Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2001-02-22
2003-03-18
Horlick, Kenneth R. (Department: 1656)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091100, C435S091200
Reexamination Certificate
active
06534269
ABSTRACT:
BACKGROUND OF THE INVENTION
This invention relates to nucleic acid polymerization and amplification. In particular, it relates to a novel and general method for nucleic acid amplification, in which pyrophosphorolysis and polymerization are serially-coupled. The method has been adapted for allele-specific amplification and can greatly increase the specificity to detect an extremely rare allele in the presence of wild type alleles. We refer to the method as pyrophosphorolysis activated polymerization (PAP).
The publications and other materials used herein to illuminate the background of the invention or provide additional details respecting the practice, are incorporated by reference, and for convenience are respectively grouped in the appended Lists of References.
A method of detecting one mutant allele in 10
6
-10
9
wild type alleles would be advantageous for many applications including detecting minimal residual disease (rare remaining cancer cells during remission, especially mutations in the p53 gene or other tumor suppressor genes previously identified within the tumors) and measurement of mutation load (the frequency of specific somatic mutations present in normal tissues, such as blood or urine). Individuals with a high mutation load may be at increased risk for cancer to either environmental exposure or endogenous defects in any of hundreds of genes necessary to maintain the integrity of the genome. For those individuals found to have a high mutation load, clues to etiology can be obtained by defining the mutation pattern.
Multiple methods for detecting mutations present in less than 10% of cells (i.e. rare alleles) have been developed including PCR amplification of specific alleles (PASA), PNA clamping blocker PCR, allele specific competitive blocker PCR, MAMA, and RFLP/PCR (1). These methods: i) amplify the rare allele selectively, ii) destroy the abundant wild type allele, or iii) spatially separate the rare allele from the wild type allele. RFLP/PCR has been reported to have the highest specificity of 10
−8
(2), but in our hands the specificity has been 10
−3
to 10
−4
(3). Methods that selectively amplify the rare allele include PASA, which routinely has a specificity of less than or equal to 1 part in 40 (4).
DNA polymerases, which are critical to DNA amplification, catalyze some or all of the following reactions: i) polymerization of deoxynucleotide triphosphates; ii) pyrophosphorolysis of duplexes of DNA in the presence of pyrophosphate (PP
i
); iii) 3′-5′ exonuclease activity and iv) 5′-3′ exonuclease activity (5, 6). For Taq and Tfl DNA polymerases, the polymerization and 5′-3′ exonuclease activity have been reported (7-9). For T7 Sequenase™ DNA polymerases, pyrophosphorolysis can lead to the degradation of specific dideoxynucleotide-terminated segments in Sanger sequencing reaction (10, 11).
There are many DNA sequencing methods and their variants, such as the Sanger sequencing using dideoxy termination and denaturing gel electrophoresis (27), Maxam-Gilber sequencing using chemical cleavage and denaturing gel electrophoresis (28), pyro-sequencing detection pyrophosphate (PPi) released during the DNA polymerase reaction (29), and sequencing by hybridization (SBH) using oligonucleotides (30-35).
Herein, we describe pyrophosphorolysis activated polymerization (PAP), an approach which has the potential to enhance dramatically the specificity of PASA. We also describe a novel method of DNA sequence determination by PAP.
SUMMARY OF THE INVENTION
The invention is a pyrophosphorolysis activated polymerization (PAP) method of synthesizing a desired nucleic acid strand on a nucleic acid template strand. The method comprises the following steps carried out serially.
(a) Annealing to the template strand a complementary activatable oligonucleotide P*. This activatable oligonucleotide has a non-extendable 3′-deoxynucleotide at its 3′ terminus. It has no nucleotides at or near its 3′ terminus that mismatch the corresponding nucleotides on the template strand. Therefore, the terminal 3′-deoxynucleotide is hybridized to the template strand when the oligonucleotide P* is annealed.
(b) Pyrophosphorolyzing the annealed activatable oligonucleotide P* with pyrophosphate and an enzyme that has pyrophosphorolysis activity. This activates the oligonucleotide P* by removal of the hybridized terminal 3′-deoxynucleotide.
(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.
The PAP method can be applied to amplify a desired nucleic acid strand by the following additional steps.
(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.
In a preferred aspect, the PAP method as described above is applied to allele-specific amplification. In this application, the nucleic acid template strand is a sense or antisense strand of one allele and is present in admixture with the corresponding (sense or antisense) nucleic acid strand of the second allele (the allelelic strand). The activatable oligonucleotide P* has at least one nucleotide at or near its 3′ terminus that mismatches the corresponding nucleotide of the allelic strand. Because of the mismatch, in step (a) of the PAP method the terminal 3′-deoxynucleotide of oligonucleotide P* is not hybridized to the allelelic strand. In step (b) the pyrophosphorolysis does not substantially remove the non-hybridized terminal 3′-deoxynucleotide from the activatable oligonucleotide P* annealed to the allelic strand. In step (c) the oligonucleotide P* is not substantially extended by polymerization on the allelic strand. As a result, the desired nucleic acid strand synthesized on the template strand is amplified preferentially over any nucleic acid strand synthesized on the allelelic strand.
The PAP method can be used to amplify either RNA or DNA. When used to amplify DNA, 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. The DNA polymerase used in step (c) can also be the enzyme having pyrophosphorolysis activity used in step (b). Preferred DNA polymerases having pyrophosphorolysis activity are thermostable Tfl, Taq, and genetically engineered DNA polymerases, such as AmpliTaqFs and ThermoSequenase™. These genetically engineered DNA polymerases have the mutation F667Y in their active sites and elimination of 5′-3′ exonuclease activity. The use of genetically engineered DNA polymerases, such as AmpliTaqFs and ThermoSequenase™, greatly improves the efficiency of PAP.
Amplification by the PAP method can be linear or exponential. Linear amplification is obtained when the activatable oligonucleotide P* is the only complementary oligonucleotide used. Exponential amplification is obtained when a second oligonucleotide is present that is complementary to the desired nucleic acid strand. The activatable oligonucleotide P* and the second oligonucleotide flank the region that is targeted for amplification. In step (a) the second oligonucleotide anneals to the separated desired nucleic acid strand product of step (d). In step (c) polymerization extends the second oligonucleotide on the desired nucleic acid strand to synthesize a copy of the nucleic acid template strand. In step (d) the synthesized nucleic acid template strand is separated from the desired nucleic acid strand. Steps (a) through (d) are repeated until the desired level exponential amplification has been achieved.
In the PAP method, a mismatch between the activatable oligonucleotide P* and the template strand results in no amplification, if the mismatch occurs in the 3&pr
Liu Qiang
Sommer Steve S.
City of Hope
Horlick Kenneth R.
Rothwell Figg Ernst & Manbeck
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