title: 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.
authors.affiliation: City of Hope National Medical Center, Duarte, CA, USA.
authors.forename: Qiang
authors.initials: Q
authors.lastname: Liu
authors.forename: Steve S
authors.initials: SS
authors.lastname: Sommer
mesh_terms: D000818:Animals; D004247:DNA / Q000032:analysis / Q000235:genetics*; D004252:DNA Mutational Analysis / Q000379:methods*; D005787:Gene Frequency / Q000235:genetics*; D051379:Mice; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA / Q000379:methods*
chemical_list: D004247:DNA