PAP: Detection of Ultra Rare Mutations Depends on P* Oligonucleotides: "Sleeping Beauties" Awakened by the Kiss of Pyrophosphorolysis

Abstract

Pyrophosphorolysis-activated polymerization (PAP) was initially developed to enhance the specificity of allele-specific 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′ blocked pyrophosphorolysis-activable oligonucleotide (P*) with extension of the unblocked, activated P*. In theory, PAP can detect a copy of a single base mutation present in 3 × 1011 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*) with one nucleotide overlap at their 3′ termini. This eliminates the problematic bypass reaction. The selectivity of Bi-PAP-A was examined using λ 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 × 109 copies of the wild-type λ 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 × 10-9. 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* oligonucleotides have the novel and unexpected property of high specificity to mismatches with the template throughout lengths of the P*. Thus, PAP also can form the basis of microarray-based scanning or resequencing methods to detect virtually all mutations. © 2004 Wiley-Liss, Inc.