Pyrophosphorolysis activated polymerization (PAP): application to allele-specific amplification and nucleic acid sequence determination
First Claim
1. A pyrophosphorolysis activated polymerization (PAP) method of synthesizing a desired nucleic acid strand on a nucleic acid template strand which comprises serially(a) annealing to the template strand a complementary activatable oligonucleotide P* that has a non-extendable 3′
- -deoxynucleotide at its 3′
terminus and that has no nucleotides at or near its 3′
terminus that mismatch the corresponding nucleotides on the template strand, so that the terminal 3′
-deoxynucleotide is hybridized to the template strand when the oligonucleotide P* is annealed, (b) pyrophosphorolyzing the resulting duplex with pyrophosphate and an enzyme that has pyrophosphorolysis activity and activates the oligonucleotide P* by removal of the hybridized 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.
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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-extendable 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 near its 3′ terminus for the wild type allele. PAP is inhibited by a mismatch in the 3′ specific subsequence 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.
39 Citations
98 Claims
-
1. A pyrophosphorolysis activated polymerization (PAP) method of synthesizing a desired nucleic acid strand on a nucleic acid template strand which comprises serially
(a) annealing to the template strand a complementary activatable oligonucleotide P* that has a non-extendable 3′ - -deoxynucleotide at its 3′
terminus and that has no nucleotides at or near its 3′
terminus that mismatch the corresponding nucleotides on the template strand, so that the terminal 3′
-deoxynucleotide is hybridized to the template strand when the oligonucleotide P* is annealed,(b) pyrophosphorolyzing the resulting duplex with pyrophosphate and an enzyme that has pyrophosphorolysis activity and activates the oligonucleotide P* by removal of the hybridized 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 (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, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42)
(d) separating the desired nucleic acid strand of step (c) from the template strand and (e) repeating steps (a)-(d) until a desired level of amplification of the desired nucleic acid strand is achieved. -
3. The pyrophosphorolysis activated polymerization method of claim 2 carried out in the presence of a second oligonucleotide that in step (a) anneals to the separated desired nucleic acid strand product of step (d), and wherein step (c) includes polymerizing by extending the second oligonucleotide on the desired nucleic acid strand to synthesize a copy of the nucleic acid template strand, and step (d) includes separating the synthesized nucleic acid template strand from the desired nucleic acid strand, so that amplification is exponential.
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4. The pyrophosphorolysis activated polymerization method claim of 2 wherein steps (a) to (c) are conducted sequentially as two or more temperature stages on a thermocycler.
-
5. The pyrophosphorolysis activated polymerization method of claim 4 carried out in the presence of a second oligonucleotide that in step (a) anneals to the separated desired nucleic acid strand product of step (d), and wherein step (c) includes polymerizing by extending the second oligonucleotide on the desired nucleic acid strand to synthesize a copy of the nucleic acid template strand, and step (d) includes separating the synthesized nucleic acid template strand from the desired nucleic acid strand, so that amplification is exponential.
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6. The pyrophosphorolysis activated polymerization method of claim 2 wherein steps (a) to (c) are conducted as one temperature stage on a thermocycler.
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7. The pyrophosphorolysis activated polymerization method of claim 6 carried out in the presence of a second oligonucleotide that in step (a) anneals to the separated desired nucleic acid strand product of step (d), and wherein step (c) includes polymerizing by extending the second oligonucleotide on the desired nucleic acid strand to synthesize a copy of the nucleic acid template strand, and step (d) includes separating the synthesized nucleic acid template strand from the desired nucleic acid strand, so that amplification is exponential.
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8. The pyrophosphorolysis activated polymerization method of claim 6 wherein the DNA polymerase is also the enzyme having pyrophosphorolysis activity.
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9. The pyrophosphorolysis activated polymerization method of claim 8 carried out in the presence of a second oligonucleotide that in step (a) anneals to the separated desired nucleic acid strand product of step (d), and wherein step (c) includes polymerizing by extending the second oligonucleotide on the desired nucleic acid strand to synthesize a copy of the nucleic acid template strand, and step (d) includes separating the synthesized nucleic acid template strand from the desired nucleic acid strand, so that amplification is exponential.
