Pyrophosphorolysis activated polymerization (PAP)
First Claim
1. A method of detecting a nucleic acid which comprises:
- (a) annealing to a nucleic acid a first oligonucleotide P*, wherein the first oligonucleotide P* has a non-extendable 3′
end, wherein the 3′
non-extendable terminus of first oligonucleotide P* is removable by pyrophosphorolysis;
(b) removing the 3′
non-extendable terminus of the first oligonucleotide P* by pyrophosphorolysis to produce an unblocked first oligonucleotide;
(c) extending the unblocked first oligonucleotide;
(d) detecting the-presence of the nucleic acid by detecting the extended first oligonucleotide.
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Abstract
A novel method of pyrophosphorolysis activated polymerization (PAP) has been developed. In PAP, pyrophosphorolysis and polymerization by DNA polymerase are coupled serially for each amplification by using an activatable oligonucleotide P* that has a non-extendible 3′-deoxynucleotide at its 3′ terminus. PAP can be applied for exponential amplification or for linear amplification. PAP can be applied to amplification of a rare allele in admixture with one or more wild-type alleles by using an activatable oligonucleotide P* that is an exact match at its 3′ end for the rare allele but has a mismatch at or rear its 3′ terminus for the wild-type allele. PAP is inhibited by a mismatch in the 3′ specific sequence as far as 16 nucleotides away from the 3′ terminus. PAP can greatly increase the specificity of detection of an extremely rare mutant allele in the presence of the wild-type allele. Specificity results from both pyrophosphorolysis and polymerization since significant nonspecific amplification requires the combination of mismatch pyrophosphorolysis and misincorporation by the DNA polymerase, an extremely rare event. Using genetically engineered DNA polymerases greatly improves the efficiency of PAP.
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Citations
72 Claims
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1. A method of detecting a nucleic acid which comprises:
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(a) annealing to a nucleic acid a first oligonucleotide P*, wherein the first oligonucleotide P* has a non-extendable 3′
end, wherein the 3′
non-extendable terminus of first oligonucleotide P* is removable by pyrophosphorolysis;
(b) removing the 3′
non-extendable terminus of the first oligonucleotide P* by pyrophosphorolysis to produce an unblocked first oligonucleotide;
(c) extending the unblocked first oligonucleotide;
(d) detecting the-presence of the nucleic acid by detecting the extended first oligonucleotide. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16)
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17. A method of synthesizing a nucleic acid which comprises:
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(a) annealing to a nucleic acid a first oligonucleotide P* which has a non-extendable 3′
end, wherein the 3′
non-extendable terminus of the first oligonucleotide P* is removable by pyrophosphorolysis;
(b) removing the 3′
non-extendable terminus of the first oligonucleotide P* annealed to the nucleic acid by pyrophosphorolysis; and
(c) extending the unblocked first oligonucleotide using a nucleic acid polymerase. - View Dependent Claims (18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31)
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32. A method of detecting a nucleic acid which comprises:
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(a) annealing to a nucleic acid two oligonucleotide P*s, wherein each oligonucleotide P* has a non-extendable 3′
end, wherein the 3′
non-extendable terminus of each oligonucleotide P* is removable by pyrophosphorolysis, wherein one oligonucleotide P* overlaps with the other oligonucleotide P* by at least one nucleotide at their respective 3′
ends, and wherein one oligonucleotide P* anneals to a first nucleic acid strand and the other oligonucleotide P* anneals to a nucleic acid strand which is the complement of the first nucleic acid strand;
(b) removing the 3′
non-extendable terminus of the annealed first and second oligonucleotide P*s by pyrophosphorolysis to produce unblocked oligonucletides;
(c) extending the unblocked oligonucleotides; and
(d) detecting the extended oligonculeotides. - View Dependent Claims (33, 34, 35, 36, 37, 38, 39)
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40. A method of nucleic acid detection which comprises:
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(a) synthesizing a template nucleic acid from the nucleic acid;
(b) annealing to the template nucleic acid a first oligonucleotide P* which has a non-extendable 3′
end, wherein the 3′
non-extendable terminus of the first oligonucleotide P* is removable by pyrophosphorolysis;
(c) removing the 3′
non-extendable terminus of the first oligonucleotide P* annealed to the template nucleic acid by pyrophosphorolysis; and
(d) extending the unblocked oligonucleotide - View Dependent Claims (41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67)
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68. A pyrophosphorolysis activated polymerization method of synthesizing a desired nucleic acid strand on a nucleic acid template strand which comprises serially
(a) annealing to the template strand an activatable oligonucleotide P* that has a non-extendable 3′ - -deoxynucleotide at its 3′
terminus and has a mismatch with respect to the corresponding nucleotide on the template strand,(b) pyrophosphorolyzing the resulting duplex with pyrophosphate and an enzyme that has pyrophosphorolysis activity and activates the oligonucleotide P* by removal of the terminal 3′
-deoxynucleotide, and(c) polymerizing by extending the activated oligonucleotide P* on the template strand in presence of four nucleoside triphosphates and a nucleic acid polymerase to synthesize the desired nucleic acid strand. - View Dependent Claims (69, 70, 71, 72)
- -deoxynucleotide at its 3′
Specification