title: Detection of ultrarare somatic mutation in the human TP53 gene by bidirectional pyrophosphorolysis-activated polymerization allele-specific amplification.
abstract: The detection of ultra-rare mutation in the presence of excess amounts of normal genomic DNA is highly advantageous in a number of circumstances, including: 1) identification of minimal residual disease for improved cancer chemotherapy; 2) measurement of mutation load to assess environmental mutagen exposure or endogenous DNA repair; and 3) prenatal diagnosis of paternally-derived mutations within fetal cells in the maternal circulation. Bidirectional pyrophosphorolysis activated polymerization allele-specific amplification (Bi-PAP-A) utilizes two opposing 3'-terminal blocked oligonucleotides (P(*)s) with 1 nucleotide overlap at their 3' termini. The selectivity of Bi-PAP-A derives from the serial coupling of pyrophosphorolysis and DNA polymerization. A total of 13 Bi-PAP-A assays were developed and validated for the human p53 gene (TP53). The sensitivity and specificity of each assay were determined with mutated and wild-type DNA templates, respectively. Bi-PAP-A has a sensitivity of one molecule for most assays and a selectivity (sensitivity:specificity) greater than 1:10(7)-1:10(9) for four of all six mutation types. Four assays with high selectivity were used to detect rare somatic mutations in blood white cells. The silent g.13147C>G (p.R156) mutation was present at an estimated frequency of 1.1 x 10(-7). The g.14523A>T (p.E285V), g.14487G>C (p.R273P), and g.14060G>C (p.G245R) mutations were undetectable with frequencies less than 2.0 x 10(-8). We conclude that Bi-PAP-A is a general and rapid method for detecting ultra-rare mutations.
authors.affiliation: Department of Molecular Genetics and Molecular Diagnosis, City of Hope National Medical Center, Duarte, California 91010-3000, USA. sommerlab@coh.org
authors.forename: Jinxiu
authors.initials: J
authors.lastname: Shi
authors.forename: Qiang
authors.initials: Q
authors.lastname: Liu
authors.forename: Steve S
authors.initials: SS
authors.lastname: Sommer
mesh_terms: D000483:Alleles; D004252:DNA Mutational Analysis / Q000379:methods*; D011756:Diphosphates / Q000378:metabolism; D016158:Genes, p53; D006801:Humans; D009154:Mutation; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D012680:Sensitivity and Specificity
chemical_list: D011756:Diphosphates