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10. The pyrophosphorolysis activated polymerization method of claim 8 wherein the DNA polymerase is thermostable Tfl or Taq.
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11. The pyrophosphorolysis activated polymerization method of claim 10 carried out in the presence of a second oligonucleotide that in step (a) anneals to the separated desired nucleic acid strand product of step (d), and wherein step (c) includes polymerizing by extending the second oligonucleotide on the desired nucleic acid strand to synthesize a copy of the nucleic acid template strand, and step (d) includes separating the synthesized nucleic acid template strand from the desired nucleic acid strand, so that amplification is exponential.
-
12. The pyrophosphorolysis activated polymerization method of claim 8 wherein the DNA polymerase is a genetically engineered DNA polymerase selected from the group consisting of AmpliTaqFS, ThermoSequenase and a DNA polymerase modified to contain a mutation equivalent to a F667Y mutation in TaqFS.
-
13. The pyrophosphorolysis activated polymerization method of claim 12 carried out in the presence of a second oligonucleotide that in step (a) anneals to the separated desired nucleic acid strand product of step (d), and wherein step (c) includes polymerizing by extending the second oligonucleotide on the desired nucleic acid strand to synthesize a copy of the nucleic acid template strand, and step (d) includes separating the synthesized nucleic acid template strand from the desired nucleic acid strand, so that amplification is exponential.
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14. The pyrophosphorolysis activated polymerization method of claim 2 applied to allele-specific amplification, wherein the nucleic acid template strand is present in admixture with a second, allelelic nucleic acid strand that differs from the template strand so that the activatable oligonucleotide P* has at least one nucleotide at or near its 3′
- terminus that mismatches the corresponding nucleotide of the alleleic strand, so that in step (a) the terminal 3′
-deoxynucleotide of oligonucleotide P* is not hybridized to the allelelic strand; and
thus in step (b) the pyrophosphate and enzyme that has pyrophosphorolysis activity do not substantially remove the non-hybridized terminal 3′
-deoxynucleotide from the activatable oligonucleotide P* and in step (c) the oligonucleotide P* is not substantially extended by polymerization on the allelic strand, whereby the desired nucleic acid strand synthesized on the template strand is amplified preferentially over any nucleic acid strand synthesized on the allelelic strand.
- terminus that mismatches the corresponding nucleotide of the alleleic strand, so that in step (a) the terminal 3′
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15. The pyrophosphorolysis activated polymerization method of claim 14 wherein the mismatch between the activatable oligonucleotide P* and the template strand occurs at the terminal 3′
- -deoxynucleotide or at the first or second nucleotide from the terminal 3′
-deoxynucleotide.
- -deoxynucleotide or at the first or second nucleotide from the terminal 3′
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16. The pyrophosphorolysis activated polymerization method of claim 14 wherein the mismatch between the activatable oligonucleotide P* and the template strand occurs at the terminal 3′
- -deoxynucleotide.
-
17. The pyrophosphorolysis activated polymerization method of claim 14 carried out in the presence of a second oligonucleotide that in step (a) anneals to the separated desired nucleic acid strand product of step (d), and wherein step (c) includes polymerizing by extending the second oligonucleotide on the desired nucleic acid strand to synthesize a copy of the nucleic acid template strand, and step (d) includes separating the synthesized nucleic acid template strand from the desired nucleic acid strand, so that amplification is exponential.
-
18. The pyrophosphorolysis activated polymerization method of claim 17 wherein the mismatch between the activatable oligonucleotide P* and the template strand occurs at the terminal 3′
- -deoxynucleotide or at the first or second nucleotide from the terminal 3′
-deoxynucleotide.
- -deoxynucleotide or at the first or second nucleotide from the terminal 3′
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19. The pyrophosphorolysis activated polymerization method of claim 17 wherein the mismatch between the activatable oligonucleotide P* and the template strand occurs at the terminal 3′
- -deoxynucleotide.
-
20. The pyrophosphorolysis activated polymerization method of claim 17, wherein the desired nucleic acid strand, the template strand, and the alleleic strand are DNA strands, the activatable oligonucleotide P* and the second oligonucleotide are both 2′
- -deoxyoligonucleotides, the terminal deoxynucleotide is a 2′
,3′
-dideoxynucleotide, the four nucleoside triphosphates are 2′
-deoxynucleoside triphosphates, and the nucleic acid polymerase is a DNA polymerase.
