Allele-Specific, Non-Extendable Primer Blocker PCR (AS-NEPB-PCR) for DNA Mutation Detection in Cancer

HY Wang, JF Jiang, Bianca Mostert, Anieta Sieuwerts, John Martens, Stefan Sleijfer, John Foekens, YX Wang

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27 Citations (Scopus)


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. (J Mol Diagn 2013, 15: 62-69;
Original languageUndefined/Unknown
Pages (from-to)62-69
Number of pages8
JournalJournal of Molecular Diagnostics
Issue number1
Publication statusPublished - 2013

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