Detection of extremely rare alleles by bidirectional pyrophosphorolysis-activated polymerization allele-specific amplification (Bi-PAP-A): measurement of mutation load in mammalian tissues

Abstract

Pyrophosphorolysis-activated polymerization (PAP) was developed to detect extremely rare mutations in complex genomes. In theory, PAP can detect a copy of a single base mutation present in 3 x 10(11) copies of the wild-type allele. In practice, the selectivity of detection is limited by a bypass reaction involving a polymerase extension error from the unblocked oligonucleotide annealed to the opposing strand. Bidirectional PAP allele-specific amplification (Bi-PAP-A) is a novel method that uses two opposing 3'-terminal blocked pyrophosphorolysis-activatable oligonucleotides (P*s) with one nucleotide overlap at their 3' termini. This eliminates the problematic bypass reaction. The selectivity of Bi-PAP-A was examined using lambda 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 x 10(9) copies of the wild-type lambda phage DNA. Bi-PAP-A was then applied to direct detection of spontaneous somatic mutations in the mouse genome at a frequency as low as 3 x 10(-9). A 370-fold variation in the frequency of a specific somatic mutation among different mouse samples was found, suggesting clonal expansion of mutation occurring during early development and a hyper-Poisson variance. Bi-PAP-A is a rapid, general, and automatable method for the detection of rare mutations.

Pyrophosphorolysis-Activated Polymerization Detects Circulating Tumor DNA in Metastatic Uveal Melanoma

“…This study shows that PAP developed over years by Sommer and Colleagues is a reliable, exquisitely sensitive, and specific technique able to detect circulating tumor DNA identified by a single nucleotide variation (16,17). An important advantage of PAP over other proposed techniques is its simplicity, allowing its implementation in most clinical molecular pathology laboratories.…”

“…However, these strategies remain delicate and expensive to conduct, requiring instrumentations and expertise beyond most diagnostic laboratory settings. A promising method developed by Sommer and colleagues relies on the use of PAP, a highly specific PCR-derived method that can detect point mutations in the presence of a great excess of wildtype DNA (16,17). The specificity of this method, which totally discriminates a single nucleotide change, derives from the serial coupling of activation of 2 opposing 3 0 -blocked pyrophosphorolysis activatable oligonucleotides (bi-PAP) with extension of the unblocked oligonucleotides (18; Fig.…”

Sensitive Detection of KIT D816V in Patients with Mastocytosis

“…The second approach used primer 3Ј end-base discrimination. Blocking amplification of the normal allele with a 3Ј-end dideoxynucleotide or locked nucleic acid has also been used to detect variations at the level of 3 ϫ 10 Ϫ9 in mouse genomes and 100-fold excess of wild-type in BRAF cell line studies, respectively (35,36 ). The cost of manufacturing primers with 3Ј modified locked nucleic acids is high, however, and primer with added 3Ј dideoxynucleotides is not widely available commercially, so this exper- tise-requiring modification must be performed in the laboratory.…”

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

“…Once the errors occur, the mutated product can be ampli¢ed exponentially in subsequent cycles, therefore limiting the selectivity. (Modi¢ed from Liu and Sommer [2004a].) B: P n s with di¡erent 3 0 -terminal blockers.…”