for the use of any of the linkers described herein in single nucleotide polymorphism detection using mass spectrometry, wherein the linker increases mass separation between different dideoxynucleotides and increases mass spectrometry resolution.
  • Labeled dideoxynucleotides are provided which comprise a chemical moiety attached via a linker to a 5-position of cytosine or thymine or to a 7-position of adenine or guanine.
  • In one embodiment, the dideoxynucleotide is selected from the group consisting of 2′,3′-dideoxyadenosine 5′-triphosphate (ddATP), 2′,3′-dideoxyguanosine 5′-triphosphate (ddGTP), 2′,3′-dideoxycytidine 5′-triphosphate (ddCTP), and 2′,3′-dideoxythymidine 5′-triphosphate (ddTTP).
  • In different embodiments, the linker can comprise a chain structure, or a structure comprising one or more rings, or a structure comprising a chain and one or more rings. In different embodiments, the linker is cleavable by a means selected from the group consisting of one or more of a physical means, a chemical means, a physical chemical means, heat, and light. In one embodiment, the linker is cleavable by ultraviolet light. In different embodiments, the linker is cleavable by ammonium hydroxide, formamide, or a change in pH -log [H+ concentration].
  • In different embodiments of the labeled dideoxynucleotide, the chemical moiety comprises biotin, streptavidin, phenylboronic acid, salicylhydroxamic acid, an antibody, or an antigen.
  • In one embodiment, the labeled dideoxynucleotide is selected from the group consisting of:
    Figure US20060252038A1-20061109-C00007
      • wherein ddNTP1, ddNTP2, ddNTP3, and ddNTP4 represent four different dideoxynucleotides, or their analogues.
  • In one embodiment, the labeled dideoxynucleotide is selected from the group consisting of:
    Figure US20060252038A1-20061109-C00008
  • In one embodiment, the labeled dideoxynucleotide is selected from the group consisting of:
    Figure US20060252038A1-20061109-C00009

    wherein ddNTP1, ddNTP2T ddNTP3, and ddNTP4 represent four different dideoxynucleotides, or their analogues.
  • In one embodiment, the labeled dideoxynucleotide is selected from the group consisting of:
    Figure US20060252038A1-20061109-C00010
  • In one embodiment, the labeled dideoxynucleotide has a molecular weight of 844, 977, 1,017, or 1,051. In one embodiment, the labeled dideoxynucleotide has a molecular weight of 1,049, 1,182, 1,222, or 1,257. Other molecular weights with sufficient mass differences to allow resolution in mass spectrometry can also be used.
  • In one embodiment the mass spectrometry is matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.
  • A system is provided for separating a chemical moiety from other components in a sample in solution, which comprises:
      • (a) a channel coated with a compound that specifically interacts with the chemical moiety at the 3′ end of the DNA fragment, wherein the channel comprises a plurality of ends;
      • (b) a plurality of wells each suitable for holding the sample;
      • (c) a connection between each end of the channel and a well; and
      • (d) a means for moving the sample through the channel between wells.
  • In one embodiment of the system, the interaction between the chemical moiety and the compound coating the surface is a biotin-streptavidin interaction, a phenylboronic acid-salicylhydroxamic acid interaction, or an antigen-antibody interaction.
  • In one embodiment, the chemical moiety is a biotinylated moiety and the channel is a streptavidin-coated silica glass channel. In one embodiment, the biotinylated moiety is a biotinylated DNA fragment.
  • In one embodiment, the chemical moiety can be freed from the surface by disrupting the interaction between the chemical moiety and the compound coating the surface. In different embodiments, the interaction can be disrupted by a means selected from the group consisting of one or more of a physical means, a chemical means, a physical chemical means, heat, and light. In different embodiments, the interaction can be disrupted by ammonium hydroxide, formamide, or a change in pH -log [H+ concentration].
  • In one embodiment, the chemical moiety is attached via a linker to another chemical compound. In one embodiment, the other chemical compound is a DNA fragment. In one embodiment, the linker is cleavable by a means selected from the group consisting of one or more of a physical means, a chemical means, a physical chemical means, heat, and light. In one embodiment, the channel is transparent to ultraviolet light and the linker is cleavable by ultraviolet light. Cleaving the linker frees the DNA fragment or other chemical compound from the chemical moiety which remains captured on the surface.
  • Multi-channel systems are provided which comprise a plurality of any of the single channel systems disclosed herein. In one embodiment, the channels are in a chip. In one embodiment, the multi-channel system comprises 96 channels in a chip. Chips can also be used with fewer or greater than 96 channels.
  • The invention provides for the use of any of the separation systems described herein for single nucleotide polymorphism detection.
  • This invention will be better understood from the Experimental Details which follow. However, one skilled in the art will readily appreciate that the specific methods and results discussed are merely illustrative of the invention as described more fully in the claims which follow thereafter.
  • Experimental Details
  • Experimental Set I
  • A. Materials and Methods
  • PCR amplification. DNA templates containing the polymorphic sites for the human hereditary hemochromatosis gene HFE were amplified from genomic DNA in a total volume of 10 μl, that contains 20 ng of genomic DNA, 500 pmol each of forward (C282Y; 5′-CTACCCCCAGAACATCACC-3′ (SEQ ID NO: 1), H63D; 5′-GCACTACCTCTTCATGGGTGCC-3′ (SEQ ID NO: 2)) and reverse (C282Y; 5′-CATCAGTCACATACCCCA-3′ (SEQ ID NO: 3), H63D; 5′-CAGTGAACATGTGATCCCACCC-3′ (SEQ ID NO: 4)) primers, 25 μM dNTPs (Amersham Biosciences, Piscataway, N.J.), 1 U Tag polymerase (Life Technologies, Rockville, Md.), and 1× PCR buffer (50 mM KCl, 1.5 mM MgCl2, 10 mM Tris-HCl). PCR amplification reactions were started at 94° C. for 4 min, followed by 45 cycles of 94° C. for 30 s, 59° C. for 30 s and 72° C. for 10 s, and finished with an additional extension step of 72° C. for 6 min. Excess primers and dNTPs were degraded by adding 2 U shrimp alkaline phosphatase (Roche Diagnostics, Indianapolis, Ind.) and E. Coli exonuclease I (Boehringer Mannheim, Indianapolis, Ind.) in 1× shrimp alkaline phosphatase buffer. The reaction mixture was incubated at 37° C. for 45 min followed by enzyme inactivation at 95° C. for 15 min.
  • Single base extension using biotin-ddNTPs. The synthetic DNA templates containing six nucleotide variations in p53 gene and the five primers for detecting these variations are shown in Table 1. These oligonucleotides and an internal mass standard (5′-TTTTTCTTTTTCT-3′ (SEQ ID NO: 5), MW=3855 Da) for MALDI-TOF MS measurement were made using an Expedite nucleic acid synthesizer (Applied Biosystems, Foster City, Calif.). SBE reactions contained 20 pmol of primer, 10 pmol of biotin-11-ddATP, 20 pmol of biotin-11-ddGTP, 40 pmol of biotin-11-ddCTP (New England Nuclear Life Science, Boston, Mass.), 80 pmol of biotin-16-ddUTP (Enzo Diagnostics, Inc., Farmingdale, N.Y.), 2 μl Thermo Sequenase reaction buffer, 1 U Thermo Sequenase in its diluted buffer (Amersham Biosciences) and 20 pmol of either synthetic template or 10 μl PCR product in a total reaction volume of 20 μl. For SBE using synthetic template 1, 10 pmol of both wild type and mutated templates were combined with 20 pmol of primers 1 and 3 or 20 pmol of primers 2 and 4. The SBE reaction of primer 5 was performed with template 2 in a separate tube. PCR products from the HFE gene were mixed with 20 pmol of the corresponding primers 5′-GGGGAAGAGCAGAGATATACGT-3′ (SEQ ID NO: 6) (C282Y) and 5′-GGGGCTCCACACGGCGACTCTC-AT-3′ (SEQ ID NO: 7) (H63D) in SBE to detect the two heterozygous genotypes. All extension reactions were thermalcycled for 35 cycles at 94° C. for 10 s and 49° C. for 30 s.
  • Solid phase purification. 20 μl of the streptavidin-coated magnetic beads (Seradyn, Ramsey, Minn.) were washed with modified binding and washing (B/W) buffer (0.5 mM Tris-HCl buffer, 2 M NH4Cl, 1 mM EDTA, pH 7.0) and resuspended in 20 μl modified B/W buffer. Extension reaction mixtures of primers 1-4 with template 1 and primer 5 with template 2 were mixed in a 2:1 ratio, while extension reaction mixtures from the PCR products of HFE gene were mixed in equal amounts. 20 μl of each mixed extension product was added to the suspended beads and incubated for 1 hour. After capture, the beads were washed twice with modified B/W buffer, twice with 0.2 M triethyl ammonium acetate (TEAA) buffer and twice with deionized water. The primer extension products were released from the magnetic beads by treatment with 8 μl 98% formamide solution containing 2% 0.2 M TEAA buffer at 94° C. for 5 min. The released primer extension products were precipitated with 100% ethanol at 4° C. for 30 min, and centrifuged at 4° C. and 14000 RPM for 35 min.
  • MALDI-TOF MS analysis. The purified primer extension is products were dried and re-suspended in 1 μl deionized water and 2 μl matrix solution. The matrix solution was made by dissolving 35 mg of 3-hydroxypicolinic acid (3-HPA; Aldrich, Milwaukee, Wis.) and 6 mg of ammonium citrate (Aldrich) in 0.8 ml of 50% acetonitrile. 10 pmol internal mass standard in 1 μl of 50% acetonitrile was then added to the sample. 0.5 μl of this mixture containing the primer extension products and internal standard was spotted on a stainless steel sample plate, air-dried and analyzed using an Applied Biosystems Voyager DE Pro MALDI-TOF mass spectrometer. All measurements were taken in linear positive ion mode with a 25 kV accelerating voltage, a 94% grid voltage and a 350 ns delay time. The obtained spectra were processed using the Voyager data analysis package.
  • B. Detection of Single Nucleotide Polymorphism Using Biotinylated Dideoxynucleotides and Mass Spectrometry
  • Solid phase capturable biotinylated dideoxynucleotides (biotin-ddNTPs) were used in single base extension for multiplex genotyping by mass spectrometry (MS). In this method, oligonucleotide primers that have different molecular weights and that are specific to the polymorphic sites in the DNA template are extended with biotin-ddNTPs by DNA polymerase to generate 3′-biotinylated DNA extension products (FIG. 1). These products are then captured by streptavidin-coated solid phase magnetic beads, while the unextended primers and other components in the reaction are washed away. The pure extension DNA products are subsequently released from the solid phase and analyzed with matrix-assisted laser desorption/ionization time-of-flight MS. The mass of the extension DNA products is determined using a stable oligonucleotide as a common internal mass standard. Since only the pure extension DNA products are introduced to MS for analysis, the resulting mass spectrum is free of non-extended primer peaks and their associated dimers, which increases the accuracy and scope of multiplexing in single nucleotide polymorphism (SNP) analysis. The solid phase purification approach also facilitates desalting of the captured oligonucleotides, which is essential for accurate mass measurement by MS.
  • Four biotin-ddNTPs with distinct molecular weights were selected to generate extension products that have a two-fold increase in mass difference compared to that with conventional ddNTPs. This increase in mass difference provides improved resolution and accuracy in detecting heterozygotes in the mass spectrum.
