PAP: Detection of ultra rare mutations depends on P* oligonucleotides: ?Sleeping Beauties? awakened by the kiss of pyrophosphorolysis

Abstract

For the Mutation Detection 2003 Special IssuePyrophosphorolysis-activated polymerization (PAP) was initially developed to enhance the specificity of allelespecific PCR for detection of known mutations in the presence of a great excess of wild-type allele. The high specificity of PAP derives from the serial coupling of activation of a 3 0 blocked pyrophosphorolysis-activable oligonucleotide (P n ) with extension of the unblocked, activated P n . In theory, PAP can detect a copy of a single base mutation present in 3 Â 10 11 copies of the wild-type allele. In practice, the selectivity of detection is limited by polymerase extension errors, a bypass reaction, from the unblocked oligonucleotide annealed to the opposing strand. Bi-directional PAP allele-specific amplification (Bi-PAP-A) is a derivative of PAP that uses two opposing pyrophosphorolysis activable oligonucleotides (P n ) with one nucleotide overlap at their 3 0 termini. This eliminates the problematic bypass reaction. The selectivity of Bi-PAP-A was examined using k phage DNA as a model system. Bi-PAP-A selectively detected two copies of a rare mutated allele in the presence of at least 2 Â 10 9 copies of the wild-type k phage DNA. We then applied Bi-PAP-A to direct detection of spontaneous somatic mutations in the lacI transgene in BigBlue transgenic mice at a frequency as low as 3 Â 10 À9. A 370-fold variation in the frequency of a specific somatic mutation among different mouse samples was found, implying hyper-Poisson variance and clonal expansion of mutation occurring during early development. Bi-PAP-A is a simple, rapid, and general method capable of automation and particularly suited to detection of ultra rare mutations. We also show that P n oligonucleotides have the novel and unexpected property of high specificity to mismatches with the template throughout lengths of the P

Sensitive Detection of KIT D816V in Patients with Mastocytosis

“…A method with high sensitivity is required in which the rare variant allele will not be overwhelmed by the large proportion of wild-type alleles. Numerous techniques have been developed to enhance the detection of the rare allele signal (19 ).…”

Approaches for analyzing human mutations and nucleotide sequence variation: A report from the Seventh International Mutation Detection meeting, 2003

“…By combining allele-specific PCR and serial coupling of pyrophosphorolysis, Liu Chiang Liu and Steve Sommer (City of Hope/Beckman Research Institute, Duarte, CA) succeeded in improving the specificity of primer extension and reported detection of one mutant SNP allele in the presence of up to 1 Â 10 9 copies of wild-type sequence in a lambda phage model system [Liu and Sommer, 2004].…”

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“…Pyrophosphorolysis activated polymerization (PAP) was a technique originally developed to enhance the specificity of allele-specific PCR for detection of known mutants in the presence of an excess of the wild type allele (Liu and Sommer, 2004). Pyrophosphorolysis is the removal of the 3 nucleotide by DNA polymerase in the presence of pyrophosphate (PPi).…”

“…If the primer has fully annealed the DNA polymerase can remove the dideoxynucleotide by pyrophosphorolysis and extension can begin. As a result of the specific annealing which is required before extension, any nucleotide mismatch (within the first 15 nucleotides of the 3 prime end of the primer) between the primer and target will prevent the extension reaction from beginning (Liu and Sommer, 2004). If this technology could be applied to assaying the small proportion of cff DNA in the presence of the great excess of cfm DNA, then PAP could be a viable technology for NIPD.…”