Massive parallel method for decoding DNA and RNA
View Patent ↗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 nucleotide analogue which comprises:
(a) a base which is an adenine, an analogue of adenine, a cytosine, an analogue of cytosine, a guanine, an analogue of guanine, a thymine, an analogue of thymine, a uracil, or an analogue of uracil;
(b) a unique label attached through a cleavable linker to the base or to the analogue of the base;
(c) a deoxyribose;
(d) a —OR group at a 3′-position of the deoxyribose, wherein R is —CH 2 OCH 3 or —CH 2 CH═CH 2; and
(e) a triphosphate group.
2. The nucleotide analogue of claim 1 , wherein the unique label is a fluorescent moiety or a fluorescent semiconductor crystal.
3. The nucleotide analogue of claim 2 , wherein the fluorescent moiety is 5-carboxyfluorescein, 6-carboxyrhodamine-6G, N,N,N′,N′-tetramethyl-6-carboxyrhodamine, or 6-carboxy-X-rhodamine.
4. The nucleotide analogue of claim 1 , wherein the unique label is a fluorescence energy transfer tag which comprises an energy transfer donor and an energy transfer acceptor.
5. The nucleotide analogue of claim 4 , wherein the energy transfer donor is 5-carboxyfluorescein or cyanine, and wherein the energy transfer acceptor is selected from the group consisting of dichiorocarboxyfluorescein, dichloro-6-carboxyrhodamine-6G, dichloro-N,N,N′,N′-tetramethyl-6-carboxyrhodamine, and dichloro-6-carboxy-X-rhodamine.
6. The nucleotide analogue of claim 1 , wherein the unique label is a mass tag that can be detected and differentiated by a mass spectrometer.
7. The nucleotide analogue of claim 6 , wherein the mass tag is a 2-nitro-α-methyl-benzyl group, a 2-nitro-α-methyl-3-fluorobenzyl group, a 2-nitro-α-methyl-3,4-difluorobenzyl group, or a 2-nitro-α-methyl-3,4-dimethoxybenzyl group.
8. The nucleotide analogue of claim 1 , wherein the unique label is attached through a cleavable linker to a 5-position of cytosine or thymine or to a 7-position of deaza-adenine or deaza-guanine.
9. The nucleotide analogue of claim 1 , wherein the cleavable linker between the unique label and the nucleotide analogue is cleavable by one or more of a physical means, a chemical means, a physical chemical means, heat, or light.
10. The nucleotide analogue of claim 9 , wherein the cleavable linker is a photocleavable linker which comprises a 2-nitrobenzyl moiety.
11. The nucleotide analogue of claim 1 , wherein the nucleotide analogue has the structure:
wherein Dye1, Dye2, Dye3, and Dye4 are four different dye labels; and wherein R is —CH 2 OCH 3 or —CH 2 CH═CH 2 .
12. The nucleotide analogue of claim 11 , wherein the nucleotide analogue has the structure:
wherein R is —CH 2 OCH 3 or —CH 2 CH═CH 2 .
13. The nucleotide analogue of claim 1 , wherein the nucleotide analogue has the structure:
wherein Tag1, Tag2, Tag3, and Tag4 are four different mass tag labels; and wherein R is —CH 2 OCH 3 or —CH 2 CH═CH 2 .
14. The nucleotide analogue of claim 13 , wherein the nucleotide analogue has the structure:
wherein R is —CH 2 OCH 3 or —CH 2 CH═CH 2 .
15. A nucleotide analogue which comprises:
(a) a base which is an adenine, an analogue of adenine, a cytosine, an analogue of cytosine, a guanine, an analogue of guanine, a thymine, an analogue of thymine, a uracil, or analogue of uracil;
(b) a unique label attached through a cleavable linker to the base or to the analogue of the base;
(c) a deoxyribose;
(d) a —OR group at a 3′-position of the deoxyribose, wherein R is a cleavable chemical group; and
(e) a triphosphate group,
wherein the nucleotide analogue has the structure:
wherein Dye1, Dye2, Dye3, and Dye4 are four different dye labels.
16. The nucleotide analogue of claim 15 , wherein the cleavable chemical group is cleavable by one or more of a physical means, a chemical means, a physical chemical means, heat, or light.
17. The nucleotide analogue of claim 15 , wherein the dye is 5-carboxyfluorescein, 6-carboxyrhodamine-6G, N,N,N′,N′-tetramethyl-6-carboxyrhodamine, or 6-carboxy-X-rhodamine.
18. The nucleotide analogue of claim 15 , wherein R is —CH 2 OCH 3 or —CH 2 CH═CH 2 .