  • The “lock and key” functionality of biotin and streptavidin is often utilized in biological sample preparation as a way to remove undesired impurities (23). In different embodiments of the methods described herein, affinity systems other than biotin-streptavidin can be used. Such affinity systems include but are not limited to phenylboronic acid-salicylhydroxamic acid (31) and antigen-antibody systems.
  • The multiplex genotyping approach was validated by detecting six nucleotide variations from synthetic DNA templates that mimic mutations in exons 7 and 8 of the p53 gene. Sequences of the templates and the corresponding primers are shown in Table 1 along with the masses of the primers and their extension products. The mass increase of the resulting single base extension products in comparison with the primers is 665 Da for addition of biotin-ddCTP, 688 Da for addition of biotin-ddATP, 704 Da for addition of biotin-ddGTP and 754 Da for addition of biotin-ddUTP. The mass data in Table 1 indicate that the smallest mass difference among any possible extensions of a primer is 16 Da (between biotin-ddATP and biotin-ddGTP). This is a substantial increase over the smallest mass difference between extension products using standard ddNTPs (9 Da between ddATP and ddTTP). This mass increase yields improved resolution of the peaks in the mass spectrum. Increased mass difference in ddNTPs fosters accurate detection of heterozygous genotypes (15), since an A/T heterozygote with a mass difference of 9 Da using conventional ddNTPs can not be well resolved in the MALDI-TOF mass spectra. The five primers for each polymorphic site were designed to produce extension products without overlapping masses. Primers extended by biotin-ddNTPs were purified and analyzed by MALDI-TOF MS according to the scheme in FIG. 1. Extension products of all five primers were well-resolved in the mass spectrum free from any unextended primers (FIG. 2A), allowing each nucleotide variation to be unambiguously identified. Unextended primers occupy the mass range in the mass spectrum decreasing the scope of multiplexing, and excess primers can dimerize to form false peaks in the mass spectrum (21). The excess primers and their associated dimers also compete for the ion current, reducing the detection sensitivity of MS for the desired DNA fragments. These complications were completely removed by carrying out SBE using biotin-ddNTPs and solid phase capture. Extension products for all four biotin-ddNTPs were clearly detected with well resolved mass values. The relative masses of the primer extension products in comparison to the internal mass standard revealed the identity of each nucleotide at the polymorphic site. In the case of heterozygous genotypes, two peaks, one corresponding to each allele (C/A), are clearly distinguishable in the mass spectrum shown in FIG. 2A.
    TABLE 1
    Oligonucleotide primers and synthetic DNA templates for detecting mutations
    in the p53 gene.
    Masses of single base
    extension products (Da)
    Biotin- Biotin- Biotin- Biotin-
    Masses ddcTP ddATP ddGTP ddUTP
    Primers Primer sequences (Da) Δ665 Δ688 Δ704 Δ754
    1 5′-AGAGGATCCAACCGAGAC-3′ 1656 2321 2344 2360 2410
    2 5′-TGGTGGTAGGTGATGTTGATGTA-3′ 3350 4015 4038 4054 4103
    3 5′-CACATTGTCAAGGACGTACCCG-3′ 2833 3498 3521 3538 3587
    4 5′-TACCCGCCGTACTTGGCCTC-3′ 2134 2799 2822 2838 2480
    5 5′-TCCACGCACAAACACGGACAG-3′ 2507 3172 3195 3211 3261
    Templates Template sequences
    1 5′-TACCCG/TGAGGCCAAGTACGGCGGGTACGTCCTTGACAATGTGTACATCAACATCACCTACCA
    CCATGTCAGTCTCGGTTGGATCCTCTATTGTGTCCGGG-3′
    (SEQ ID NO:13)
    2 GAAGGAGACACGCGGCCAGAGAGGGTCCTGTCCGTGTTTGTGCGTGGAGTTTCGACAAGGCAGGGTC
    ATCTAATGGTGATGAGTCCTATCCTTTTCTCTTCGTTCTCCGT-3′
    (SEQ ID NO:14)

    (Top) The sequences and the calculated masses of primers and the four possible single base extension products relative to the internal mass standard are listed. The bold numbers refer to the nucleotide variations detected in the p53 gene. (Bottom) The six nucleotide variations in template 1 and 2 are shown in bold letters. Template 1 contains a heterozygous genotype (G/T). Primers 1-5 = SEQ ID NOs: 8-12, respectively.
  • One advantage of MALDI-TOF MS in comparison to other detection techniques is its ability to simultaneously measure masses of DNA fragments over a certain range.
  • In order to explore this feature to detect multiple SNPs in a single spectrum, if unextended primers are not removed, masses of all primers and their extension products must have sufficient differences to yield adequately resolved peaks in the mass spectrum. Ross et al. simultaneously detected multiple SNPs by carefully tuning the masses of all primers and extension products so that they would lie in the range of 4.5 kDa and 7.6 kDa without overlapping (14). Since the unextended primers occupy the mass range in the mass spectrum, by eliminating them, the approach disclosed herein will significantly increase the scope of multiplexing in SNP analysis.
  • To demonstrate the ability of this method to discriminate SNPs in genomic DNA, two disease associated SNPs were genotyped in the human hereditary hemochromatosis (HHC) gene HFE. HHC is a common genetic condition in Caucasians with approximately 1/400 Caucasians homozygous for the C282Y mutation leading to iron overload and potentially liver failure, diabetes and depression (22). A subset of individuals who are compound heterozygotes for the C282Y and H63D mutations also manifest iron overload. Because of the high prevalence of these mutations and the ability to prevent disease manifestations by phlebotomy, accurate methods for genotyping these two SNPs will foster genetic screening for this condition. Two PCR products were generated from human genomic DNA for the C282Y and H63D polymorphic sites of the HFE gene and then used these products for SBE with biotin-ddNTPs. After the extension reaction, products were purified using solid phase capture according to the scheme in FIG. 1 and analyzed by MALDI-TOF MS. The mass spectrum obtained from this experiment is shown in FIG. 2B. Extension products of each primer were readily identified by their mass relative to the internal mass standard. Heterozygous genotypes of A/G and C/G with a mass difference of 16 Da and 39 Da respectively were accurately detected at the C282Y and H63D polymorphic sites.
  • These results indicate that the use of solid phase capturable biotin-ddNTPs in SBE, coupled with MALDI-TOF MS detection, provides a rapid and accurate method for multiplex SNP detection over broad mass ranges and should greatly increase the number of SNPs that can be detected simultaneously. In multiplex SBE reactions, the oligonucleotide primers and their dideoxynucleotide extension products differ by only one base pair, which requires analytical techniques with high resolution to resolve. In addition, a primer designed to detect one polymorphism and an extension product from another polymorphic site may have the same size, which can not be separated by electrophoresis and other conventional chromatographic or size exclusion methods. Methods for purifying DNA samples using the strong interaction of biotin and streptavidin are widely used (23-27). By introducing the biotin moiety at the 3′ end of DNA, the solid phase based affinity purification approach described here is a unique and effective method to remove the oligonucleotide primers from the dideoxynucleotide extension products.
  • To increase the stability of DNA fragments for MALDI-TOF MS measurement in multiplex SNP analysis, nucleotide analogues (28) and peptide nucleic acid (9) can be used in the construction of the oligonucleotide primers. It has been shown that MALDI-TOF MS could detect DNA fragments up to 100 bp with sufficient resolution (29). The mass difference between each adjacent DNA fragment is approximately 300 Da. Thus, with a mass difference of 100 Da for each primer in designing a multiplex SNP analysis project, at least 300 SNPs can be analyzed in a single spot of the sample plate by MS. It is a routine method now to place 384 spots in each sample plate in MS analysis. Thus, each plate can produce over 100,000 SNPs, which is roughly the entire SNPs in all the coding regions of the human genome. This level of multiplexing should be achievable by mass tagging the primers with stable chemical groups in SBE using biotin-ddNTPs. For SNP sites of interest, a master database of primers and the resulting masses of all four possible extension products can be constructed. The experimental data from MALDI-TOF MS can then be compared with this database to precisely identify the library of SNPs automatically. This method coupled with future improvements in mass spectrometer detector sensitivity (30) will provide a platform for high-throughput SNP identification unrivaled in speed and accuracy.
  • C. Design and Synthesis of Biotinylated Dideoxynucleotides with Mass Tags
  • The ability to distinguish various bases in DNA using mass spectrometry is dependent on the mass differences of the bases in the spectra. For the above work, the smallest difference in mass between any two nucleotides is 16 daltons (see Table 1). Fei et al. (15) have shown that using dye-labeled ddNTP paired with a regular dNTP to space out the mass difference, an increase in the detection resolution in a single nucleotide extension assay can be achieved. To enhance the ability to distinguish peaks in the spectra, the current application discloses systematic modification of the biotinylated dideoxynucleotides by incorporating mass linkers assembled using 4-aminomethyl benzoic acid derivatives to increase the mass separation of the individual bases. The mass linkers can be modified by incorporating one or two fluorine atoms to further space out the mass differences between the nucleotides. The structures of four biotinylated ddNTPs are shown in FIG. 3. ddCTP-11-biotin is commercially available (New England Nuclear, Boston). ddTTP-Linker I-11-Biotin, ddATP-Linker II-11-Biotin and ddGTP-Linker III-11-Biotin are synthesized as shown, for example, for ddATP-Linker II-11-Biotin in FIG. 5. In designing these mass tag linker modified biotinylated ddNTPs, the linkers are attached to the 5-position on the pyrimidine bases (C and T), and to the 7-position on the purines (A and G) for subsequent conjugation with biotin. It has been established that modification of these positions on the bases in the nucleotides, even with bulky energy transfer fluorescent dyes, still allows efficient incorporation of the modified nucleotides into the DNA strand by DNA polymerase (32, 33). Thus, the ddNTPs-Linker-11-biotin can be incorporated into the growing strand by the polymerase in DNA sequencing reactions. Larger mass separations will greatly aid in longer read lengths where signal intensity is smaller and resolution is lower. The smallest mass difference between two individual bases is over three times as great in the mass tagged biotinylated ddNTPs compared to normal ddNTPs and more than double that achieved by the standard biotinylated ddNTPs as shown in Table 2.
    TABLE 2
    Relative mass differences (daltons) of dideoxynucleotides
    using ddCTP as a reference.
    Commercial Biotinylated
    Standard Biotinylated ddNTP with
    Base ddNTP ddNTP mass tag linker
    C relative to C  0  0  0 (no linker)
    T relative to C 15 89 (16 linker) 125 (Linker I)
    A relative to C 24 24 165 (Linker II)
    G relative to C 40 40 200 (Linker III)
    Smallest relative  9 16  35
    difference
  • Three 4-aminomethyl benzoic acid derivatives Linker I, Linker II and Linker III are designed as mass tags as well as linkers-for bridging biotin to the corresponding dideoxynucleotides. The synthesis of Linker II (FIG. 4) is described here to illustrate the synthetic procedure. 3-Fluoro-4-aminomethyl benzoic acid that can be easily prepared via published procedures (41, 42) is first protected with trifluoroacetic anhydride, then converted to N-hydroxysuccinimide (NHS) ester with disuccinimidylcarbonate in the presence of diisopropylethylamine. The resulting NHS ester is subsequently coupled with commercially available propargylamine to form the desired compound, Linker II. Using an analogous procedure, Linker I and Linker III can be easily constructed.
