Four-color DNA sequencing by synthesis using cleavable fluorescent nucleotide reversible terminators
This invention provides a process for sequencing single-stranded DNA employing modified nucleotides.
1. A method, comprising:
a) providing a plurality of primer strands bound to nucleic acid templates, a fraction of said plurality of primer strands comprising a free 3′-OH and another fraction of said plurality of primer strands comprising an incorporated labeled nucleotide analogue reversible terminator, wherein each reversible terminator comprises a cleavable moiety bound to the 3-oxygen of the deoxyribose, wherein a label is bound via a chemically cleavable linker; and
b) extending with a polymerase one or more of said primer strands that has a free 3′-OH with an unlabeled nucleotide analogue reversible terminator, wherein each unlabeled analogue comprises a cleavable moiety bound to the 3′-oxygen of the deoxyribose.
2. The method of claim 1 , further comprising the step of detecting the incorporated labeled reversible terminator.
3. The method of claim 2 , further comprising the step of chemically cleaving the chemically cleavable linker of said incorporated labeled reversible terminator so as to remove the label.
4. The method of claim 1 , wherein the moiety cleavably linked to the 3′-oxygen of the deoxyribose in said incorporated labeled reversible terminator is chemically cleavable.
5. The method of claim 1 , wherein the moiety cleavably linked to the 3′-oxygen of the deoxyribose in said incorporated labeled reversible terminator is photocleavable.
6. The method of claim 1 , wherein the cleavable moiety bound to the 3′-oxygen of the deoxyribose of the incorporated labeled nucleotide analogue reversible terminator is an allyl moiety or a 2-nitrobenzyl moiety.
7. The method of claim 1 , wherein the cleavable moiety bound to the 3′-oxygen of the deoxyribose of the unlabeled nucleotide analogue reversible terminator is an allyl moiety or a 2-nitrobenzyl moiety.
8. The method of claim 1 , wherein the cleavable moiety attached to the 3′-oxygen of the deoxyribose of the incorporated labeled nucleotide analogue reversible terminator is chemically cleavable with Na 2 PdCl 4 /P(PhSO 3 Na) 3 .
9. The method of claim 1 , wherein the cleavable moiety attached to the 3′-oxygen of the deoxyribose of the unlabeled nucleotide analogue reversible terminator is chemically cleavable with Na 2 PdCl 4 /P(PhSO 3 Na) 3 .
10. The method of claim 1 , wherein the label is bound by a chemically cleavable linker which is chemically cleavable with Na 2 PdCl 4 /P(PhSO 3 Na) 3 .
11. The method of claim 1 , where the polymerase is a 9° N polymerase or a variant thereof.
12. The method of claim 1 , wherein the primers are self-priming moieties.
13. The method of claim 1 , wherein the nucleic acid templates are bound to a solid substrate.
14. The method of claim 13 , wherein the nucleic acid templates are bound to the solid substrate via 1,3-dipolar azide-alkyne cycloaddition chemistry.
15. The method of claim 13 , wherein about 1000 or fewer copies of the nucleic acid template are bound to the solid substrate.
16. The method of claim 13 , wherein the nucleic acid templates are bound to the solid substrate via a polyethylene glycol molecule and the solid substrate is azide-functionalized.
17. The method of claim 13 , wherein the nucleic acid templates are immobilized on the solid substrate via an azido linkage, an alkynyl linkage, or biotin streptavidin interaction.
18. The method of claim 13 , wherein the solid substrate is in the form of a chip, a bead, or a slide.
19. The method of claim 13 , wherein the solid substrate is plastic, or glass.