Stabilizing a nucleic acid for nucleic acid sequencing

• US 7,220,549 B2
• Filed: 12/30/2004
• Issued: 05/22/2007
• Est. Priority Date: 12/30/2004
• Status: Expired due to Fees

• Current Assignee: Fluidigm Corporation
• Original Assignee: Helicos Biosciences Corporation
• Inventors: Buzby, Philip Richard
• Primary Examiner(s): Benzion; Gary
• Assistant Examiner(s): Thomas; David C.
• Application Number:
  US11/027,165
• Publication Number:
  US 20060147942A1
• Time in Patent Office: 873 Days
• Field of Search: None
• US Class Current: 435/6.1
• CPC Class Codes: C12Q 1/68 involving nucleic acids
• CPC Class Codes: C12Q 1/6834 Enzymatic or biochemical coupling of nucleic acids to a solid phase
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2533/101 Primer extension
• 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 2563/131 the label being a member of a cognate binding pair, i.e. extends to antibodies, haptens, avidin

Abstract

The invention provides methods for sequencing a nucleic acid comprising stabilizing a primer/target nucleic acid duplex on a substrate. Methods of the invention generally contemplate the use of a dual-anchored primer/target nucleic acid duplex, or a stabilizing molecule in a single molecule sequencing reaction.

Claims

1. A method for stabilizing a nucleic acid duplex on a surface, the method comprising the steps of:

contacting a surface with a nucleic acid duplex comprising a first and a second nucleic acid member, wherein each nucleic acid member comprises a binding pair member and at least one of said nucleic acid members comprises a binding pair member at its 3′

terminus, wherein the surface comprises a binding partner for each of said binding pair members, and wherein said binding pair is a carbohydrate/lectin pair or an antigen/antibody pair,

thereby to stabilize said duplex on said surface.

2. The method of claim 1, wherein each of said binding pair members is the same molecular species.

3. The method of claim 1, wherein each said member of said binding pair is a different molecular species.

4. The method of claim 1, wherein said binding pair is selected from the group consisting or biotin/avidin, biotin/streptavidin, digoxigenin/anti-digoxigenin, and dinitrophenol/anti-dinitrophenol.

5. The method of claim 1, wherein said nucleic acid duplex is a template/primer duplex.

6. The method of claim 1, wherein each of said binding pair members is located at the 5′

terminus of said template and the 3′

terminus of said primer.

7. The method of claim 1, wherein one of said binding pair members is located at the 5′

terminus of said primer.

8. The method of claim 5, further comprising the steps of contacting a surface-bound nucleic acid duplex to a nucleotide base and a polymerase under conditions sufficient for said nucleotide base to be incorporated into said primer if said nucleotide base is complementary to a corresponding base in said template.

9. The method of claim 8, further comprising the step of compiling a nucleic acid sequence of said template by detecting sequential incorporations of nucleotides into said primer.

10. The method of claim 5, wherein said primer comprises a locked nucleic acid base.

11. The method of claim 5, wherein said primer comprises a peptide nucleic acid base.

13. The method of claim 1, wherein a plurality of said nucleic acid duplexes is attached to a substrate such that each of said nucleic acid duplexes is individually optically resolvable.

12. A method for performing a nucleic acid sequencing reaction, the method comprising:

contacting a mixture comprising a nucleic acid template, a polymerase, and a primer, wherein said primer comprises a locked nucleic acid, with a nucleotide under conditions wherein said nucleotide is capable of incorporation into said primer, wherein said nucleic acid template and said primer each comprises a binding pair member, and wherein said template and said primer form a stabilized nucleic acid duplex on a surface, the surface comprising a binding partner for each said binding pair member, and wherein said binding pair is a carbohydrate/lectin pair or an antigen/antibody pair,

thereby performing a nucleic acid sequencing reaction.

14. The method of claim 12, further comprising detecting ordered template-dependent nucleotide incorporation into the primer and compiling a sequence of the template based upon the order or incorporated nucleotides.