  • FIG. 5 describes the scheme required to prepare biotinylated ddATP-Linker II-11-Biotin using well-established procedures (34-36). 7-I-ddA is coupled with linker II in the presence of tetrakis(triphenylphosphine) palladium(0) to produce 7-Linker II-ddA, which is phosphorylated with POCl3 in butylammonium pyrophosphate (37). After removing the trifluoroacetyl group with ammonium hydroxide, 7-Linker II-ddATP is produced, which then couples with sulfo-NHS-LC-Biotin (Pierce, Rockford Ill.) to yield the desired ddATP-Linker II-11-Biotin. Similarly, ddTTP-Linker I-11-Biotin, and ddGTP-Linker III-11-Biotin can be synthesized.
  • D. Design and Synthesis of Mass Tagged ddNTPs Containing Photocleavable Biotin
  • A schematic of capture and cleavage of the photocleavable linker on the streptavidin coated porous surface is shown in FIG. 6. At the end of the reaction, the reaction mixture consists of excess primers, enzymes, salts, false stops, and the desired DNA fragment. This reaction mixture is passed over a streptavidin-coated surface and allowed to incubate. The biotinylated fragments are captured by the streptavidin surface, while everything else in the mixture is washed away. Then the fragments are released into solution by cleaving the photocleavable linker with near ultraviolet (UV) light, while the biotin remains attached to the streptavidin that is covalently bound to the surface. The pure DNA fragments can then be crystallized in matrix solution and analyzed by mass spectrometry. It is advantageous to cleave the biotin moiety since it contains sulfur which has several relatively abundant isotopes. The rest of the DNA fragments and linkers contain only carbon, nitrogen, hydrogen, oxygen, fluorine and phosphorous, whose dominant isotopes are found with a relative abundance of 99% to 100%. This allows high resolution mass spectra to be obtained. The photocleavage mechanism (38, 39) is shown in FIG. 7. Upon irradiation with ultraviolet light at 300-350 nm, the light sensitive o-nitroaromatic carbonamide functionality on DNA fragment 1 is cleaved, producing DNA fragment 2, PC-biotin and carbon dioxide. The partial chemical linker remaining on DNA fragment 2 is stable for detection by mass spectrometry.
  • Four new biotinylated ddNTPs disclosed here, ddCTP-PC-Biotin, ddTTP-Linker I-PC-Biotin, ddATP-Linker II-PC-Biotin and ddGTP-Linker III-PC-Biotin are shown in FIG. 8. These compounds are synthesized by a similar chemistry as shown for the synthesis of ddATP-Linker II-11-Biotin in FIG. 6. The only difference is that in the final coupling step NHS-PC-LC-Biotin (Pierce, Rockford Ill.) is used, as shown in FIG. 9. The photocleavable linkers disclosed here allow the use of solid phase capturable terminators and mass spectrometry to be turned into a high throughput technique for DNA analysis.
  • E. Overview of Capturing a DNA Fragment Terminated With a ddNTP on a Surface and Freeing the ddNTP and DNA Fragment
  • The DNA fragment is terminated with a dideoxynucleoside monophosphate (ddNMP). The ddNMP is attached via a linker to a chemical moiety (“X” in FIG. 10). The DNA fragment terminated with ddNMP is captured on the surface through interaction between chemical moiety “X” and a compound on or attached to the surface (“Y” in FIG. 10). The present application discloses two methods for freeing the captured DNA fragment terminated with ddNMP. In the situation illustrated in the lower part of FIG. 10, the DNA fragment terminated with ddNMP is freed from the surface by disrupting or breaking the interaction between chemical moiety “X” and compound “Y”. In the upper part of FIG. 10, the DNA fragment terminated with ddNMP is attached to chemical moiety “X” via a cleavable linker which can be cleaved to free the DNA fragment terminated with ddNMP.
  • Different moieties and compounds can be used for the “X”-“Y” affinity system, which include but are not limited to, biotin-streptavidin, phenylboronic acid-salicylhydroxamic acid (31), and antigen-antibody systems.
  • In different embodiments, the cleavable linker can be cleaved and the “X”-“Y” interaction can be disrupted by a means selected from the group consisting of one or more of a physical means, a chemical means, a physical chemical means, heat, and light. In one embodiment, ultraviolet light can be used to cleave the cleavable linker. Chemical means include, but are not limited to, ammonium hydroxide (40), formamide, or a change in pH (-log H+ concentration) of the solution.
  • F. High Density Streptavidin-Coated, Porous Silica Channel System.
  • Streptavidin coated magnetic beads are not ideal for using the photocleavable biotin capture and release process for DNA fragments, since they are not transparent to UV light. Therefore, the photocleavage reaction is not efficient. For efficient capture of the biotinylated fragments, a high-density surface coated with streptavidin is essential. It is known that the commercially available 96-well streptavidin coated plates cannot provide a sufficient surface area for efficient capture of the biotinylated DNA fragments. Disclosed in this application is a porous silica channel system designed to-overcome this limitation.
  • To increase the surface area available for solid phase capture, porous channels are coated with a high density of streptavidin. For example, ninety-six (96) porous silica glass channels can be etched into a silica chip (FIG. 11). The surfaces of the channels are modified to contain streptavidin as shown in FIG. 12. The channel is first treated with 0.5 M NaOH, washed with water, and then briefly pre-etched with dilute hydrogen fluoride. Upon cleaning with water, the capillary channel is coated with high density 3-aminopropyltrimethoxysilane in aqueous ethanol (43). An excess of disuccinimidyl glutarate in N,N-dimethylformamide (DMF) is then introduced into the capillary to ensure a highly efficient conversion of the surface end group to a succinimidyl ester. Streptavidin is then conjugated with the succinimidyl ester to form a high-density surface using excess streptavidin solution. The resulting 96-channel chip is used as a purification cassette.
  • A 96-well plate that can be used with biotinylated terminators for DNA analysis is shown in FIG. 11. In the example shown, each end of a channel is connected to a single well. However, for other applications, the end of a channel could be connected to a plurality of wells. Pressure is applied to drive the samples through a glass capillary into the channels on the chip. Inside the channels the biotin is captured by the covalently bound streptavidin. After passing through the channel, the sample enters into a clean plate in the other end of the chip. Pressure applied in reverse drives the sample through the channel multiple times and ensures a highly efficient solid phase capture. Water is similarly added to drive out the reaction mixture and thoroughly wash the captured fragments. After washing, the chip is irradiated with ultraviolet light to cleave the photosensitive linker and release the DNA fragments. The fragment solution is then driven out of the channel and into a collection plate. After matrix solution is added, the samples are spotted on a chip and allowed to crystallize for detection by MALDI-TOF mass spectrometry. The purification cassette is cleaned by chemically cleaving the biotin-streptavidin linkage, and is then washed and reused.
  • Experimental Set II
  • A. Synopsis
  • The following experiments show the simultaneous genotyping of 30 nucleotide variations in the p53 gene from human tumors in one tube, by using solid phase capturable dideoxynucleotides to generate single base extension products which are detected by mass spectrometry. Both homozygous and heterozygous genotypes are accurately determined with digital resolution. This is the highest level of SNP multiplexing reported thus far using mass spectrometry, indicating the approach will have wide applications in screening a repertoire of genotypes in candidate genes as potential markers for cancer and other diseases.
  • B. Introduction
  • With the completion of the Human Genome Project, a stage has been set to screen genetic mutations for identifying disease genes in a genomewide scale (44). Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), which allows rapid DNA sample measurement yielding digital data, has been explored to detect single nucleotide polymorphisms (SNPs) using invasive cleavage (11) and primer-directed base extension (14, 45). Conventional single base extension (SBE) methods using MS to measure multiplex SNPs require unambiguous simultaneous detection of a library of primers and their extension products. However, limitations in resolution and sensitivity of MALDI-TOF MS for longer DNA molecules make it difficult to simultaneously measure DNA fragments over a large mass range. The requirement to measure both primers and their extension products in this range limits the scope of multiplexing. The use of MALDI-TOF MS and molecular affinity for multiplex digital SNP detection using solid phase capturable (SPC) dideoxynucleotides and SBE has recently been explored, establishing the feasibility of simultaneously measuring 20 SNPs in synthetic DNA templates (46). This study shows the simultaneous genotyping of 30 nucleotide variations, corresponding to known sites of cancer-associated somatic mutations, in exons 5, 7 and 8 of the p53 gene from human tumors in one tube using the SPC-SBE method. This is the highest level of multiplexing reported thus far using mass spectrometry for SNP analysis.
  • C. Materials and Methods
  • Multiplex PCR and single base extension reactions Multiplex PCR was performed to amplify 3 regions in exons 5, 7 and 8 of the p53 gene. The primers for each region were 5′-TATCTGTTCACTTGTGCCC-3′ (exon 5, forward), 5′-CAGAGGCCTGGGGA-CCCTG-3′(exon 5, reverse), 5′-CTGCTTGCCACAGGTCTC-3′(exon 7, forward), 5′-CACAGCAG-GCCAGTGTGC-3′ (exon 7, reverse), 51-GGACCTGATTTCCTTAC-TG-3′ (exon 8, forward), and 5′-TGAATCTGAGGCATAACTG-3′ (exon 8, reverse). The 45 1 PCR reaction consisted of 180 ng genomic DNA, 1.5 nmol dNTP, 4.5 1 10× PCR buffer, 15 mM MgCl2, 4 pmol of forward and reverse primers for exons 5 and 7, 6 pmol of forward and reverse primers for exon 8, and 1.0 U of JumpStart RedAccuTaq DNA Polymerase. After a 5 min 96° C. hot start, the touchdown PCR program was performed with 10 cycles of 96° C. (30 sec), 67° C. to 57° C. (−1.0° C. per cycle, 30 sec) and 72° C. (30 sec), an additional 30 cycles of 96° C. (30 sec), 57° C. (30 sec) and 72° C. (30 sec), and a final extension at 72° C. for 7 min. The 30 SBE primers (Table 3) were designed to yield extension products with a sufficient mass difference and to be extended simultaneously in a single tube. Primer sequences were designed to avoid any overlap in mass, and the formation of secondary structures. To evenly separate the masses of such a large number of primers for SBE, some primers were synthesized using methyl-dC and dU phosphoramidites (Glen Research) to replace dC and dT respectively. Substitution of dC by methyl-dC increased the primer mass by 14 Da whereas a change from dT to dU decreased the mass by 14 Da. Primers were synthesized using an Applied Biosystems DNA synthesizer. The procedures for the SBE, solid phase purification and MALDI-TOF MS measurement were performed as described (Kim et al., Analytical Biochemistry 2003, 316, 251). Direct DNA sequencing was conducted using energy transfer terminator chemistry and a MegaBACE 1000 capillary DNA sequencer (Amersham Bioscience).
  • D. Discussion
  • Thirty polymorphic sites, including the most frequently mutated p53 codons, were chosen to explore the high multiplexing scope of the SPC-SBE method (FIG. 1). Thirty primers specific to each polymorphic site were designed to yield SBE products with sufficient mass differences. This was achieved by tuning the mass of some primers using methyl-dc and dU to replace dC and dT, respectively. Human genomic DNA was amplified by multiplex PCR to produce amplicons of three p53 exons. The 30 primers were mixed with the PCR products and biotinylated dideoxynucleotides for SBE to generate 3′-biotinylated extension DNA products. These products were then captured by streptavidin-coated solid phase magnetic beads, while the unextended primers and other components in the reaction were washed away. The pure DNA products were subsequently released from the solid phase and analyzed by MALDI-TOF MS. The nucleotide at the polymorphic site is accurately identified by the mass of the DNA extension product in a mass spectrum. Since only the DNA extension products are isolated for MS analysis, the resulting mass spectrum is free of non-extended primer peaks and their associated dimers, increasing accuracy and scope of multiplexing. The solid phase purification also facilitates desalting of the captured DNA, a process that is critical for accurate mass measurement by MALDI-TOF MS.
