Methods for nucleic acid amplification and sequence determination
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.
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.