- -deoxyoligonucleotides, the terminal deoxynucleotide is a 2′
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21. The pyrophosphorolysis activated polymerization method of claim 20 wherein the mismatch between the activatable 2′
- -deoxyoligonucleotide P* and the template strand occurs at the terminal 2′
,3′
-deoxynucleotide or at the first or second 2′
-deoxynucleotide from the terminal 2′
,3′
-deoxynucleotide.
- -deoxyoligonucleotide P* and the template strand occurs at the terminal 2′
-
22. The pyrophosphorolysis activated polymerization method of claim 20 wherein the mismatch between the activatable 2′
- -deoxyoligonucleotide P* and the template strand occurs at the terminal 2′
,3′
-deoxynucleotide.
- -deoxyoligonucleotide P* and the template strand occurs at the terminal 2′
-
23. The pyrophosphorolysis activated polymerization method of claim 14, wherein the desired nucleic acid strand, the template strand, and the alleleic strand are DNA strands, the activatable oligonucleotide P* is a 2′
- -deoxyoligonucleotide, the terminal deoxynucleotide is a 2′
,3′
-dideoxynucleotide, the four nucleoside triphosphates are 2′
-deoxynucleoside triphosphates, and the nucleic acid polymerase is a DNA polymerase.
- -deoxyoligonucleotide, the terminal deoxynucleotide is a 2′
-
24. The pyrophosphorolysis activated polymerization method of claim 23 wherein the mismatch between the activatable oligonucleotide P* and the template strand occurs at the terminal 3′
- -deoxynucleotide or at the first or second nucleotide from the terminal 3′
-deoxynucleotide.
- -deoxynucleotide or at the first or second nucleotide from the terminal 3′
-
25. The pyrophosphorolysis activated polymerization method of claim 23 wherein the mismatch between the activatable oligonucleotide P* and the template strand-occurs at the terminal 3′
- -deoxynucleotide.
-
26. The pyrophosphorolysis activated polymerization method of claim 23 carried out in the presence of a second oligonucleotide that in step (a) anneals to the separated desired nucleic acid strand product of step (d), and wherein step (c) includes polymerizing by extending the second oligonucleotide on the desired nucleic acid strand to synthesize a copy of the nucleic acid template strand, and step (d) includes separating the synthesized nucleic acid template strand from the desired nucleic acid strand, so that amplification is exponential.
-
27. The pyrophosphorolysis activated polymerization method of claim 26 wherein the mismatch between the activatable oligonucleotide P* and the template strand occurs at the terminal 3′
- -deoxynucleotide or at the first or second nucleotide from the terminal 3′
-deoxynucleotide.
- -deoxynucleotide or at the first or second nucleotide from the terminal 3′
-
28. The pyrophosphorolysis activated polymerization method of claim 26 wherein the mismatch between the activatable oligonucleotide P* and the template strand occurs at the terminal 3′
- -deoxynucleotide.
-
29. The method of claim 1 wherein the nucleic acid polymerase used in step (c) is a genetically modified DNA polymerase selected from the group consisting of AmpliTaqFS, ThermoSequenase and DNA polymerase modified to contain a mutation in its active site equivalent to a F667Y mutation in TaqFS.
-
30. The method of claim 29 wherein PAP efficiency is enhanced or PAP efficiency is less discriminated against any kind of dideoxynucleotide at the 3′
- terminus of P*.
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31. The method of claim 29 wherein the non-extendable 3′
- -deoxynucleotide at the 3′
terminus of P* is a dideoxynucleotide.
- -deoxynucleotide at the 3′
-
32. The method of claim 31 wherein the dideoxynucleotide is labeled by dyes.
-
33. The method of claim 29 wherein the nucleoside triphosphates are dideoxynucleotide triphosphates as substrates of DNA polymerase.
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34. The method of claim 33 wherein the dideoxynucleotide triphosphates are labeled by dyes.