  • The SPC-SBE genotyping approach was used to analyze nucleotide variations in 30 codons of 3 exons of the p53 gene from 30 Wilms' tumors, 19 head and neck squamous carcinomas and 3 colorectal carcinomas. Primer sequences are shown in Table 3 along with the masses of the primers and their extension products. Extension products of all 30 primers were resolved in the mass spectrum, free from any unextended primers, yielding digital data to unambiguously determine each nucleotide variation (FIGS. 13A-13C). Unextended primers occupy the mass range in the mass spectrum decreasing the scope of multiplexing, and excess primers can dimerize to form false peaks in the mass spectrum (21). The excess primers and their associated dimers also compete for the ion current, reducing the detection sensitivity of MS for the desired DNA fragments. These complications were completely removed in the SPC-SBE method. When using conventional ddNTPs, the mass difference between ddATP and ddTTP is 9 Da, which is difficult to resolve by MALDI-TOF MS (15). In the SPC-SBE method using biotinylated ddNTPs, the difference between A and T is increased to 66 Da, which fosters accurate detection of heterozygous genotypes.
  • None of the 30 Wilms' tumor samples showed somatic mutations for the 30 polymorphic sites tested, yielding 30 distinct peaks corresponding to the wild type p53 sequences in a mass spectrum (FIG. 13A). In contrast, two of the 19 head and neck tumor samples contained a genetic variation; one at codon 157 (G/T heterozygous configuration; primary tumor biopsy; FIG. 13B) and the other at codon 151 (C to T homozygous; squamous carcinoma cell line; FIG. 14). In the three colorectal tumor cell lines tested, one (HCT-116) had 30 wild type p53 sequences for the 30 sites, yielding a mass spectrum similar to the one shown in FIG. 13A, while the other two (HT-29 and SW-480) had a G to A homozygous mutation in codon 273 (FIG. 13C). Both heterozygous and homozygous genotypes were clearly detected in the 30 codons with great accuracy. The G/T heterozygote (4684/4734 Da) was shown with two peaks corresponding to the wild type and mutant alleles, respectively (FIG. 13B). These data, confirmed by direct DNA sequencing, are consistent with the known paucity of the p53 mutations in Wilms' tumor, and the known occurrence of such mutations in squamous carcinomas and colorectal carcinomas.
  • It has been reported that MALDI-TOF MS could detect DNA sequencing fragments up to 100 bp with sufficient resolution using cleavable primers (29). The mass difference between each adjacent DNA sequencing fragment is approximately 300 Da. In principle, with a mass difference of 100 Da for each primer in designing a multiplex SNP analysis project using the SPC-SBE method, at least 300 SNPs can be analyzed in a single spot of an MS sample plate. Thus, each MS sample plate with 384 spots can produce over 100,000 SNPs, which is roughly the number of tag SNPs required to identify all the haplotypes in the human genome. This level of multiplexing should be achievable by mass tuning the primers with nucleotide analogues containing stable chemical groups (28). It is anticipated that the SPC-SBE high-throughput digital SNP detection approach will have wide applications in screening a repertoire of genotypes in candidate genes as potential markers for cancer and other diseases.
    TABLE 3
    Thirty p53 codons and the corresponding 30 SBE primers.
    Mass of Single Base
    Primer Primer Extention Products (Da)
    Number Exon Codon Sequences (5′-3′) Modification Mass (Da) ddATP-B ddCTP-B ddGTP-B ddUTP-B
    1 5 179 (CAT) GCGCTGCCCCCAC None 3857 4545 4522 4561 4611
    2 5 157 (GTC) GCCC GGCACCCGC methyl C 3980 4668 4645 4684 4734
    3 5 179 (CAT) GCGCTGCCCCCACC None 4146 4834 4811 4850 4900
    4 5 163 (TAC) CGCCATGGCCATCT methyl C 4270 4958 4935 4974 5024
    5 5 158 (CGC) CCGGCACCCGCGTCC None 4475 5163 5140 5179 5229
    6 7 248 (CGG) TGGGCGGCATGAACC None 4618 5306 5283 5322 5372
    7 5 132 (AAG) TCCCCTGCCCTCAACA methyl C 4736 5424 5401 5440 5490
    8 8 298 (GAG) AGGGGAGCCTCACCAC None 4876 5564 5541 5580 5630
    9 8 285 (GAG) GAGAGACCGGCGCACA methyl C 4995 5683 5660 5699 5749
    10 5 161 (GCC) CCCGCGTCCGCGCCATG None 5108 5796 5773 5812 5862
    11 7 249 (AGG) GGCGGCATGAACCGGAG methyl C 5341 6029 6006 6045 6095
    12 8 266 (GGA) GTAGTGGTAATCTACTGG dU 5486 6174 6151 6190 6240
    13 8 286 (GAA) AGAGACCGGCGCACAGAG methyl C 5638 6326 6303 6342 6392
    14 7 258 (GAA) CCTCACCATCATCACACTG methyl C 5765 6453 6430 6469 6519
    15 5 176 (TGC) ACGGAGGTTGTGAGGCGCT dU 5897 6585 6562 6601 6651
    16 5 152 (CCG) GTGGGTTGATTCCACACCCC dU 6041 6729 6706 6745 6795
    17 8 273 (CGT) ACGGAACAGCTTTGAGGTGC None 6182 6870 6847 6886 6936
    18 7 234 (TAC) CTGACTGTACCACCATCCACT None 6286 6974 6951 6990 7040
    19 7 248 (CGG) TCCTGCATGGGCGGCATGAAC dU 6405 7093 7070 7109 7159
    20 7 249 (AGG) GCATGGGCGGCATGAACCGGA None 6521 7209 7186 7225 7275
    21 8 282 (CGG) TTGTGCCTGTCCTGGGAGAGAC dU 6698 7386 7363 7402 7452
    22 8 278 (CCT) TGAGGTGCGTGTTTGTGCCTGT None 6819 7507 7484 7523 7573
    23 5 135 (TGC) CCCTGCCCTCAACAAGATGTTTT None 6935 7623 7600 7639 7689
    24 7 245 (GGC) TGTGTAACAGTTCCTGCATGGGC dU 7043 7731 7708 7747 7797
    25 7 237 (ATG) TACCACCATCCACTACAACTACAT None 7170 7858 7835 7874 7924
    26 7 242 (TGC) ACAAC TACATGTGTAACAGTTCCT dU 7282 7970 7947 7986 8036
    27 7 241 (TCC) ACTACAACTACATGTGTAACAGTT methyl C 7390 8078 8055 8094 8144
    28 8 275 (TGT) GGAACAGCTTTGAGGTGCGTGTTT methyl C 7497 8185 8162 8201 8251
    29 5 141 (TGC) ATGTTTTGCCAACTGGCCAAGACCT None 7617 8305 8282 8321 8371
    30 5 175 (CGC) CAGCACATGACGGAGGTTGTGAGGC None 7772 8460 8437 8476 8526

    The position of the nucleotide variation tested in each codon is shown in bold. The primer sequence and modification is specified and the modified nucleotides are shown in bold. The mass of each primer is indicated along with the mass of all four possible SBE products. The mass values in bold specify the wild type nucleotide sequences (ddNTP-B = Biotinylated dideoxynucleotides).
  • REFERENCES
    • 1) Kwok, P.-Y. (2000) Pharmacogenomics 1, 95-100.
    • 2) Roses A. (2000) Pharmacogenetics and the practice of medicine. Nature. 405: 857-865.
    • 3) The International SNP Map Working Group (2001) Nature 409, 928-933.
    • 4) Beavis, R. C. & Chait, B. T. (1989) Rapid Commun. Mass Spectrom. 3, 436-439.
    • 5) Li, J., Butler, J. M., Tan, Y., Lin, H., Royer, S., Ohler, L., Shaler, T. A., Hunter, J. A., Pollart, D. J., Monforte, J. A. & Becker, C. H. (1999) Electrophoresis 20, 1258-1265.
    • 6) Griffin, T. J. & Smith, L. M. (2000) Trends. Biotechnol. 18, 77-84.
    • 7) Graber, J. H., Smith, C. L. & Cantor, C. R. (1999) Genetic Analysis: Biomol. Eng. 14, 215-219.
    • 8) Stoerker, J., Mayo, J. D., Tetzlaff, C. N., Sarracino, D. A., Schwope, I. & Richert, C. (2000) Nat. Biotechnol. 18, 1213-1216.
    • 9) Ross, P. L., Lee, K. & Belgrader, P. (1997) Anal. Chem. 69, 4197-4202.
    • 10) Jiang-Baucom, P., Girard, J. E., Butler, J. & Belgrader, P. (1997) Anal. Chem. 69, 4894-4898.
    • 11) Griffin, T. J., Hall, J. G., Prudent, J. R. & Smith, L. M. (1999) Proc. Natl. Acad. Sci. USA. 96, 6301-6306.
    • 12) Lyamichev, V., Mast, A. L., Hall, J. G., Prudent, J. R., Kaiser, M. W., Takova, T., Kwiatkowski, R. W., Sander, T. J., de Arruda, M., Arco, D. A., Neri, B. P. & Brow, M. A. D. (1999) Nat. Biotechnol. 17, 292-296.
    • 13) Haff, L. A. & Smirnov, I. P. (1997) Nucleic Acids Res. 25, 3749-3750.
    • 14) Ross, P., Hall, L., Smirnov, I. P. & Haff, L. (1998) Nat. Biotechncl. 16, 1347-1351.
    • 15) Fei Z, Ono T, Smith L M. (1998) MALDI-TOF mass spectrometric typing of single nucleotide polymorphisms with mass-tagged ddNTPs. Nucleic Acids Res. 26: 2827-2828.
    • 16) Tang K, Fu D J, Julien D, Braun A, Cantor C R, Koster H. (1999) Chip-based genotyping by mass spectrometry. Proc. Natl. Acad. Sci. USA. 96: 10016-10020.
    • 17) Taranenko, N. I., Allman, S. L., Golovlev, V. V., Taranenko, N. V., Isola, N. R. & Chen, C. H. (1998) Nucleic Acids Res. 26, 2488-2490.
    • 18) Ju J. Nucleic Acid Sequencing with Solid Phase Capturable Terminators. U.S. Pat. No. 5,876,936, issued Mar. 2, 1999.
    • 19) Edwards, J. R., Itagaki, Y. & Ju, J. (2001) Nucleic Acids Res. 29, e104 (p1-5).
    • 20) Tong, A. K. & Ju, J. (2002) Single nucleotide polymorphism detection by combinatorial fluorescence energy transfer tags and biotinylated dideoxynucleotides. Nucleic Acids Res. 30(5):e19.
    • 21) Roskey M T, Juhasz P, Smirnov I P, Takach E J, Martin S A, Haff L A. (1996) DNA sequencing by delayed extraction-matrix-assisted laser desorption/ionization time of flight mass spectrometry. Proc. Natl. Acad. Sci. USA. 93: 4724-4729.
