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 thymine deoxyribonucleotide analogue having the structure:
wherein R (a) represents a small, chemically cleavable, chemical group capping the oxygen at the 3′ position of the deoxyribose of the deoxyribonucleotide analogue, (b) does not interfere with recognition of the analogue as a substrate by a DNA polymerase, (c) is stable during a DNA polymerase reaction, (d) does not contain a ketone group, and (e) is not a —CH 2 CH═CH 2 group;
wherein OR is not a methoxy group or an ester group;
wherein the covalent bond between the 3′-oxygen and R is stable during a DNA polymerase reaction;
wherein tag represents a detectable fluorescent moiety;
wherein Y represents a chemically cleavable, chemical linker which (a) does not interfere with recognition of the analogue as a substrate by a DNA polymerase and (b) is stable during a DNA polymerase reaction; and
wherein the thymine deoxyribonucleotide analogue:
i) is recognized as a substrate by a DNA polymerase,
ii) is incorporated at the end of a growing strand of DNA during a DNA polymerase reaction,
iii) produces a 3′-OH group on the deoxyribose upon cleavage of R,
iv) no longer includes a tag on the base upon cleavage of Y, and
v) is capable of forming hydrogen bonds with adenine or an adenine nucleotide analogue.
2. A thymine deoxyribonucleotide analogue having the structure:
wherein R (a) represents a small, chemically cleavable, chemical group capping the oxygen at the 3′ position of the deoxyribose of the deoxyribonucleotide analogue, (b) does not interfere with recognition of the analogue as a substrate by a DNA polymerase, (c) is stable during a DNA polymerase reaction, and (d) does not contain a ketone group;
wherein OR is not a methoxy group, an ester group, or an allyl ether group;
wherein the covalent bond between the 3′-oxygen and R is stable during a DNA polymerase reaction;
wherein tag represents a detectable fluorescent moiety;
wherein Y represents a chemically cleavable, chemical linker which (a) does not interfere with recognition of the analogue as a substrate by a DNA polymerase and (b) is stable during a DNA polymerase reaction; and
wherein the thymine deoxyribonucleotide analogue:
i) is recognized as a substrate by a DNA polymerase,
ii) is incorporated at the end of a growing strand of DNA during a DNA polymerase reaction,
iii) produces a 3′-OH group on the deoxyribose upon cleavage of R,
iv) no longer includes a tag on the base upon cleavage of Y, and
v) is capable of forming hydrogen bonds with adenine or an adenine nucleotide analogue.