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.
Claims22
A method of detecting a fluorescently labeled nucleotide incorporated into a DNA coupled to a phosphine moiety, comprising:
- a) reacting a DNA comprising an azido group with a compound comprising a phosphine moiety to produce the DNA coupled to the phosphine moiety;
- b) contacting the DNA coupled to the phosphine moiety of step a) with a polymerase and a fluorescently labeled nucleotide so as to incorporate the fluorescently labeled nucleotide into the DNA coupled to the phosphine moiety;
- and
- c) detecting the fluorescently labeled nucleotide incorporated into the DNA coupled to the phosphine moiety.
A method of detecting a fluorescently labeled nucleotide incorporated into a DNA fragment coupled to a phosphine moiety, comprising:
- a) reacting a DNA fragment having an azido group attached thereto through a linker with a compound comprising a phosphine moiety to produce the DNA fragment coupled to the phosphine moiety;
- b) contacting the DNA fragment coupled to the phosphine moiety of step a) with a polymerase and a fluorescently labeled nucleotide so as to thereby incorporate the fluorescently labeled nucleotide into the DNA fragment coupled to the phosphine moiety;
- and
- c) detecting the fluorescently labeled nucleotide incorporated into the DNA fragment coupled to the phosphine moiety.
A method comprising:
- a) providing a nucleic acid template hybridized to a primer, the primer comprising an azido group;
- and
- b) reacting said azido group with a phosphine.
A complex comprising a primer hybridized to a nucleic acid template, the primer comprising an azido group, and the nucleic acid template derived from genomic nucleic acid.
Dependencies are inferred from the claim text.