    • 22) Hanson, E. H., Imperatore, G. & Burke, W. (2001) Am. J. Epidem. 154, 193-206.
    • 23) Langer P R, Waldrop A A, Ward D C. (1981) Enzymatic synthesis of biotin-labeled polynucleotides: novel nucleic acid affinity probes. Proc. Natl. Acad. Sci. USA. 78: 6633-6637.
    • 24) Hawkins, T. L., O'Connor-Morin, T., Roy, A. & Santillan, C. (1994) Nucleic Acids Res. 22, 4543-4544.
    • 25) Uhlen, M. (1989) Nature, 340, 733-734.
    • 26) Wahlberg, J., Lunderberg, J., Hultman, T. & Uhlen, M. (1990) Proc. Natl. Acad. Sci. USA. 87, 6569-6573.
    • 27) Tong, X., Smith L M (1992) Solid-Phase Method for the Purification of DNA Sequencing Reactions. Anal. Chem. 64: 2672-2677.
    • 28) Schneider K, Chait B T. (1995) Increased stability of nucleic acids containing 7-deaza-guanosine and 7-deaza-adenosine may enable rapid DNA sequencing by matrix-assisted laser desorption mass spectrometry. Nucleic Acids Res. 23: 1570-1575.
    • 29) Monforte J A, Becker C H (1997) High-throughput DNA analysis by time-of-flight mass spectrometry. Nat Medicine. 3(3): 360-362.
    • 30) Hilton, G. C., Martinis, J. M., Wollman, D. A., Irwin, K. D., Dulcie, L. L., Gerber, D., Gillevet, P. M. & Twerenbold, D. (1998) Nature 391, 672-675.
    • 31) Bergseid M, Baytan A R, Wiley J P, Ankener W M, Stolowitz, Hughs K A, Chestnut J D (November 2000) Small-molecule base chemical affinity system for the purification of proteins. BioTechniques 29: 1126-1133.
    • 32) Rosenblum B B, Lee L G, Spurgeon S L, Khan S H, Menchen S M, Heiner C R, Chen S M. (1997) New dye-labeled terminators for improved DNA sequencing patterns. Nucleic Acids Res. 25: 4500-4504.
    • 33) Zhu Z, Chao J, Yu H, Waggoner A S. (1994) Directly labeled DNA probes using fluorescent nucleotides with different length linkers. Nucleic Acids Res. 22: 3418-3422.
    • 34) Prober J M, Trainor G L, Dam R J, Hobbs F W, Robertson C W, Zagursky R J, Cocuzza A J, Jensen M A, Baumeister K. (1987) A system for rapid DNA sequencing with fluorescent chain-terminating dideoxynucleotides. Science 238: 336-341.
    • 35) Lee L G, Connell C R, Woo S L, Cheng R D, Mcardle B F, Fuller C W, Halloran N D, Wilson R K. (1992) DNA sequencing with dye-labeled terminators and T7 DNA-polymerase-effect of dyes and dNTPs on incorporation of dye-terminators and probability analysis of termination fragments. Nucleic Acids Res. 20: 2471-2483.
    • 36) Hobbs F W Jr, Cocuzza A J. Alkynylamino-Nucleotides. U.S. Pat. No. 5,047,519, issued Sep. 10, 1991.
    • 37) Burgess K, Cook D. (2000) Chemical Reviews. 100: 2047-2060.
    • 38) Olejnik J, Sonar S, Krzymanska-Olejnik E, Rothschild K J. (1995) Photocleavable biotin derivatives: a versatile approach for the isolation of biomolecules. Proc. Natl. Acad. Sci. USA. 92: 7590-7594.
    • 39) Olejnik J, Ludemann H C, Krzymanska-Olejnik E, Berkenkamp S, Hillenkamp F, Rothschild K J. (1999) Photocleavable peptide-DNA conjugates: synthesis and applications to DNA analysis using MALDI-MS. Nucleic Acids Res. 27: 4626-4631.
    • 40) Jurinke C, van de Boom D, Collazo V, Luchow A, Jacob A, Koster H. (1997) Recovery of nucleic acids from immobilized biotin-streptavidin complexes using ammonium hydroxide and applications in MALDI-TOF mass spectrometry. Anal. Chem. 69: 904-910.
    • 41) Maudling D R, Lotts K D, Robinson S A. (1983) New procedure for making 2-(chloromethyl)-4-nitrotoluene. J. Org. Chem. 48: 2938.
    • 42) Rolla F. (1982) Sodium-borohydride reactions under phase-transfer conditions—reduction of azides to amines. J. Org. Chem. 47: 4327-4329.
    • 43) Woolley A T, Mathies R A. (1994) Ultra-high-speed DNA fragment separations using microfabricated capillary array electrophoresis chips. Proc. Natl. Acad. Sci. USA. 91: 11348-11352.
    • 44) Collins F S, Green E D, Guttmacher, A E and Guyer M S. (2003) Nature 422: 835-847.
    • 45) Jurinke C, van den Boom D, Cantor C R and Koster H. (2002) Methods Mol. Biol.187: 179-192
    • 46) Kim S et al. (2003) Analytical Biochemistry 316: 251-258

Claims (25)

1. A method for determining the identity of a nucleotide present at a predetermined site in a DNA whose sequence immediately 3′ of such predetermined site is known which comprises:
(a) treating the DNA with an oligonucleotide primer whose sequence is complementary to such known sequence so that the oligonucleotide primer hybridizes to the DNA and forms a complex in which the 3′ end of the oligonucleotide primer is located immediately adjacent to the predetermined site in the DNA;
(b) simultaneously contacting the complex from step (a) with four different labeled dideoxynucleotides, in the presence of a polymerase under conditions permitting a labeled dideoxynucleotide to be added to the 3′ end of the primer so as to generate a labeled single base extended primer, wherein each of the four different labeled dideoxynucleotides (i) is complementary to one of the four nucleotides present in the DNA and (ii) has a molecular weight which can be distinguished from the molecular weight of the other three labeled dideoxynucleotides using mass spectrometry; and
(c) determining the difference in molecular weight between the labeled single base extended primer and the oligonucleotide primer so as to identify the dideoxynucleotide incorporated into the single base extended primer and thereby determine the identity of the nucleotide present at the predetermined site in the DNA.
2. The method of claim 1, wherein each of the four labeled dideoxynucleotides comprises a chemical moiety attached to the dideoxynucleotide by a different linker which has a molecular weight different from that of each other linker.
3. The method of claim 1 which further comprises after step (b) the steps of:
(i) contacting the labeled single base extended primer with a surface coated with a compound that specifically interacts with a chemical moiety attached to the dideoxynucleotide by a linker so as to thereby capture the extended primer on the surface; and
(ii) treating the labeled single base extended primer so as to release it from the surface.
4. The method of claim 3 which further comprises after step (i) the step of treating the surface to remove primers that have not been extended by a labeled dideoxynucleotide.
5. The method of claim 1, wherein step (c) comprises determining the difference in mass between the labeled single base extended primer and an internal mass calibration standard added to the extended primer.
6. The method of claim 3, wherein the interaction between the chemical moiety attached to the dideoxynucleotide by the linker and the compound on the surface comprises a biotin-streptavidin interaction, a phenylboronic acid-salicylhydroxamic acid interaction, or an antigen-antibody interaction.
7. The method of claim 3, wherein the step of releasing the labeled single base extended primer from the surface comprises disrupting the interaction between the chemical moiety attached to the dideoxynucleotide by the linker and the compound on the surface.
8. The method of claim 7, wherein the interaction is disrupted by a means selected from the group consisting of one or more of a physical means, a chemical means, a physical chemical means, heat, and light.
9. The method of claim 2, wherein the linker is attached to the dideoxynucleotide at the 5-position of cytosine or thymine or at the 7-position of adenine or guanine.
10. The method of claim 3, wherein the step of releasing the labeled single base extended primer from the surface comprises cleaving the linker between the chemical moiety and the dideoxynucleotide.
11. The method of claim 10, where the linker is cleaved by a means selected from the group consisting of one or more of a physical means, a chemical means, a physical chemical means, heat, and light.
12. The method of claim 11, wherein the linker is cleaved by light.
13. The method of claim 2, wherein the linker comprises a derivative of 4-aminomethyl benzoic acid, a 2-nitrobenzyl group, or a derivative of a 2-nitrobenzyl group.
14. The method of claim 13, wherein the linker comprises one or more fluorine atoms.
15. The method of claim 14, wherein the linker is selected from the group consisting of:
Figure US20060252038A1-20061109-C00011
16. The method of claim 3, wherein the chemical moiety comprises biotin, the labeled dideoxynucleotide is a biotinylated dideoxynucleotide, the labeled single base extended primer is a biotinylated single base extended primer, and the surface is a streptavidin-coated solid surface.
17. The method of claim 16, wherein the biotinylated dideoxynucleotide is selected from the aroup consisting of ddATP-11-biotin, ddCTP-11-biotin, ddGTP-11-biotin, and ddTTP-16-biotin.
18. The method of claim 16, wherein the biotinylated dideoxynucleotide is selected from the group consisting of:
Figure US20060252038A1-20061109-C00012
wherein ddNTP1, ddNTP2, ddNTP3, and ddNTP4 represent four different dideoxynucleotides.
19. The method of claim 18, wherein the biotinylated dideoxynucleotide is selected from the group consisting of:
Figure US20060252038A1-20061109-C00013
20. The method of claim 16, wherein the biotinylated dideoxynucleotide is selected from the group consisting of:
Figure US20060252038A1-20061109-C00014
wherein ddNTP1, ddNTP2, ddNTF3, and ddNTF4 represent four different dideoxynucleotides.
21. The method of claim 20, wherein the biotinylated dideoxynucleotide is selected from the group consisting of:
Figure US20060252038A1-20061109-C00015
22. The method of claim 16, wherein the streptavidin-coated solid surface is a streptavidin-coated magnetic bead or a streptavidin-coated silica glass.
23. The method of claim 1, wherein steps (a) and (b) are performed in a single container or in a plurality of connected containers.
24. A method for determining the identity of nucleotides present at a plurality of predetermined sites, which comprises carrying out the method of claim 3 using a plurality of different primers each having a molecular weight different from that of each other primer, wherein a different primer hybridizes adjacent to a different predetermined site.
25. The method of claim 24, wherein different linkers each having a molecular weight different from that of each other linker are attached to the different dideoxynucleotides to increase mass separation between different labeled single base extended primers and thereby increase mass spectrometry resolution.
