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 for sequencing a DNA template of at least 30 nucleotides which comprises the stepwise detection of the identities of nucleotide analogues of adenine, guanine, cytosine, and thymine incorporated into the end of a growing strand of complementary DNA synthesized stepwise using a DNA polymerase in a DNA polymerase reaction, wherein the nucleotide analogues act as reversible terminators in the DNA polymerase reaction and wherein one such nucleotide analogue is any of the following:
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 the DNA polymerase or with incorporation of the analogue into the growing DNA strand, during the DNA polymerase reaction, (c) is stable during the 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 the DNA polymerase reaction;
wherein Tag represents a detectable fluorescent moiety;
wherein (i) the nucleotide analogue is incorporated into the growing DNA strand as a result of the DNA polymerase reaction, (ii) the Tag is detected and thereby the incorporated analogue is detected, and (iii) the covalent bond between the 3′-oxygen and R is chemically cleaved under conditions enabling the growing DNA strand to survive the washing, detection and cleavage processes and to remain annealed to the DNA template so as to allow the incorporation and detection of the next nucleotide analogue;
wherein Y represents a chemically cleavable, chemical linker which (a) does not interfere with recognition of the analogue as a substrate by the DNA polymerase or with incorporation of the analogue into the growing DNA strand during the DNA polymerase reaction and (b) is stable during the DNA polymerase reaction;
wherein the nucleotide analogue:
i) is recognized as a substrate by the DNA polymerase for incorporation into the growing DNA strand during the DNA polymerase reaction,
ii) is efficiently and faithfully incorporated at the end of the growing DNA strand during the DNA polymerase reaction,
iii) produces a 3′—OH group on the deoxyribose upon cleavage of R under conditions compatible with DNA sequencing, and
iv) no longer includes a tag on the base upon cleavage of Y under conditions compatible with DNA sequencing;
and wherein if the nucleotide analogue is: (A), it is capable of forming hydrogen bonds with cytosine or a cytosine nucleotide analogue; (B), it is capable of forming hydrogen bonds with thymine or a thymine nucleotide analogue; (C), it is capable of forming hydrogen bonds with guanine or a guanine nucleotide analogue; or (D), it is capable of forming hydrogen bonds with adenine or an adenine nucleotide analogue.
2. A method for simultaneously sequencing a plurality of different nucleic acid templates which comprises simultaneously applying the method of claim 1 to the plurality of different nucleic acid templates.
3. A method for sequencing a DNA template of at least 30 nucleotides which comprises the stepwise detection of the identities of nucleotide analogues of adenine, guanine, cytosine, and thymine incorporated into the end of a growing strand of complementary DNA synthesized stepwise using a DNA polymerase in a DNA polymerase reaction, wherein the nucleotide analogues act as reversible terminators in the DNA polymerase reaction and wherein one such nucleotide analogue is any of the following:
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 the DNA polymerase or with incorporation of the analogue into the growing DNA strand, during the DNA polymerase reaction, (c) is stable during the 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 the DNA polymerase reaction;
wherein Tag represents a detectable fluorescent moiety;
wherein (i) the nucleotide analogue is incorporated into the growing DNA strand as a result of the DNA polymerase reaction, (ii) the Tag is detected and thereby the incorporated analogue is detected, and (iii) the covalent bond between the 3′-oxygen and R is chemically cleaved under conditions enabling the growing DNA strand to survive the washing, detection and cleavage processes and to remain annealed to the DNA template so as to allow the incorporation and detection of the next nucleotide analogue;
wherein Y represents a chemically cleavable, chemical linker which (a) does not interfere with recognition of the analogue as a substrate by the DNA polymerase or with incorporation of the analogue into the growing DNA strand during the DNA polymerase reaction and (b) is stable during the DNA polymerase reaction;
wherein the nucleotide analogue:
i) is recognized as a substrate by the DNA polymerase for incorporation into the growing DNA strand during the DNA polymerase reaction,
ii) is efficiently and faithfully incorporated at the end of the growing DNA strand during the DNA polymerase reaction,
iii) produces a 3′—OH group on the deoxyribose upon cleavage of R under conditions compatible with DNA sequencing, and
iv) no longer includes a tag on the base upon cleavage of Y under conditions compatible with DNA sequencing;
and wherein if the nucleotide analogue is: (A), it is capable of forming hydrogen bonds with cytosine or a cytosine nucleotide analogue; (B), it is capable of forming hydrogen bonds with thymine or a thymine nucleotide analogue; (C), it is capable of forming hydrogen bonds with guanine or a guanine nucleotide analogue; or (D), it is capable of forming hydrogen bonds with adenine or an adenine nucleotide analogue.
4. A method for simultaneously sequencing a plurality of different nucleic acid templates which comprises simultaneously applying the method of claim 3 to the plurality of different nucleic acid templates.