Allele-specific, non-extendable primer blocker PCR (AS-NEPB-PCR) for DNA mutation detection in cancer

Allele-specific amplification, combined with TaqMan probe real-time polymerase chain reaction (real-time AS-PCR), has been widely used for detecting genetic variants, single nucleotide polymorphisms, and genetic mutations. In addition, several probe-blocking methods have been introduced in real-time AS-PCR to block amplification of wild-type templates and to increase detection sensitivity and specificity. However, most of these methods provide a limited sensitivity of no better than 1% and are complex in design of blockers, and thus cannot be readily adapted for different mutation assays. We have developed a modified non-extendable primer blocker (NEPB) for real-time AS-PCR (AS-NEPB-PCR). The NEPB method provides an easy design of allele-specific primer and corresponding primer blocker that can be used in any single nucleotide polymorphism or mutation detection, specifically in the detection of low-frequency mutations. The method is straight-forward in assay optimization and can achieve 0.1% sensitivity with 100% specificity (95% confidence interval, 92-100%) in detecting K-Ras, B-Raf, and EGFR mutations in cancer cells.