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US9041420B2 (en) 2010-02-08 2015-05-26 Genia Technologies, Inc. Systems and methods for characterizing a molecule
US9110478B2 (en) 2011-01-27 2015-08-18 Genia Technologies, Inc. Temperature regulation of measurement arrays
US9115163B2 (en) 2007-10-19 2015-08-25 The Trustees Of Columbia University In The City Of New York DNA sequence with non-fluorescent nucleotide reversible terminators and cleavable label modified nucleotide terminators
US9175342B2 (en) 2007-10-19 2015-11-03 The Trustees Of Columbia University In The City Of New York Synthesis of cleavable fluorescent nucleotides as reversible terminators for DNA sequencing by synthesis
US9255292B2 (en) 2005-10-31 2016-02-09 The Trustees Of Columbia University In The City Of New York Synthesis of four-color 3′-O-allyl modified photocleavable fluorescent nucleotides and related methods
US9322062B2 (en) 2013-10-23 2016-04-26 Genia Technologies, Inc. Process for biosensor well formation
US9494554B2 (en) 2012-06-15 2016-11-15 Genia Technologies, Inc. Chip set-up and high-accuracy nucleic acid sequencing
US9551697B2 (en) 2013-10-17 2017-01-24 Genia Technologies, Inc. Non-faradaic, capacitively coupled measurement in a nanopore cell array
US9605309B2 (en) 2012-11-09 2017-03-28 Genia Technologies, Inc. Nucleic acid sequencing using tags
US9624539B2 (en) 2011-05-23 2017-04-18 The Trustees Of Columbia University In The City Of New York DNA sequencing by synthesis using Raman and infrared spectroscopy detection
US9678055B2 (en) 2010-02-08 2017-06-13 Genia Technologies, Inc. Methods for forming a nanopore in a lipid bilayer
US9708358B2 (en) 2000-10-06 2017-07-18 The Trustees Of Columbia University In The City Of New York Massive parallel method for decoding DNA and RNA
US9759711B2 (en) 2013-02-05 2017-09-12 Genia Technologies, Inc. Nanopore arrays
US20180312916A1 (en) * 2017-04-26 2018-11-01 Mike Joseph Pugia Digital sequencing using mass labels
US10240195B2 (en) 2014-03-24 2019-03-26 The Trustees Of Columbia University In The City Of New York Chemical methods for producing tagged nucleotides
US10421995B2 (en) 2013-10-23 2019-09-24 Genia Technologies, Inc. High speed molecular sensing with nanopores
US10648026B2 (en) 2013-03-15 2020-05-12 The Trustees Of Columbia University In The City Of New York Raman cluster tagged molecules for biological imaging
US10732183B2 (en) 2013-03-15 2020-08-04 The Trustees Of Columbia University In The City Of New York Method for detecting multiple predetermined compounds in a sample
US10738072B1 (en) 2018-10-25 2020-08-11 Singular Genomics Systems, Inc. Nucleotide analogues
US10822653B1 (en) 2019-01-08 2020-11-03 Singular Genomics Systems, Inc. Nucleotide cleavable linkers and uses thereof
US11085076B2 (en) 2015-09-28 2021-08-10 The Trustees Of Columbia University In The City Of New York Synthesis of novel disulfide linker based nucleotides as reversible terminators for DNA sequencing by synthesis
US11266673B2 (en) 2016-05-23 2022-03-08 The Trustees Of Columbia University In The City Of New York Nucleotide derivatives and methods of use thereof
US11591647B2 (en) 2017-03-06 2023-02-28 Singular Genomics Systems, Inc. Nucleic acid sequencing-by-synthesis (SBS) methods that combine SBS cycle steps
US11608523B2 (en) 2012-06-20 2023-03-21 The Trustees Of Columbia University In The City Of New York Nucleic acid sequencing by nanopore detection of tag molecules

Families Citing this family (35)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20060057565A1 (en) * 2000-09-11 2006-03-16 Jingyue Ju Combinatorial fluorescence energy transfer tags and uses thereof
US7153699B2 (en) * 2001-12-21 2006-12-26 Cytonome, Inc. Microfabricated two-pin system for biomolecule crystallization
US7057026B2 (en) * 2001-12-04 2006-06-06 Solexa Limited Labelled nucleotides
GB0129012D0 (en) * 2001-12-04 2002-01-23 Solexa Ltd Labelled nucleotides
US7776524B2 (en) 2002-02-15 2010-08-17 Genzyme Corporation Methods for analysis of molecular events
US7074597B2 (en) * 2002-07-12 2006-07-11 The Trustees Of Columbia University In The City Of New York Multiplex genotyping using solid phase capturable dideoxynucleotides and mass spectrometry
US7414116B2 (en) 2002-08-23 2008-08-19 Illumina Cambridge Limited Labelled nucleotides
DK3587433T3 (en) 2002-08-23 2020-05-18 Illumina Cambridge Ltd MODIFIED NUCLEOTIDES
US11008359B2 (en) 2002-08-23 2021-05-18 Illumina Cambridge Limited Labelled nucleotides
US20050032081A1 (en) * 2002-12-13 2005-02-10 Jingyue Ju Biomolecular coupling methods using 1,3-dipolar cycloaddition chemistry
US8637650B2 (en) 2003-11-05 2014-01-28 Genovoxx Gmbh Macromolecular nucleotide compounds and methods for using the same
EP2436778A3 (en) * 2004-03-03 2012-07-11 The Trustees of Columbia University in the City of New York Photocleavable fluorescent nucleotides for DNA sequencing on chip constructed by site-specific coupling chemistry
WO2006073436A2 (en) * 2004-04-29 2006-07-13 The Trustees Of Columbia University In The City Of New York Mass tag pcr for multiplex diagnostics
WO2005111244A2 (en) * 2004-05-10 2005-11-24 Exact Sciences Corporation Methods for detecting a mutant nucleic acid
WO2006026654A2 (en) 2004-08-27 2006-03-09 Exact Sciences Corporation Method for detecting a recombinant event
US9109256B2 (en) 2004-10-27 2015-08-18 Esoterix Genetic Laboratories, Llc Method for monitoring disease progression or recurrence
WO2006097320A2 (en) * 2005-03-17 2006-09-21 Genovoxx Gmbh Macromolecular nucleotide links and methods for their use
US9777314B2 (en) 2005-04-21 2017-10-03 Esoterix Genetic Laboratories, Llc Analysis of heterogeneous nucleic acid samples
GB0517097D0 (en) * 2005-08-19 2005-09-28 Solexa Ltd Modified nucleosides and nucleotides and uses thereof
CA2630544A1 (en) * 2005-11-21 2007-05-31 The Trustees Of Columbia University In The City Of New York Multiplex digital immuno-sensing using a library of photocleavable mass tags
US7959876B2 (en) * 2006-07-17 2011-06-14 Industrial Technology Research Institute Fluidic device
WO2008042067A2 (en) 2006-09-28 2008-04-10 Illumina, Inc. Compositions and methods for nucleotide sequencing
EP2527501A1 (en) 2006-11-15 2012-11-28 The University Of British Columbia Polymorphisms predictive of anthracycline-induced cardiotoxicity
GB201016484D0 (en) 2010-09-30 2010-11-17 Geneseque As Method
CN107083421A (en) 2010-12-17 2017-08-22 纽约哥伦比亚大学理事会 The DNA detected using the nucleotides through modification and nano-pore is sequenced in synthesis
CN110564819A (en) 2011-05-19 2019-12-13 基纳生物技术有限公司 Products and methods for multiplex nucleic acid identification
CA2869753A1 (en) 2012-04-09 2013-10-17 Jingyue Ju Method of preparation of nanopore and uses thereof
EP3388442A1 (en) 2013-03-15 2018-10-17 Illumina Cambridge Limited Modified nucleosides or nucleotides
US10337049B2 (en) 2013-06-17 2019-07-02 The Trustees Of Columbia University In The City Of New York Universal methylation profiling methods
GB201413929D0 (en) 2014-08-06 2014-09-17 Geneseque As Method
US10233489B2 (en) 2015-04-24 2019-03-19 Agena Bioscience, Inc. Multiplexed method for the identification and quantitation of minor alleles and polymorphisms
CN114350766A (en) * 2015-04-24 2022-04-15 基纳生物技术有限公司 Parallel method for detecting and quantifying minor variants
WO2017075421A1 (en) * 2015-10-29 2017-05-04 Temple University-Of The Commonwealth System Of Higher Education Modification of 3' terminal ends of nucleic acids by dna polymerase theta
CN110144399B (en) * 2019-04-09 2021-12-03 中源维康(天津)医学检验所有限公司 Primer group and kit for detecting lung cancer related gene mutation in human circulating tumor DNA and using method
CN114223705B (en) * 2021-11-02 2022-10-11 青岛海洋科学与技术国家实验室发展中心 Device for preparing low-fluorine euphausia superba kernels on land

Citations (46)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4824775A (en) * 1985-01-03 1989-04-25 Molecular Diagnostics, Inc. Cells labeled with multiple Fluorophores bound to a nucleic acid carrier
US5118605A (en) * 1984-10-16 1992-06-02 Chiron Corporation Polynucleotide determination with selectable cleavage sites
US5174962A (en) * 1988-06-20 1992-12-29 Genomyx, Inc. Apparatus for determining DNA sequences by mass spectrometry
US5302509A (en) * 1989-08-14 1994-04-12 Beckman Instruments, Inc. Method for sequencing polynucleotides
US5599675A (en) * 1994-04-04 1997-02-04 Spectragen, Inc. DNA sequencing by stepwise ligation and cleavage
US5654419A (en) * 1994-02-01 1997-08-05 The Regents Of The University Of California Fluorescent labels and their use in separations
US5728528A (en) * 1995-09-20 1998-03-17 The Regents Of The University Of California Universal spacer/energy transfer dyes
US5763594A (en) * 1994-09-02 1998-06-09 Andrew C. Hiatt 3' protected nucleotides for enzyme catalyzed template-independent creation of phosphodiester bonds
US5770367A (en) * 1993-07-30 1998-06-23 Oxford Gene Technology Limited Tag reagent and assay method
US5789167A (en) * 1993-09-10 1998-08-04 Genevue, Inc. Optical detection of position of oligonucleotides on large DNA molecules
US5804386A (en) * 1997-01-15 1998-09-08 Incyte Pharmaceuticals, Inc. Sets of labeled energy transfer fluorescent primers and their use in multi component analysis
US5808045A (en) * 1994-09-02 1998-09-15 Andrew C. Hiatt Compositions for enzyme catalyzed template-independent creation of phosphodiester bonds using protected nucleotides
US5843203A (en) * 1996-03-22 1998-12-01 Grantek, Inc. Agricultural carrier
US5849542A (en) * 1993-11-17 1998-12-15 Amersham Pharmacia Biotech Uk Limited Primer extension mass spectroscopy nucleic acid sequencing method
US5853992A (en) * 1996-10-04 1998-12-29 The Regents Of The University Of California Cyanine dyes with high-absorbance cross section as donor chromophores in energy transfer labels
US5869255A (en) * 1994-02-01 1999-02-09 The Regents Of The University Of California Probes labeled with energy transfer couples dyes exemplified with DNA fragment analysis
US5872244A (en) * 1994-09-02 1999-02-16 Andrew C. Hiatt 3' protected nucleotides for enzyme catalyzed template-independent creation of phosphodiester bonds
US5876936A (en) * 1997-01-15 1999-03-02 Incyte Pharmaceuticals, Inc. Nucleic acid sequencing with solid phase capturable terminators
US5885775A (en) * 1996-10-04 1999-03-23 Perseptive Biosystems, Inc. Methods for determining sequences information in polynucleotides using mass spectrometry
US5945283A (en) * 1995-12-18 1999-08-31 Washington University Methods and kits for nucleic acid analysis using fluorescence resonance energy transfer
