SHORT CYCLE METHODS FOR SEQUENCING POLYNUCLEOTIDES
The invention provides methods for sequencing a polynucleotide comprising stopping an extension cycle in a sequence by synthesis reaction before the reaction has run to near or full completion.
1 - 26 . (canceled)
27 . A method for obtaining sequence information from a target nucleic acid, comprising:
a) immobilizing a plurality of target nucleic acid/primer duplexes at discrete locations on a surface, wherein each of said target nucleic acids comprise the same adaptor sequence, and wherein each of said primers are hybridized to said adaptor sequence;
b) contacting said plurality of immobilized target nucleic acid/primer duplexes with a solution comprising a single nucleotide species in the presence of a polymerase under conditions wherein the polymerase catalyses template-dependent addition of a nucleotide to the 3′ terminus of at least one of said primers in said plurality of different target nucleic acid/primer duplexes; and
c) analyzing said discrete locations on said surface to detect the addition of said nucleotide, wherein addition of said nucleotide is indicative of the presence of a complementary nucleotide in the target nucleic acid.
28 . The method of claim 27 , further comprising:
d) washing said surface;
e) in the presences of a polymerase, contacting said plurality of different target nucleic acid/primer duplexes with a solution comprising a second single nucleotide species under conditions wherein the polymerase catalyses template-dependent addition of a nucleotide to the 3′ terminus at least one of said primers; and
f) analyzing said discrete locations to detect the addition of said second nucleotide to said duplex.
29 . The method of claim 27 , wherein said surface is selected from the group consisting of spheres, microparticles, beads, membranes, slides, plates, micro-machined chips, tubes, microwells, microfluidic devices, and filters.
30 . The method of claim 27 , wherein said analyzing comprises detecting a label.
31 . The method of claim 39 , wherein said target nucleic acid in said target nucleic acid/primer duplexes are attached to said surface.
32 . The method of claim 27 , wherein said primers in said target nucleic acid/primer duplexes are attached to said surface.
33 . The method of claim 27 , wherein said target nucleic acid/primer duplexes are formed prior to immobilization at said discrete locations in said flow cell.
34 . The method of claim 3 , wherein said target nucleic acid/primer duplexes are formed prior to immobilization on said one or more surfaces.