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35. The method of claim 1 wherein P* has a 3′
- specific subsequence with length n >
3 nucleotides, and P* is not substantially amplified when one or more mismatches to its template strand is located within the 3′
specific subsequence, while P* is substantially amplified with its perfectly matched template strand within the 3′
specific subsequence.
- specific subsequence with length n >
-
36. The method of claim 35 wherein the mismatch in the 3′
- specific subsequence is within 16 nucleotides of the 3′
terminus of P*.
- specific subsequence is within 16 nucleotides of the 3′
-
37. The method of claim 35 wherein PAP is applied with one P* or two oligonucleotides.
-
38. The method of claim 1 to compare two DNA sequences or to monitor gene expression profiling, wherein a set of P*s with different 3′
- specific subsequences are applied for PAP.
-
39. The method of claim 38 wherein each P* has a 3′
- specific subsequence.
-
40. The method of claim 38 wherein the set of P* is incomplete with different 3′
- specific subsequences.
-
41. The method of claim 38 wherein a list of the specific PAP amplifications with the set of P*s are scored and then a DNA sequence complementary to the template strand of the nucleic acid is determined by ordering the 3′
- specific subsequences.
-
42. The method of claim 41 wherein PAP is applied with one P* or two oligonucleotides.
- -deoxynucleotide at its 3′
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43. A pyrophosphorolysis activated polymerization method for exponential amplification of a mutant allele that is present in admixture with a wild-type allele, which comprises preparing single-stranded DNA for each allele, one single stranded DNA for each allele being a template strand and the other being a complementary strand and then serially
(a) annealing to the template strand of each allele a complementary activatable 2′ - -deoxyoligonucleotide P* that has a non-extendable 2′
,3′
-deoxynucleotide at its 3′
terminus and that has no 2′
-deoxynucleotides at or near its 3′
terminus that mismatch the corresponding 2′
-deoxynucleotides on the template strand of the mutant allele but that has at least one 2′
-deoxynucleotide at or near its 3′
terminus that mismatches the corresponding 2′
-deoxynucleotide on the template stand of the wild-type allele, so that the terminal 2′
,3′
-deoxynucleotide is hybridized to the mutant template strand but not to the wild-type template strand when the oligonucleotide P* is annealed, and simultaneously annealing to the complementary strand of each allele a second, complementary 2′
-deoxyoligonucleotide, where the activatable 2′
-deoxyoligonucleotide P* and the second 2′
-deoxyoligonucleotide flank the region of the gene to be amplified;
(b) pyrophosphorolyzing the activatable 2′
-deoxyoligonucleotide P* that is annealed to a mutant template strand with pyrophosphate and an enzyme that has pyrophosphorolysis activity to activate the 2′
-deoxyoligonucleotide P* by removal of the hybridized terminal 2′
,3′
-deoxynucleotide, and(c) polymerizing by extending the activated oligonucleotide P* on the mutant template strand in presence of four nucleoside triphosphates and a DNA polymerase and simultaneously extending the second 2′
-deoxyoligonucleotide on both mutant and wild-type complementary strands,(d) separating the extension products of step (c); and
(e) repeating steps (a)-(d) until the desired level of exponential amplification of the mutant allele has been achieved. - View Dependent Claims (44, 45, 46, 47, 48, 49, 50, 51, 52)
- -deoxyoligonucleotide P* that has a non-extendable 2′
-
53. A pyrophosphorolysis activated polymerization method which comprises serially
(a) annealing to a template nucleic acid strand a complementary activatable oligonucleotide P* that has a non-extendable 3′ - -deoxynucleotide at its 3′
terminus and that has no nucleotides at or near its 3′
terminus that mismatch the corresponding nucleotides on the template strand, so that the terminal 3′
-deoxynucleotide is hybridized to the template strand when the oligonucleotide P* is annealed,(b) pyrophosphorolyzing the resulting duplex with pyrophosphate and an enzyme that has pyrophosphorolysis activity and activates the oligonucleotide P* by removal of the hybridized terminal 3′
-deoxynucleotide, and(c) extending the activated oligonucleotide P* on the template strand in presence of a non-extendable 3′
-deoxynucleoside triphosphate and a nucleic acid polymerase.- View Dependent Claims (54, 55, 56, 57, 58, 59, 60)
- -deoxynucleotide at its 3′
-
61. A method of scanning for unknown sequence variants in a nucleic acid sequence or re-sequencing of a predetermined sequence in a nucleic acid by pyrophosphorolysis activated polymerization (PAP) which comprises