US6028190A (en) * 1994-02-01 2000-02-22 The Regents Of The University Of California Probes labeled with energy transfer coupled dyes
US6046005A (en) * 1997-01-15 2000-04-04 Incyte Pharmaceuticals, Inc. Nucleic acid sequencing with solid phase capturable terminators comprising a cleavable linking group
US6074823A (en) * 1993-03-19 2000-06-13 Sequenom, Inc. DNA sequencing by mass spectrometry via exonuclease degradation
US6136543A (en) * 1997-01-31 2000-10-24 Hitachi, Ltd. Method for determining nucleic acids base sequence and apparatus therefor
US6197557B1 (en) * 1997-03-05 2001-03-06 The Regents Of The University Of Michigan Compositions and methods for analysis of nucleic acids
US6214987B1 (en) * 1994-09-02 2001-04-10 Andrew C. Hiatt Compositions for enzyme catalyzed template-independent formation of phosphodiester bonds using protected nucleotides
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
US6232465B1 (en) * 1994-09-02 2001-05-15 Andrew C. Hiatt Compositions for enzyme catalyzed template-independent creation of phosphodiester bonds using protected nucleotides
US6312893B1 (en) * 1996-01-23 2001-11-06 Qiagen Genomics, Inc. Methods and compositions for determining the sequence of nucleic acid molecules
US6316230B1 (en) * 1999-08-13 2001-11-13 Applera Corporation Polymerase extension at 3′ terminus of PNA-DNA chimera
US6361940B1 (en) * 1996-09-24 2002-03-26 Qiagen Genomics, Inc. Compositions and methods for enhancing hybridization and priming specificity
US20020168642A1 (en) * 1994-06-06 2002-11-14 Andrzej Drukier Sequencing duplex DNA by mass spectroscopy
US20030008285A1 (en) * 2001-06-29 2003-01-09 Fischer Steven M. Method of DNA sequencing using cleavable tags
US20030022225A1 (en) * 1996-12-10 2003-01-30 Monforte Joseph A. Releasable nonvolatile mass-label molecules
US20030027140A1 (en) * 2001-03-30 2003-02-06 Jingyue Ju High-fidelity DNA sequencing using solid phase capturable dideoxynucleotides and mass spectrometry
US20030044871A1 (en) * 2001-08-27 2003-03-06 Pharmanetics Incorporated Coagulation assay reagents containing lanthanides and a protein C assay using such a lanthanide-containing reagent
US20030099972A1 (en) * 2001-07-13 2003-05-29 Ambergen, Inc. Nucleotide compositions comprising photocleavable markers and methods of preparation thereof
US6613508B1 (en) * 1996-01-23 2003-09-02 Qiagen Genomics, Inc. Methods and compositions for analyzing nucleic acid molecules utilizing sizing techniques
US6627748B1 (en) * 2000-09-11 2003-09-30 The Trustees Of Columbia University In The City Of New York Combinatorial fluorescence energy transfer tags and their applications for multiplex genetic analyses
US6664399B1 (en) * 1999-09-02 2003-12-16 E. I. Du Pont De Nemours & Company Triazole linked carbohydrates
US6664079B2 (en) * 2000-10-06 2003-12-16 The Trustees Of Columbia University In The City Of New York Massive parallel method for decoding DNA and RNA
US20050032081A1 (en) * 2002-12-13 2005-02-10 Jingyue Ju Biomolecular coupling methods using 1,3-dipolar cycloaddition chemistry
US20060003352A1 (en) * 2004-04-29 2006-01-05 Lipkin W I Mass tag PCR for mutliplex diagnostics
US20060057565A1 (en) * 2000-09-11 2006-03-16 Jingyue Ju Combinatorial fluorescence energy transfer tags and uses thereof
US7074597B2 (en) * 2002-07-12 2006-07-11 The Trustees Of Columbia University In The City Of New York Multiplex genotyping using solid phase capturable dideoxynucleotides and mass spectrometry
USRE39663E1 (en) * 2000-02-07 2007-05-29 Applera Corporation Electron-deficient nitrogen heterocycle-substituted fluorescein dyes

Family Cites Families (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1991006678A1 (en) 1989-10-26 1991-05-16 Sri International Dna sequencing
US5834203A (en) * 1997-08-25 1998-11-10 Applied Spectral Imaging Method for classification of pixels into groups according to their spectra using a plurality of wide band filters and hardwire therefore
US5876036A (en) * 1997-11-10 1999-03-02 Mathis; Darryl One-on-one basketball game apparatus
JP2002537858A (en) 1999-03-10 2002-11-12 エーエスエム サイエンティフィック, インコーポレイテッド Methods for direct sequencing of nucleic acids
GB0013276D0 (en) 2000-06-01 2000-07-26 Amersham Pharm Biotech Uk Ltd Nucleotide analogues
AU8911101A (en) 2000-09-11 2002-03-26 Univ Columbia Combinatorial fluorescence energy transfer tags and uses thereof
US6887690B2 (en) * 2001-06-22 2005-05-03 Pe Corporation Dye-labeled ribonucleotide triphosphates

Patent Citations (49)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5118605A (en) * 1984-10-16 1992-06-02 Chiron Corporation Polynucleotide determination with selectable cleavage sites
US4824775A (en) * 1985-01-03 1989-04-25 Molecular Diagnostics, Inc. Cells labeled with multiple Fluorophores bound to a nucleic acid carrier
US5174962A (en) * 1988-06-20 1992-12-29 Genomyx, Inc. Apparatus for determining DNA sequences by mass spectrometry
US5302509A (en) * 1989-08-14 1994-04-12 Beckman Instruments, Inc. Method for sequencing polynucleotides
US6074823A (en) * 1993-03-19 2000-06-13 Sequenom, Inc. DNA sequencing by mass spectrometry via exonuclease degradation
US5770367A (en) * 1993-07-30 1998-06-23 Oxford Gene Technology Limited Tag reagent and assay method
US5789167A (en) * 1993-09-10 1998-08-04 Genevue, Inc. Optical detection of position of oligonucleotides on large DNA molecules
US5849542A (en) * 1993-11-17 1998-12-15 Amersham Pharmacia Biotech Uk Limited Primer extension mass spectroscopy nucleic acid sequencing method
US5654419A (en) * 1994-02-01 1997-08-05 The Regents Of The University Of California Fluorescent labels and their use in separations
US6028190A (en) * 1994-02-01 2000-02-22 The Regents Of The University Of California Probes labeled with energy transfer coupled dyes
US5869255A (en) * 1994-02-01 1999-02-09 The Regents Of The University Of California Probes labeled with energy transfer couples dyes exemplified with DNA fragment analysis
US5599675A (en) * 1994-04-04 1997-02-04 Spectragen, Inc. DNA sequencing by stepwise ligation and cleavage
US20020168642A1 (en) * 1994-06-06 2002-11-14 Andrzej Drukier Sequencing duplex DNA by mass spectroscopy
US6214987B1 (en) * 1994-09-02 2001-04-10 Andrew C. Hiatt Compositions for enzyme catalyzed template-independent formation of phosphodiester bonds using protected nucleotides
US5763594A (en) * 1994-09-02 1998-06-09 Andrew C. Hiatt 3' protected nucleotides for enzyme catalyzed template-independent creation of phosphodiester bonds
US6232465B1 (en) * 1994-09-02 2001-05-15 Andrew C. Hiatt Compositions for enzyme catalyzed template-independent creation of phosphodiester bonds using protected nucleotides
US5808045A (en) * 1994-09-02 1998-09-15 Andrew C. Hiatt Compositions for enzyme catalyzed template-independent creation of phosphodiester bonds using protected nucleotides
US5872244A (en) * 1994-09-02 1999-02-16 Andrew C. Hiatt 3' protected nucleotides for enzyme catalyzed template-independent creation of phosphodiester bonds
US5728528A (en) * 1995-09-20 1998-03-17 The Regents Of The University Of California Universal spacer/energy transfer dyes
US5945283A (en) * 1995-12-18 1999-08-31 Washington University Methods and kits for nucleic acid analysis using fluorescence resonance energy transfer
US6312893B1 (en) * 1996-01-23 2001-11-06 Qiagen Genomics, Inc. Methods and compositions for determining the sequence of nucleic acid molecules
US6613508B1 (en) * 1996-01-23 2003-09-02 Qiagen Genomics, Inc. Methods and compositions for analyzing nucleic acid molecules utilizing sizing techniques
US5843203A (en) * 1996-03-22 1998-12-01 Grantek, Inc. Agricultural carrier
US6361940B1 (en) * 1996-09-24 2002-03-26 Qiagen Genomics, Inc. Compositions and methods for enhancing hybridization and priming specificity
US5885775A (en) * 1996-10-04 1999-03-23 Perseptive Biosystems, Inc. Methods for determining sequences information in polynucleotides using mass spectrometry
US5853992A (en) * 1996-10-04 1998-12-29 The Regents Of The University Of California Cyanine dyes with high-absorbance cross section as donor chromophores in energy transfer labels
US20030022225A1 (en) * 1996-12-10 2003-01-30 Monforte Joseph A. Releasable nonvolatile mass-label molecules
US5814454A (en) * 1997-01-15 1998-09-29 Incyte Pharmaceuticals, Inc. Sets of labeled energy transfer fluorescent primers and their use in multi component analysis
US6046005A (en) * 1997-01-15 2000-04-04 Incyte Pharmaceuticals, Inc. Nucleic acid sequencing with solid phase capturable terminators comprising a cleavable linking group
US5952180A (en) * 1997-01-15 1999-09-14 Incyte Pharmaceuticals, Inc. Sets of labeled energy transfer fluorescent primers and their use in multi component analysis
US5876936A (en) * 1997-01-15 1999-03-02 Incyte Pharmaceuticals, Inc. Nucleic acid sequencing with solid phase capturable terminators
US5804386A (en) * 1997-01-15 1998-09-08 Incyte Pharmaceuticals, Inc. Sets of labeled energy transfer fluorescent primers and their use in multi component analysis
US6136543A (en) * 1997-01-31 2000-10-24 Hitachi, Ltd. Method for determining nucleic acids base sequence and apparatus therefor
US6197557B1 (en) * 1997-03-05 2001-03-06 The Regents Of The University Of Michigan Compositions and methods for analysis of nucleic acids
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
US6316230B1 (en) * 1999-08-13 2001-11-13 Applera Corporation Polymerase extension at 3′ terminus of PNA-DNA chimera
US6664399B1 (en) * 1999-09-02 2003-12-16 E. I. Du Pont De Nemours & Company Triazole linked carbohydrates
USRE39663E1 (en) * 2000-02-07 2007-05-29 Applera Corporation Electron-deficient nitrogen heterocycle-substituted fluorescein dyes
US6627748B1 (en) * 2000-09-11 2003-09-30 The Trustees Of Columbia University In The City Of New York Combinatorial fluorescence energy transfer tags and their applications for multiplex genetic analyses
US20060057565A1 (en) * 2000-09-11 2006-03-16 Jingyue Ju Combinatorial fluorescence energy transfer tags and uses thereof
US20040185466A1 (en) * 2000-10-06 2004-09-23 The Trustees Of Columbia University In The City Of New York. Massive parallel method for decoding DNA and RNA
US6664079B2 (en) * 2000-10-06 2003-12-16 The Trustees Of Columbia University In The City Of New York Massive parallel method for decoding DNA and RNA
US20030027140A1 (en) * 2001-03-30 2003-02-06 Jingyue Ju High-fidelity DNA sequencing using solid phase capturable dideoxynucleotides and mass spectrometry
US20030008285A1 (en) * 2001-06-29 2003-01-09 Fischer Steven M. Method of DNA sequencing using cleavable tags
US20030099972A1 (en) * 2001-07-13 2003-05-29 Ambergen, Inc. Nucleotide compositions comprising photocleavable markers and methods of preparation thereof
