Massive parallel method for decoding DNA and RNA
This invention provides methods for attaching a nucleic acid to a solid surface and for sequencing nucleic acid by detecting the identity of each nucleotide analogue after the nucleotide analogue is incorporated into a growing strand of DNA in a polymerase reaction. The invention also provides nucleotide analogues which comprise unique labels attached to the nucleotide analogue through a cleavable linker, and a cleavable chemical group to cap the —OH group at the 3′-position of the deoxyribose.
1. A method of sequencing nucleic acid, comprising:
a) reacting a DNA strand containing an azido group with a phosphine;
b) growing a double-stranded DNA containing said DNA strand by incorporating a fluorescently labeled nucleotide into said strand; and
c) identifying the fluorescently labeled nucleotide, so as to sequence the nucleic acid.
2. The method of claim 1 , wherein said fluorescently labeled nucleotide has the label linked to the base and a blocking group on the 3′-hydroxyl group.
3. The method of claim 2 , wherein the label is attached to the base via a cleavable linker.
4. The method of claim 1 , wherein the phosphine is a triarylphosphine.
5. The method of claim 1 , wherein the azido group is attached to the deoxyribose at a 5′ position.
6. The method of claim 1 , wherein the azido group is attached to the deoxyribose via a linker.
7. The method of claim 1 , wherein the nucleotide analogue comprises a deazapurine base.
8. A method of sequencing nucleic acid comprising:
a) providing a nucleic acid template hybridized to a primer, the primer comprising an azido group;
b) reacting said azido group with a phosphine;
c) extending the primer hybridized to said nucleic acid template with a fluorescently labeled nucleotide or nucleotide analogue,
wherein said fluorescently labeled nucleotide or nucleotide analogue has the label linked to the base and a blocking group on the 3′-hydroxyl group; and
d) identifying the fluorescently labeled nucleotide, so as to sequence the nucleic acid.
9. The method of claim 8 , wherein said fluorescently labeled nucleotide or nucleotide analogue has the label on the base and a blocking group on the 3′-hydroxyl group.
10. The method of claim 9 , wherein the label is attached to the base via a cleavable linker.
11. The method of claim 8 , wherein the phosphine is a triarylphosphine.
12. The method of claim 8 , wherein the azido group is attached to the deoxyribose at a 5′ position.
13. The method of claim 8 , wherein the azido group is attached to the deoxyribose via a linker.
14. The method of claim 8 , wherein the nucleotide analogue comprises a deazapurine base.
15. A method of simultaneously sequencing a plurality of different nucleic acids, comprising:
a) reacting each of a plurality of different DNA strands containing an azido group with a phosphine;
b) growing a plurality of double-stranded DNA, each of which comprises one of said DNA strands, by incorporating a fluorescently labeled nucleotide; and
c) identifying each fluorescently labeled nucleotide, so as to simultaneously sequence the plurality of different nucleic acids.
16. The method of claim 15 , wherein said fluorescently labeled nucleotide or nucleotide analogue has the label linked to the base and a blocking group on the 3′-hydroxyl group.
17. The method of claim 16 , wherein the label is attached to the base via a cleavable linker.
18. The method of claim 15 , wherein the phosphine is a triarylphosphine.
19. The method of claim 15 , wherein the azido group is attached to the deoxyribose at a 5′ position via a linker.
20. The method of claim 15 , wherein the nucleotide analogue comprises a deazapurine base.