(a) mixing under hybridization conditions a template strand of the nucleic acid with multiple sets of four activatable oligonucleotides P* which are sufficiently complementary to the template strand to hybridize therewith and which, within each set differ, from each other in having a different 3′ - -terminal non-extendable nucleotide, so that the 3′
terminal non-extendable nucleotide is hybridized to the template strand if the template strand is complementary to the 3′
terminal non-extendable nucleotide, the number of sets corresponding to the number of nucleotides in the sequence;
(b) treating the resulting duplexes with pyrophosphate and an enzyme that has pyrophosphorolysis activity to activate by pyrophosphorolysis only those oligonucleotides P* which have a 3′
terminal non-extendable nucleotide that is hybridized to the template strand,(c) polymerizing by extending the activated oligonucleotides P* on the template strand in presence of four nucleoside triphosphates and a nucleic acid polymerase, (d) separating the nucleic acid strands synthesized in step (c) from the template strand, (e) repeating steps (a)-(d) until a desired level of amplification is achieved, and (f) arranging the nucleic acid sequence in order by analyzing overlaps of oligonuclotides P* that produced amplifications. - View Dependent Claims (62, 63, 64, 65, 66, 67, 68)
- -terminal non-extendable nucleotide, so that the 3′
-
69. A method of determining de novo the sequence of a nucleic acid by pyrophosphorolysis activated polymerization (PAP) which comprises
(a) mixing under hybridization conditions a template strand of the nucleic acid with multiple activatable oligonucleotides P*, all having the same number n of nucleotides and constituting collectively all possible sequences having n nucleotides, and all having a non-extendable nucleotide at the 3′ - terminus, whereby any oligonucleotides P* that are sufficiently complementary will hybridize to the template strand, and the 3′
terminal non-extendable nucleotide will hybridize to the template strand only if the template strand is complementary at the position corresponding to the 3′
terminus;
(b) treating the resulting duplexes with pyrophosphate and an enzyme that has pyrophosphorolysis activity to activate only those hybridized oligonucleotides P* which have a 3′
terminal non-extendable nucleotide that is hybridized to the template strand, by pyrophosphorolysis of those hybridized 3′
terminal non-extendable nucleotides;
(c) polymerizing by extending the activated oligonucleotides P* on the template strand in presence of four nucleoside triphosphates and a nucleic acid polymerase, (d) separating the nucleic acid strands synthesized in step (c) from the template strand, (e) repeating steps (a)-(d) until a desired level of amplification has been achieved, and (f) determining the sequence of oligonucleotides P* that produced amplifications, then arranging the nucleic acid sequence in order by analyzing overlaps of these oligonucleotides. - View Dependent Claims (70, 71, 72, 73, 74, 75, 76, 77, 78)
- terminus, whereby any oligonucleotides P* that are sufficiently complementary will hybridize to the template strand, and the 3′
-
79. 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 a complementary activatable oligonucleotide P* that has a non-extendable 3′ - -deoxynucleotide at its 3′
terminus and has a mismatch at its 3′
terminus or at the first or second nucleotide from its 3′
terminus 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 hybridized 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 (80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98)
(d) separating the desired nucleic acid strand of step (c) from the template strand and (e) repeating steps (a)-(d) until a desired level of amplification of the desired nucleic acid strand is achieved. -
81. The pyrophosphorolysis activated polymerization method of claim 80 carried out in the presence of a second oligonucleotide that in step (a) anneals to the separated desired nucleic acid strand product of step (d), and wherein step (c) includes polymerizing by extending the second oligonucleotide on the desired nucleic acid strand to synthesize a copy of the nucleic acid template strand, and step (d) includes separating the synthesized nucleic acid template strand from the desired nucleic acid strand, so that amplification is exponential.
-
82. The pyrophosphorolysis activated polymerization method of claim 81 wherein the mismatch between the activatable oligonucleotide P* and the template strand occurs at the terminal 3′
- -deoxynucleotide.