US20030044871A1 (en) * 2001-08-27 2003-03-06 Pharmanetics Incorporated Coagulation assay reagents containing lanthanides and a protein C assay using such a lanthanide-containing reagent
US7074597B2 (en) * 2002-07-12 2006-07-11 The Trustees Of Columbia University In The City Of New York Multiplex genotyping using solid phase capturable dideoxynucleotides and mass spectrometry
US20050032081A1 (en) * 2002-12-13 2005-02-10 Jingyue Ju Biomolecular coupling methods using 1,3-dipolar cycloaddition chemistry
US20060003352A1 (en) * 2004-04-29 2006-01-05 Lipkin W I Mass tag PCR for mutliplex diagnostics

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US9169510B2 (en) 2005-06-21 2015-10-27 The Trustees Of Columbia University In The City Of New York Pyrosequencing methods and related compositions
US20090325154A1 (en) * 2005-06-21 2009-12-31 The Trustees Of Columbia University In The City Of New York Pyrosequencing Methods and Related Compositions
US9297042B2 (en) 2005-10-31 2016-03-29 The Trustees Of Columbia University In The City Of New York Chemically cleavable 3′-O-allyl-dNTP-allyl-fluorophore fluorescent nucleotide analogues and related methods
US20090263791A1 (en) * 2005-10-31 2009-10-22 Jingyue Ju Chemically Cleavable 3'-O-Allyl-DNTP-Allyl-Fluorophore Fluorescent Nucleotide Analogues and Related Methods
US10907194B2 (en) 2005-10-31 2021-02-02 The Trustees Of Columbia University In The City Of New York Synthesis of four-color 3′-O-allyl modified photocleavable fluorescent nucleotides and related methods
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US11098353B2 (en) 2006-12-01 2021-08-24 The Trustees Of Columbia University In The City Of New York Four-color DNA sequencing by synthesis using cleavable fluorescent nucleotide reversible terminators
US7883869B2 (en) 2006-12-01 2011-02-08 The Trustees Of Columbia University In The City Of New York Four-color DNA sequencing by synthesis using cleavable fluorescent nucleotide reversible terminators
US8298792B2 (en) 2006-12-01 2012-10-30 The Trustees Of Columbia University In The City Of New York Four-color DNA sequencing by synthesis using cleavable fluorescent nucleotide reversible terminators
US20100092952A1 (en) * 2006-12-01 2010-04-15 Jingyue Ju Four-color dna sequencing by synthesis using cleavable fluorescent nucleotide reversible terminators
US10144961B2 (en) 2007-10-19 2018-12-04 The Trustees Of Columbia University In The City Of New York Synthesis of cleavable fluorescent nucleotides as reversible terminators for DNA sequencing by synthesis
US11208691B2 (en) 2007-10-19 2021-12-28 The Trustees Of Columbia University In The City Of New York Synthesis of cleavable fluorescent nucleotides as reversible terminators for DNA sequencing by synthesis
US10260094B2 (en) 2007-10-19 2019-04-16 The Trustees Of Columbia University In The City Of New York DNA sequencing with non-fluorescent nucleotide reversible terminators and cleavable label modified nucleotide terminators
US11242561B2 (en) 2007-10-19 2022-02-08 The Trustees Of Columbia University In The City Of New York DNA sequencing with non-fluorescent nucleotide reversible terminators and cleavable label modified nucleotide terminators
US9670539B2 (en) 2007-10-19 2017-06-06 The Trustees Of Columbia University In The City Of New York Synthesis of cleavable fluorescent nucleotides as reversible terminators for DNA sequencing by synthesis
US9175342B2 (en) 2007-10-19 2015-11-03 The Trustees Of Columbia University In The City Of New York Synthesis of cleavable fluorescent nucleotides as reversible terminators for DNA sequencing by synthesis
US9115163B2 (en) 2007-10-19 2015-08-25 The Trustees Of Columbia University In The City Of New York DNA sequence with non-fluorescent nucleotide reversible terminators and cleavable label modified nucleotide terminators
US11027502B2 (en) 2010-02-08 2021-06-08 Roche Sequencing Solutions, Inc. Systems and methods for forming a nanopore in a lipid bilayer
US20110193249A1 (en) * 2010-02-08 2011-08-11 Genia Technologies, Inc. Systems and methods for forming a nanopore in a lipid bilayer
US9041420B2 (en) 2010-02-08 2015-05-26 Genia Technologies, Inc. Systems and methods for characterizing a molecule
US20110192723A1 (en) * 2010-02-08 2011-08-11 Genia Technologies, Inc. Systems and methods for manipulating a molecule in a nanopore
US9678055B2 (en) 2010-02-08 2017-06-13 Genia Technologies, Inc. Methods for forming a nanopore in a lipid bilayer
US9605307B2 (en) 2010-02-08 2017-03-28 Genia Technologies, Inc. Systems and methods for forming a nanopore in a lipid bilayer
US10926486B2 (en) 2010-02-08 2021-02-23 Roche Sequencing Solutions, Inc. Systems and methods for forming a nanopore in a lipid bilayer
US10343350B2 (en) 2010-02-08 2019-07-09 Genia Technologies, Inc. Systems and methods for forming a nanopore in a lipid bilayer
US10371692B2 (en) 2010-02-08 2019-08-06 Genia Technologies, Inc. Systems for forming a nanopore in a lipid bilayer
US9377437B2 (en) 2010-02-08 2016-06-28 Genia Technologies, Inc. Systems and methods for characterizing a molecule
US10920271B2 (en) 2010-12-22 2021-02-16 Roche Sequencing Solutions, Inc. Nanopore-based single DNA molecule characterization, identification and isolation using speed bumps
US9121059B2 (en) 2010-12-22 2015-09-01 Genia Technologies, Inc. Nanopore-based single molecule characterization
US10400278B2 (en) 2010-12-22 2019-09-03 Genia Technologies, Inc. Nanopore-based single DNA molecule characterization, identification and isolation using speed bumps
US8845880B2 (en) 2010-12-22 2014-09-30 Genia Technologies, Inc. Nanopore-based single DNA molecule characterization, identification and isolation using speed bumps
US9617593B2 (en) 2010-12-22 2017-04-11 Genia Technologies, Inc. Nanopore-based single DNA molecule characterization, identification and isolation using speed bumps
US8962242B2 (en) 2011-01-24 2015-02-24 Genia Technologies, Inc. System for detecting electrical properties of a molecular complex
US9581563B2 (en) 2011-01-24 2017-02-28 Genia Technologies, Inc. System for communicating information from an array of sensors
US10156541B2 (en) 2011-01-24 2018-12-18 Genia Technologies, Inc. System for detecting electrical properties of a molecular complex
US10010852B2 (en) 2011-01-27 2018-07-03 Genia Technologies, Inc. Temperature regulation of measurement arrays
US9110478B2 (en) 2011-01-27 2015-08-18 Genia Technologies, Inc. Temperature regulation of measurement arrays
CN102304565A (en) * 2011-04-29 2012-01-04 广州益善生物技术有限公司 Specific primers and liquid phase chip for polymorphic detection of hemochromatosis (HFE) gene
US9624539B2 (en) 2011-05-23 2017-04-18 The Trustees Of Columbia University In The City Of New York DNA sequencing by synthesis using Raman and infrared spectroscopy detection
US8986629B2 (en) 2012-02-27 2015-03-24 Genia Technologies, Inc. Sensor circuit for controlling, detecting, and measuring a molecular complex
US11275052B2 (en) 2012-02-27 2022-03-15 Roche Sequencing Solutions, Inc. Sensor circuit for controlling, detecting, and measuring a molecular complex
US9494554B2 (en) 2012-06-15 2016-11-15 Genia Technologies, Inc. Chip set-up and high-accuracy nucleic acid sequencing
US11608523B2 (en) 2012-06-20 2023-03-21 The Trustees Of Columbia University In The City Of New York Nucleic acid sequencing by nanopore detection of tag molecules
US10526647B2 (en) 2012-11-09 2020-01-07 The Trustees Of Columbia University In The City Of New York Nucleic acid sequences using tags
US11674174B2 (en) 2012-11-09 2023-06-13 The Trustees Of Columbia University In The City Of New York Nucleic acid sequences using tags
US10822650B2 (en) 2012-11-09 2020-11-03 Roche Sequencing Solutions, Inc. Nucleic acid sequencing using tags
US9605309B2 (en) 2012-11-09 2017-03-28 Genia Technologies, Inc. Nucleic acid sequencing using tags
US9759711B2 (en) 2013-02-05 2017-09-12 Genia Technologies, Inc. Nanopore arrays
US10012637B2 (en) 2013-02-05 2018-07-03 Genia Technologies, Inc. Nanopore arrays
US10809244B2 (en) 2013-02-05 2020-10-20 Roche Sequencing Solutions, Inc. Nanopore arrays
US10648026B2 (en) 2013-03-15 2020-05-12 The Trustees Of Columbia University In The City Of New York Raman cluster tagged molecules for biological imaging
US10732183B2 (en) 2013-03-15 2020-08-04 The Trustees Of Columbia University In The City Of New York Method for detecting multiple predetermined compounds in a sample
US10393700B2 (en) 2013-10-17 2019-08-27 Roche Sequencing Solutions, Inc. Non-faradaic, capacitively coupled measurement in a nanopore cell array
US9551697B2 (en) 2013-10-17 2017-01-24 Genia Technologies, Inc. Non-faradaic, capacitively coupled measurement in a nanopore cell array
US11021745B2 (en) 2013-10-23 2021-06-01 Roche Sequencing Solutions, Inc. Methods for forming lipid bilayers on biochips
US10421995B2 (en) 2013-10-23 2019-09-24 Genia Technologies, Inc. High speed molecular sensing with nanopores
US9322062B2 (en) 2013-10-23 2016-04-26 Genia Technologies, Inc. Process for biosensor well formation
US9567630B2 (en) 2013-10-23 2017-02-14 Genia Technologies, Inc. Methods for forming lipid bilayers on biochips
US10240195B2 (en) 2014-03-24 2019-03-26 The Trustees Of Columbia University In The City Of New York Chemical methods for producing tagged nucleotides
US11396677B2 (en) 2014-03-24 2022-07-26 The Trustees Of Columbia University In The City Of New York Chemical methods for producing tagged nucleotides
US11085076B2 (en) 2015-09-28 2021-08-10 The Trustees Of Columbia University In The City Of New York Synthesis of novel disulfide linker based nucleotides as reversible terminators for DNA sequencing by synthesis
US11266673B2 (en) 2016-05-23 2022-03-08 The Trustees Of Columbia University In The City Of New York Nucleotide derivatives and methods of use thereof
US11773439B2 (en) 2017-03-06 2023-10-03 Singular Genomics Systems, Inc. Nucleic acid sequencing-by-synthesis (SBS) methods that combine SBS cycle steps
US11591647B2 (en) 2017-03-06 2023-02-28 Singular Genomics Systems, Inc. Nucleic acid sequencing-by-synthesis (SBS) methods that combine SBS cycle steps
US20180312916A1 (en) * 2017-04-26 2018-11-01 Mike Joseph Pugia Digital sequencing using mass labels
US10738072B1 (en) 2018-10-25 2020-08-11 Singular Genomics Systems, Inc. Nucleotide analogues
US11878993B2 (en) 2018-10-25 2024-01-23 Singular Genomics Systems, Inc. Nucleotide analogues
US10822653B1 (en) 2019-01-08 2020-11-03 Singular Genomics Systems, Inc. Nucleotide cleavable linkers and uses thereof

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