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 method of determining the identity of a nucleotide analogue incorporated into a nucleic acid primer extension strand, comprising: a) contacting a nucleic acid template attached to a solid surface with a nucleic acid primer which hybridizes to the template; b) simultaneously contacting the product of step a) with a polymerase and four nucleotide analogues which are either (i) aA, aC, aG, and aT, or (ii) aA, aC, aG, and aU, so as to incorporate one of the nucleotide analogues onto the nucleic acid primer and form a nucleic acid primer extension strand, wherein each nucleotide analogue within (i) or (ii) comprises a base labeled with a unique label and contains a small removable chemical moiety capping the 3′-OH group of the sugar of the nucleotide analogue, wherein said small cleavable chemical group does not interfere with the recognition of the nucleotide analogue by polymerase as a substrate; and c) detecting the unique label of the incorporated nucleotide analogue, so as to thereby determine the identity of the nucleotide analogue incorporated into the nucleic acid primer extension strand.
2. The method of claim 1 , further comprising removing the chemical moiety capping the 3′-OH group of the sugar of the incorporated nucleotide analogue, thereby permitting the incorporation of a further nucleotide analogue so as to create a growing annealed nucleic acid primer extension strand.
3. The method of claim 1 , wherein each of the four unique labels is a fluorescent label.
4. The method of claim 3 , wherein one of said four unique labels comprises ROX.
5. The method of claim 3 , wherein one of said four unique labels comprises R6G.
6. The method of claim 1 , wherein said base of at least one of said nucleotide analogues is a deazapurine.