Coamplification at Lower Denaturation Temperature-PCR Increases Mutation-Detection Selectivity of TaqMan-Based Real-Time PCR

摘要: Background: DNA genotyping with mutation-specific TaqMan® probes (Applied Biosystems) is broadly used in detection of single-nucleotide polymorphisms but less so for somatic mutations because its limited selectivity low-level mutations. We recently described coamplification at lower denaturation temperature-PCR (COLD-PCR), a method that amplifies minority alleles selectively from mixtures wild-type and mutation-containing sequences during the PCR. demonstrate combining COLD-PCR TaqMan technology provides needed to detect mutations. Methods: Minor-groove binder-based or common were designed contain nucleotide matches desired mutation approximately middle probe. The critical temperature ( T c) each amplicon was then experimentally determined. COLD-PCR/TaqMan performed 2 steps: c, followed by annealing extension single (fast COLD-PCR). threshold cycle identify on basis serial dilutions mutant into TP53 (tumor protein p53) EGFR [epidermal growth factor receptor (erythroblastic leukemia viral (v-erb-b) oncogene homolog, avian)] tumors. Results: identified G>A within exon 8 (codon 273 hot spot) C>T gene (drug-resistance T790M) improvement 15- 30-fold over regular PCR/TaqMan genotyping. A second round improved another enabled 1 2000 alleles. Use allowed quantitative identification T790 colon tumor samples non-small-cell lung cancer cell lines treated kinase inhibitors. Conclusions: major provided enables popular become powerful tool detecting clinical samples.