-
83. The pyrophosphorolysis activated polymerization method claim of 80 wherein steps (a) to (c) are conducted sequentially as two or more temperature stages on a thermocycler.
-
84. The pyrophosphorolysis activated polymerization method of claim 83 carried out in the presence of a second oligonucleotide that in step (a) anneals to the separated desired nucleic acid strand product of step (d), and wherein step (c) includes polymerizing by extending the second oligonucleotide on the desired nucleic acid strand to synthesize a copy of the nucleic acid template strand, and step (d) includes separating the synthesized nucleic acid template strand from the desired nucleic acid strand, so that amplification is exponential.
-
85. The pyrophosphorolysis activated polymerization method of claim 84 wherein the mismatch between the activatable oligonucleotide P* and the template strand occurs at the terminal 3′
- -deoxynucleotide.
-
86. The pyrophosphorolysis activated polymerization method of claim 80 wherein steps (a) to (c) are conducted as one temperature stage on a thermocycler.
-
87. The pyrophosphorolysis activated polymerization method of claim 86 carried out in the presence of a second oligonucleotide that in step (a) anneals to the separated desired nucleic acid strand product of step (d), and wherein step (c) includes polymerizing by extending the second oligonucleotide on the desired nucleic acid strand to synthesize a copy of the nucleic acid template strand, and step (d) includes separating the synthesized nucleic acid template strand from the desired nucleic acid strand, so that amplification is exponential.
-
88. The pyrophosphorolysis activated polymerization method of claim 87 wherein the mismatch between the activatable oligonucleotide P* and the template strand occurs at the terminal 3′
- -deoxynucleotide.
-
89. The pyrophosphorolysis activated polymerization method of claim 86 wherein the DNA polymerase is also the enzyme having pyrophosphorolysis activity.
-
90. The pyrophosphorolysis activated polymerization method of claim 89 wherein the mismatch between the activatable oligonucleotide P* and the template strand occurs at the terminal 3′
- -deoxynucleotide.
-
91. The pyrophosphorolysis activated polymerization method of claim 89 carried out in the presence of a second oligonucleotide that in step (a) anneals to the separated desired nucleic acid strand product of step (d), and wherein step (c) includes polymerizing by extending the second oligonucleotide on the desired nucleic acid strand to synthesize a copy of the nucleic acid template strand, and step (d) includes separating the synthesized nucleic acid template strand from the desired nucleic acid strand, so that amplification is exponential.
-
92. The pyrophosphorolysis activated polymerization method of claim 91 wherein the mismatch between the activatable oligonucleotide P* and the template strand occurs at the terminal 3′
- -deoxynucleotide.
-
93. The pyrophosphorolysis activated polymerization method of claim 89 wherein the DNA polymerase is thermostable Tfl or Taq.
-
94. The pyrophosphorolysis activated polymerization method of claim 93 wherein the mismatch between the activatable oligonucleotide P* and the template strand occurs at the terminal 3′
- -deoxynucleotide.
-
95. The pyrophosphorolysis activated polymerization method of claim 93 carried out in the presence of a second oligonucleotide that in step (a) anneals to the separated desired nucleic acid strand product of step (d), and wherein step (c) includes polymerizing by extending the second oligonucleotide on the desired nucleic acid strand to synthesize a copy of the nucleic acid template strand, and step (d) includes separating the synthesized nucleic acid template strand from the desired nucleic acid strand, so that amplification is exponential.
-
96. The pyrophosphorolysis activated polymerization method of claim 95 wherein the mismatch between the activatable oligonucleotide P* and the template strand occurs at the terminal 3′
- -deoxynucleotide.
-
97. The pyrophosphorolysis activated polymerization method of claim 89 wherein the DNA polymerase is a genetically engineered DNA polymerase selected from the group consisting of AmpliTaqFS, ThermoSequenase and a DNA polymerase modified to contain a mutation equivalent to a F667Y mutation in TaqFS.
-
98. The pyrophosphorolysis activated polymerization method of claim 97 wherein the mismatch between the activatable oligonucleotide P* and the template strand occurs at the terminal 3′
- -deoxynucleotide.
- -deoxynucleotide at its 3′
Specification