Massive parallel method for decoding DNA and RNA

• US 9,133,511 B2
• Filed: 08/05/2013
• Issued: 09/15/2015
• Est. Priority Date: 10/06/2000
• Status: Expired due to Fees

• Current Assignee: Trustees Of Columbia University In The City Of New York (Columbia University)
• Original Assignee: Trustees Of Columbia University In The City Of New York (Columbia University)
• Inventors: Ju, Jingyue, Li, Zengmin, Edwards, John Robert, Itagaki, Yasuhiro
• Primary Examiner(s): Riley, Jezia
• Application Number:
  US13/959,660
• Publication Number:
  US 20140206553A1
• Time in Patent Office: 771 Days
• Field of Search: 435/6.1, 435/91.1, 536/24.3, 536/26.6
• US Class Current: 1/1
• CPC Class Codes: C07B 2200/11 Compounds covalently bound to a solid support
• CPC Class Codes: C07H 19/10 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• CPC Class Codes: C07H 19/14 Pyrrolo-pyrimidine radicals
• CPC Class Codes: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: C12Q 1/68 involving nucleic acids
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 1/6872 involving mass spectrometry
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 1/6876 Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
• CPC Class Codes: C12Q 2525/117 incorporating modified base
• CPC Class Codes: C12Q 2525/186 incorporating a non-extendable or blocking moiety
• CPC Class Codes: C12Q 2535/101 Sanger sequencing method, i.e. oligonucleotide sequencing using primer elongation and dideoxynucleotides as chain terminators
• CPC Class Codes: C12Q 2535/122 Massive parallel sequencing
• CPC Class Codes: C12Q 2563/107 fluorescence
• CPC Class Codes: C12Q 2565/501 being an array of oligonucleotides
• CPC Class Codes: C40B 40/00 Libraries per se, e.g. arrays, mixtures

Abstract

This invention provides methods for attaching a nucleic acid to a solid surface and for sequencing nucleic acid by detecting the identity of each nucleotide analogue after the nucleotide analogue is incorporated into a growing strand of DNA in a polymerase reaction. The invention also provides nucleotide analogues which comprise unique labels attached to the nucleotide analogue through a cleavable linker, and a cleavable chemical group to cap the —OH group at the 3′-position of the deoxyribose.

Claims

1. A method of sequencing nucleic acid, comprising:

a) reacting a DNA strand containing an azido group with a phosphine;

b) growing a double-stranded DNA containing said DNA strand by incorporating a fluorescently labeled nucleotide into said strand; and

c) identifying the fluorescently labeled nucleotide, so as to sequence the nucleic acid.

2. The method of claim 1, wherein said fluorescently labeled nucleotide has the label linked to the base and a blocking group on the 3′

-hydroxyl group.

3. The method of claim 2, wherein the label is attached to the base via a cleavable linker.

4. The method of claim 1, wherein the phosphine is a triarylphosphine.

5. The method of claim 1, wherein the azido group is attached to the deoxyribose at a 5′

position.

6. The method of claim 1, wherein the azido group is attached to the deoxyribose via a linker.

7. The method of claim 1, wherein the nucleotide analogue comprises a deazapurine base.

8. A method of sequencing nucleic acid comprising:

a) providing a nucleic acid template hybridized to a primer, the primer comprising an azido group;

b) reacting said azido group with a phosphine;

c) extending the primer hybridized to said nucleic acid template with a fluorescently labeled nucleotide or nucleotide analogue,

wherein said fluorescently labeled nucleotide or nucleotide analogue has the label linked to the base and a blocking group on the 3′

-hydroxyl group; and

d) identifying the fluorescently labeled nucleotide, so as to sequence the nucleic acid.

9. The method of claim 8, wherein said fluorescently labeled nucleotide or nucleotide analogue has the label on the base and a blocking group on the 3′

-hydroxyl group.

10. The method of claim 9, wherein the label is attached to the base via a cleavable linker.

11. The method of claim 8, wherein the phosphine is a triarylphosphine.

12. The method of claim 8, wherein the azido group is attached to the deoxyribose at a 5′

position.

13. The method of claim 8, wherein the azido group is attached to the deoxyribose via a linker.

14. The method of claim 8, wherein the nucleotide analogue comprises a deazapurine base.

15. A method of simultaneously sequencing a plurality of different nucleic acids, comprising:

a) reacting each of a plurality of different DNA strands containing an azido group with a phosphine;

b) growing a plurality of double-stranded DNA, each of which comprises one of said DNA strands, by incorporating a fluorescently labeled nucleotide; and

c) identifying each fluorescently labeled nucleotide, so as to simultaneously sequence the plurality of different nucleic acids.

16. The method of claim 15, wherein said fluorescently labeled nucleotide or nucleotide analogue has the label linked to the base and a blocking group on the 3′

-hydroxyl group.

17. The method of claim 16, wherein the label is attached to the base via a cleavable linker.

18. The method of claim 15, wherein the phosphine is a triarylphosphine.

19. The method of claim 15, wherein the azido group is attached to the deoxyribose at a 5′

position via a linker.

20. The method of claim 15, wherein the nucleotide analogue comprises a deazapurine base.