Stabilizing a nucleic acid for nucleic acid sequencing
View Patent ↗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.
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.
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.
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.
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.