Sequencing Performance With Additives
Methods are described which utilize additives that ease secondary structure of DNA to improve sequencing performance. In one embodiment, said additive is betaine. The addition of betaine leads to decreased error rates.
1 . A method for sequencing a nucleic acid by detecting the identity of a nucleotide analogue incorporated into primer extension strand in a polymerase reaction, comprising:
a) providing i) an additive that eases secondary structure of DNA; ii) sequencing primer; (iii) single-stranded template attached to a solid surface; (iv) polymerase and v) a mixture of different nucleotide analogues, wherein each different nucleotide analogue comprises a base selected from the group consisting of adenine, guanine, cytosine, thymine, and uracil, and their analogues; and a unique label attached through a cleavable linker to the base or to an analogue of the base; a deoxyribose; and a cleavable chemical group at the 3′-position of the deoxyribose;
b) hybridizing the sequencing primer to the template so as to create a primer-template complex;
c) adding said additive to said mixture of different nucleotide analogues so as to create an extension solution;
d) exposing said primer-template complex to said extension solution under conditions such that the sequencing primer is extended by incorporating a first nucleotide analogue therein with said polymerase in a polymerase reaction so as to create a primer extension strand, wherein the incorporated nucleotide analogue terminates the polymerase reaction; and
e) 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.
2 . The method of claim 1 , further comprising f) cleaving the cleavable linker between the nucleotide analogue that was incorporated into primer extension strand and the unique label; and cleaving the cleavable chemical group at the 3′-position of the deoxyribose to leave an —OH group.
3 . The method of claim 2 , further comprises g) extending the sequencing primer by incorporating a second nucleotide analogue therein.
4 . The method of claim 3 , further comprising h) detecting the unique label attached to said second nucleotide analogue so as to thereby identity of the second incorporated nucleotide.
5 . The method of claim 1 , wherein said additive is betaine.
6 . A method for sequencing a nucleic acid by detecting the identity of a nucleotide analogue incorporated into primer extension strand in a polymerase reaction, comprising:
a) providing i) sequencing primer, said sequencing primer hybridized to (ii) single-stranded template attached to a solid surface; (iii) polymerase and iv) an extension solution comprising an additive that eases secondary structure of DNA and one or more different nucleotide analogues, wherein each different nucleotide analogue comprises a base selected from the group consisting of adenine, guanine, cytosine, thymine, and uracil, and their analogues; and a unique label attached through a cleavable linker to the base or to an analogue of the base; a deoxyribose; and a cleavable chemical group at the 3′-position of the deoxyribose;
b) exposing said primer hybridized to said template to said extension solution and said polymerase under conditions such that the sequencing primer is extended by incorporating a first nucleotide analogue therein with said polymerase in a polymerase reaction so as to create a primer extension strand, wherein the incorporated nucleotide analogue terminates the polymerase reaction; and
c) 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.
7 . The method of claim 6 , further comprising d) cleaving the cleavable linker between the nucleotide analogue that was incorporated into primer extension strand and the unique label; and cleaving the cleavable chemical group at the 3′-position of the deoxyribose to leave an —OH group.
8 . The method of claim 7 , further comprising e) extending the sequencing primer by incorporating a second nucleotide analogue therein.
9 . The method of claim 8 , further comprising f) detecting the unique label attached to said second nucleotide analogue so as to thereby identity of the second incorporated nucleotide.
10 . The method of claim 6 , wherein said additive is betaine.