Pyrophosphorolysis activated polymerization (PAP): application to allele-specific amplification and nucleic acid sequence determination
First Claim
1. 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 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 phosphorolyis 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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Accused Products
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.
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96 Claims
-
1. 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 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 phosphorolyis 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, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54)
- -deoxynucleotide at its 3′
-
55. The pyrophosphorolysis activated polymerization method for exponential amplification of a mutant allele that is present in admixture with a wild-type allele, which comprises separating the strands of the alleles to provide single-stranded DNA and then serially
(a) annealing to the sense or antisense strands 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 mutant strand but that has at least one 2′
-deoxynucleotide at or near its 3′
terminus that mismatches the corresponding 2′
-deoxynucleotide on the wild type stand, so that the terminal 2′
,3′
-deoxynucleotide is hybridized to the mutant strand but not to the wild-type strand when the oligonucleotide P* is annealed, and simultaneously annealing to the anti-parallel strands 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 strand with pyrophosphate and an enzyme that has phosphorolyis 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 strand in presence of four nucleoside triphosphates and a DNA polymerase and simultaneously extending the second 2′
-deoxyoligonucleotide on both mutant and wild-type anti-parallel 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 (56, 57, 58, 59, 60, 61, 62, 63)
- -deoxyoligonucleotide P* that has a non-extendable 2′
-
64. 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 phosphorolyis 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 (65, 66, 67, 68, 69, 70, 71)
- -deoxynucleotide at its 3′
-
72. A process which comprises serial coupling of two reactions, the second reaction being amplification of a nucleic acid by extension of an oligonucleotide on a nucleic acid template in the presence of four nucleoside triphosphates and a nucleic acid polymerase, the first reaction being activation of the oligonucleotide by removal of a 3′
- end block which, if not removed, would prevent the oligonucleotide from being extended on the template.
- View Dependent Claims (73, 74, 75, 76, 77, 78, 80, 81, 82, 83, 84, 85, 86, 87)
-
79. 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 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 phosphorolyis 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.
- -terminal non-extendable nucleotide, so that the 3′
-
88. A method of determining de novo the sequence of a nucleic acid by pyrophosphorolysis activated polymerization 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 phosphorolyis 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 (89, 90, 91, 92, 93, 94, 95, 96)
- terminus, whereby any oligonucleotides P* that are sufficiently complementary will hybridize to the template strand, and the 3′
Specification