Methods and compositions for improving fidelity in a nucleic acid synthesis reaction
First Claim
1. A method for inhibiting nucleotide misincorporation in a nucleic acid synthesis reaction, the method comprising conducting a polymerization reaction on a nucleic acid duplex comprising a template and a primer in the presence ofa nucleic acid polymerase, a first detectably labeled nucleotide corresponding to a first nucleotide species complementary to the position opposite the incorporation site at the 3′
- terminus of the primer,a second nucleotide of a different species, andat least one nucleotide derivative, each of the nucleotide derivatives corresponding to a different nucleotide species than the first nucleotide species,wherein each nucleotide derivative comprises a modification that inhibits formation of a phosphodiester bond between the nucleotide derivative and a free 3′
hydroxyl on a primer nucleotide.
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Abstract
The invention provides methods and compositions for improving the fidelity of a sequencing-by-synthesis reaction by using a nucleotide derivative that forms a hydrogen bond with a complementary nucleotide on a template, but fails to form a phosphodiester bond with the 3′ hydroxyl group of a primer under conditions otherwise suitable for a polymerization reaction; thereby blocking incorporation of a mismatched nucleotide.
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Citations
30 Claims
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1. A method for inhibiting nucleotide misincorporation in a nucleic acid synthesis reaction, the method comprising conducting a polymerization reaction on a nucleic acid duplex comprising a template and a primer in the presence of
a nucleic acid polymerase, a first detectably labeled nucleotide corresponding to a first nucleotide species complementary to the position opposite the incorporation site at the 3′ - terminus of the primer,
a second nucleotide of a different species, and at least one nucleotide derivative, each of the nucleotide derivatives corresponding to a different nucleotide species than the first nucleotide species, wherein each nucleotide derivative comprises a modification that inhibits formation of a phosphodiester bond between the nucleotide derivative and a free 3′
hydroxyl on a primer nucleotide. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 16, 30)
- terminus of the primer,
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11. A method for sequencing a nucleic acid, the method comprising the steps of:
- (a) exposing a nucleic acid duplex comprising a template and a primer to a nucleic acid polymerase, a first labeled nucleotide, and at least one nucleotide derivative corresponding to a nucleotide species different from said first nucleotide;
wherein said nucleotide derivative comprises a modification that inhibits formation of a phosphodiester bond between the nucleotide derivative and a 3′
hydroxyl group of the primer, and (b) removing nucleotide derivatives and labeled nucleotides that are not incorporated into the primer;
(c) determining incorporation of the labeled nucleotide and thereby identifying a nucleotide that is complementary to a nucleotide at a position on said template opposite the incorporated labeled nucleotide; and
(d) repeating steps (a), (b) and (c) with a second labeled nucleotide and at least one nucleotide derivative corresponding to a nucleotide species different from said second labeled nucleotide. - View Dependent Claims (12, 13, 14, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29)
- (a) exposing a nucleic acid duplex comprising a template and a primer to a nucleic acid polymerase, a first labeled nucleotide, and at least one nucleotide derivative corresponding to a nucleotide species different from said first nucleotide;
Specification