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 3x10(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 2x10(9) copies of the wild-type lambda 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 3x10(-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.
Liu
Qiang
Q
Department of Molecular Genetics, City of Hope National Medical Center, Duarte, California 91010-3000, USA.
Sommer
Steve S
SS
D000483:Alleles; D000818:Animals; D001483:Base Sequence; D004252:DNA Mutational Analysis / Q000379:methods*; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D011756:Diphosphates / Q000378:metabolism; D006801:Humans; D051379:Mice; D021141:Nucleic Acid Amplification Techniques; D009693:Nucleic Acid Hybridization; D009841:Oligonucleotides / Q000737:chemistry* / Q000378:metabolism; D016133:Polymerase Chain Reaction
D011756:Diphosphates; D009841:Oligonucleotides; D004259:DNA-Directed DNA Polymerase