Massive parallel method for decoding DNA and RNA
AssigneeThe Trustees of Columbia University in the City of New York
InventorsJingyue Ju, Zengmin Li, John Robert Edwards, Yasuhiro Itagaki
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Abstract
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.
Claims28
A method for sequencing a nucleic acid by detecting the identity of a nucleotide analogue after the nucleotide analogue is incorporated into a growing strand of DNA in a polymerase reaction, which comprises the following steps:
- (i) attaching a 5′ end of the nucleic acid to a solid surface;
- (ii) attaching a primer to the nucleic acid attached to the solid surface;
- (iii) adding a polymerase and one or more different nucleotide analogues to the nucleic acid to thereby incorporate a nucleotide analogue into the growing strand of DNA, wherein the incorporated nucleotide analogue terminates the polymerase reaction and wherein each different nucleotide analogue comprises (a) a base selected from the group consisting of adenine, guanine, cytosine, thymine, and uracil, and their analogues;
- (b) a unique label attached through a cleavable linker to the base or to an analogue of the base;
- (c) a deoxyribose;
- and (d) a cleavable chemical group to cap an —OH group at a 3′-position of the deoxyribose;
- (iv) washing the solid surface to remove unincorporated nucleotide analogues;
- (v) detecting the unique label attached to the nucleotide analogue that has been incorporated into the growing strand of DNA, so as to thereby identify the incorporated nucleotide analogue;
- (vi) adding one or more chemical compounds to permanently cap any unreacted —OH group on the primer attached to the nucleic acid or on a primer extension strand formed by adding one or more nucleotides or nucleotide analogues to the primer;
- (vii) cleaving the cleavable linker between the nucleotide analogue that was incorporated into the growing strand of DNA and the unique label;
- (viii) cleaving the cleavable chemical group capping the —OH group at the 3′-position of the deoxyribose to uncap the —OH group, and washing the solid surface to remove cleaved compounds;
- and
- (ix) repeating steps (iii) through (viii) so as to detect the identity of a newly incorporated nucleotide analogue into the growing strand of DNA;
- wherein if the unique label is a dye, the order of steps (v) through (vii) is:
- (v), (vi), and (vii);
- and
- wherein if the unique label is a mass tag, the order of steps (v) through (vii) is:
- (vi), (vii), and (v).
Dependencies are inferred from the claim text.