Methods for nucleic acid amplification and sequence determination

• US 20060024711A1
• Filed: 06/23/2005
• Published: 02/02/2006
• Est. Priority Date: 07/02/2004
• Status: Abandoned Application

• Current Assignee: Fluidigm Corporation
• Original Assignee: Helicos Biosciences Corporation
• Inventors: Buzby, Philip R., Lapidus, Stanley N.
• Application Number:
  US11/167,046
• Publication Number:
  US 20060024711A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2531/125 Rolling circle
• CPC Class Codes: C12Q 2565/501 being an array of oligonucleotides

Abstract

The invention provides methods for sequencing a nucleic acid comprising conducting rolling circle amplification on a circular nucleic acid template, wherein the resulting amplicon is optionally anchored to a substrate in an individually optically resolvable manner, and performing a sequencing reaction.

Claims

1. A method of determining a sequence of a nucleic acid, the method comprising the steps of:

(a) conducting rolling circle amplification of a nucleic acid to produce an amplicon comprising about two to about one hundred linked complements of a nucleic acid;

wherein said amplicon is anchored to a substrate, such that said amplicon is individually optically resolvable; and

(b) determining a sequence of at least a portion of said nucleic acid.

2. The method of claim 1, wherein said nucleic acid is genomic DNA, cDNA, or RNA.

3. The method of claim 1, wherein said amplification step comprises circularizing said nucleic acid thereby forming a circular template;

combining said circular template with a primer, a polymerizing agent, and nucleotides; and

producing an amplicon comprising multiple linked complements of said circular template.

4. The method of claim 3, wherein said primer binds to at least one of the 3′

end and the 5′

end of the nucleic acid.

5. The method of claim 1, wherein said nucleic acid is single stranded.

6. The method of claim 1, wherein said nucleic acid is double stranded, and wherein said amplification step further includes the step of denaturing said double stranded nucleic acid prior to combining with said primer.

7. The method of claim 1, wherein said amplicon is anchored to said substrate after completion of said amplification step.

8. The method of claim 1, wherein said amplicon is covalently bound to said substrate.

9. The method of claim 1, wherein said amplicon is anchored to said substrate via a biotin-streptavidin complex.

10. The method of claim 1, wherein said amplicon comprises a number of linked complements of the nucleic acid, said number determined by a concentration of nucleotides available for incorporation into said amplicon.

11. The method of claim 1, wherein said substrate comprises an accumulation of negative charge.

12. The method of claim 1, wherein said substrate comprises a plurality of loci for anchoring said amplicon.

13. The method of claim 1, wherein said substrate is selected from the group consisting of glass, fused silica, epoxy, plastic, metal, gel matrix, and composites.

14. The method of claim 13, wherein said substrate has a chemically modified surface comprising a polyelectrolyte multilayer.

15. The method of claim 1, wherein said determining step comprises exposing said amplicon to a sequencing primer, a polymerizing agent, and at least one nucleotide;

allowing incorporation of said nucleotide(s) into a synthesis strand;

detecting incorporation of said nucleotide(s); and

repeating said determining step at least once, thereby determining said sequence of said nucleic acid.

16. The method of claim 15, wherein said determining step results in incorporation of about one or about two nucleotides into said synthesis strand.

17. The method of claim 15, wherein said nucleotide is labeled with a fluorescent moiety.

18. The method of claim 1, wherein said amplification step is performed with a primer that is anchored to said substrate such that upon completion of said amplification step, said amplicon is anchored to said substrate.

19. The method of claim 1, wherein said amplification step is performed with a primer that is not anchored to said substrate such that upon completion of said amplification step, said amplicon is not attached to said substrate.