NUCLEIC ACID SEQUENCING-BY-SYNTHESIS (SBS) METHODS THAT COMBINE SBS CYCLE STEPS
The present disclosure provides improved nucleic acid sequencing-by-synthesis (SBS) methods, related kits and reagents, and systems for performing such methods using such kits and reagents.
1 - 33 . (canceled)
34 . A method of detecting a labeled nucleotide analogue, the method comprising:
contacting a solid support comprising a primer hybridized to a template DNA molecule with a plurality of labeled nucleotide analogues; and binding a labeled nucleotide analogue to a complementary nucleotide of the template DNA molecule;
removing unbound labeled nucleotide analogues;
imaging said solid support and detecting the bound labeled nucleotide analogue; and, while imaging, contacting said solid support with a plurality of blocked nucleotide analogues, wherein each blocked nucleotide analogue comprises a blocking moiety.
35 . The method of claim 34 , wherein the solid support further comprises a second template DNA molecule, wherein a second primer is hybridized to the second template DNA molecule.
36 . The method of claim 34 , wherein the labeled nucleotide analogue comprises a first label moiety and a blocking moiety.
37 . The method of claim 36 , wherein the blocking moiety comprises an allyl moiety, a 2-nitrobenzyl moiety, an azido moiety, or a disulfide moiety.
38 . The method of claim 36 , wherein the blocking moiety comprises an allyl moiety, an azido moiety, or a disulfide moiety.
39 . The method of claim 36 , wherein the first label moiety is a fluorophore.
40 . The method of claim 36 , wherein the labeled nucleotide analogue is covalently linked to the first label moiety via a cleavable linker.
41 . The method of claim 34 , wherein the labeled nucleotide analogue comprises a first label moiety, and the blocked nucleotide analogue comprises a second label moiety, wherein the first label moiety and the second label moiety are different.
42 . The method of claim 34 , wherein the blocked nucleotide analogue does not comprise a label.
43 . The method of claim 34 , wherein the labeled nucleotide analogue is incorporated with a polymerase into the primer to form an extension product.
44 . The method of claim 34 , wherein the labeled nucleotide analogue is removed and the blocked nucleotide analogue is incorporated into the primer to form a blocked extension product.
45 . The method of claim 35 , wherein the blocked nucleotide analogue is incorporated with a polymerase into the second primer to form a blocked extension product.
46 . The method of claim 44 , the method further comprises removing a blocking moiety linked to a 3′-oxygen atom of the blocked extension product to generate an extendible extension product.
47 . The method of claim 45 , the method further comprises removing a blocking moiety linked to a 3′-oxygen atom of the blocked extension product to generate an extendible extension product.
48 . The method of claim 34 , wherein imaging comprises detecting and identifying the labeled nucleotide analogue.
49 . The method of claim 46 , further comprising contacting the extendible extension product with a second labeled nucleotide analogue.
50 . The method of claim 47 , further comprising contacting the extendible extension product with a second labeled nucleotide analogue.
51 . The method of claim 34 , wherein imaging occurs for
about 1 minute to about 10 minutes;
about 1 minute to about 5 minutes; or
about 1 minute to about 3 minutes.
52 . The method of claim 34 , wherein imaging occurs for less than 15 minutes.
53 . The method of claim 34 , wherein said solid support is glass or plastic.