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

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 x 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*) 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. We then applied Bi-PAR-A to direct detection of spontaneous somatic mutations in the lacI transgene in BigBlue transgenic mice 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, implying hyper-Poisson variance and clonal expansion of mutation occurring during early development. Bi-PAR-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. (C) 2004 Wiley-Liss, Inc.