Nucleotides and primers with removable blocking groups
First Claim
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1. A method of amplifying nucleic acids comprising:
- a) providing a nucleic acid template, an amplification enzyme, an oligonucleotide primer and a plurality of nucleotide precursors, wherein the plurality of nucleotide precursors comprises a plurality of modified nucleotides, each modified nucleotide comprising a 3′
blocking group, wherein the 3′
blocking group is not thermolabile;
b) removing the 3′
blocking group from the plurality of modified nucleotides in the presence of the amplification enzyme to produce a plurality of unblocked nucleotides; and
c) amplifying the nucleic acid template using the plurality of unblocked nucleotides and the amplification enzyme present during the removing of the 3′
blocking group from the plurality of modified nucleotides, wherein the amplifying the nucleic acid template comprises two or more cycles of exponential amplification of the nucleic acid template.
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Abstract
Provided herein is a method of amplifying nucleic acids using a plurality of modified nucleotides one or more of the nucleotides comprising a 3′ blocking group. Also provided is a method of amplifying nucleic acids using oligonucleotide primers one or both of the primers comprising a 3′ blocking group on one or more of the nucleotides of the primers.
11 Citations
50 Claims
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1. A method of amplifying nucleic acids comprising:
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a) providing a nucleic acid template, an amplification enzyme, an oligonucleotide primer and a plurality of nucleotide precursors, wherein the plurality of nucleotide precursors comprises a plurality of modified nucleotides, each modified nucleotide comprising a 3′
blocking group, wherein the 3′
blocking group is not thermolabile;b) removing the 3′
blocking group from the plurality of modified nucleotides in the presence of the amplification enzyme to produce a plurality of unblocked nucleotides; andc) amplifying the nucleic acid template using the plurality of unblocked nucleotides and the amplification enzyme present during the removing of the 3′
blocking group from the plurality of modified nucleotides, wherein the amplifying the nucleic acid template comprises two or more cycles of exponential amplification of the nucleic acid template. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 49)
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27. A method of amplifying a target nucleic acid comprising:
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a) providing a target nucleic acid template, an amplification enzyme, a plurality of nucleotide precursors, and a pair of oligonucleotide primers, one or both of the oligonucleotide primers comprising a 3′
blocking group on one or more nucleotides of the oligonucleotide primers, wherein the 3′
blocking group is not thermolabile;b) removing the 3′
blocking group from the one or more nucleotides of the oligonucleotide primers in the presence of the amplification enzyme to produce unblocked oligonucleotide primers; andc) amplifying the target nucleic acid template using the unblocked oligonucleotide primers and the amplification enzyme present during the removing of the 3′
blocking group from the one or more nucleotides of the oligonucleotide primers, wherein the amplifying the nucleic acid template comprises two or more cycles of exponential amplification of the nucleic acid template. - View Dependent Claims (28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 50)
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Specification