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. A method of sequencing a DNA molecule, comprising:
(a) contacting a solid support comprising a primed template DNA molecule with a deoxyribonucleotide triphosphate (dNTP) analogue species, wherein said dNTP analogue species is complementary to a nucleotide of the primed template DNA molecule, and wherein said dNTP analogue species is associated with a detectable label;
(b) after step (a), contacting said primed template DNA molecule with a plurality of blocked dNTP analogue species, and incorporating with a polymerase at least one of said plurality of blocked dNTP analogue species, thereby forming a blocked extension product; and
(c) imaging said solid support during steps (a) and (b), wherein the combined steps (a)-(b) occurs for about 1 minute to about 10 minutes.
2. The method of claim 1 , wherein after step (a), the method further comprises removing the dNTP analogue species.
3. The method of claim 1 , wherein step (b) comprises adding at least four different unlabeled, blocked dNTP analogue species.
4. The method of claim 1 , wherein after step (c), the method further comprises (d) removing a blocking moiety linked to a 3′-oxygen atom of the blocked extension product to generate an extendible extension product.
5. The method of claim 4 , wherein the blocking moiety comprises an allyl moiety, a 2-nitrobenzyl moiety, an azido moiety, or a disulfide moiety.
6. The method of claim 4 , wherein the blocking moiety comprises an allyl moiety, an azido moiety, or a disulfide moiety.
7. The method of claim 1 , wherein step (c) further comprises detecting the detectable label and identifying the dNTP analogue species associated with the detectable label.
8. The method of claim 7 , further comprising assigning the complement of the dNTP analogue species associated with the detectable label determined in step (c) as the identity of the nucleotide residue in the template DNA molecule.
9. The method of claim 8 , further comprising iterating steps (a)-(c) one or more times, thereby determining a nucleotide sequence of at least a portion of the template DNA molecule.
10. The method of claim 1 , comprising iterating steps (a)-(c) one or more times, wherein after step (c) of each iteration, the blocked product is treated to generate an extendible extension product, with the proviso that after the last iteration, treatment of the blocked extension product can be omitted.
11. The method of claim 1 , wherein solid support comprises a plurality of DNA template molecules.
12. The method of claim 11 , wherein each of the plurality of template DNA molecules is attached to the solid support via a linker.
13. The method of claim 1 , wherein the detectable label comprises a fluorophore.
14. The method of claim 1 , wherein said dNTP analogue species is covalently linked to said detectable label via a cleavable linker.
15. The method of claim 1 , wherein the combined steps (a)-(b) occurs for about 1 minute to about 5 minutes or about 1 minute to about 3 minutes.
16. The method of claim 1 , wherein the combined steps (a)-(b) occurs for about 1 minute to about 2 minutes.
17. The method of claim 1 , wherein step (c) occurs at a temperature of about 60° C.
18. The method of claim 1 , wherein said solid support is glass or plastic.
19. The method of claim 18 , wherein said solid support further includes a polymer.
20. The method of claim 1 , wherein the DNA molecule is 50, 75, 100, 150, 200, 250, or more nucleotides in length.