ACB-PCR Quantification of Low-Frequency Hotspot Cancer-Driver Mutations.

Abstract

Allele-specific competitive blocker PCR (ACB-PCR) is a sensitive and quantitative approach for the selective amplification of a specific base substitution. Using the ACB-PCR technique, hotspot cancer-driver mutations (tumor-relevant mutations in oncogenes and tumor suppressor genes, which confer a selective growth advantage) are being developed as quantitative biomarkers of cancer risk. ACB-PCR employs a mutant-specific primer (with a 3'-penultimate mismatch relative to the mutant DNA sequence, but a double 3'-terminal mismatch relative to the wild-type DNA sequence) to selectively amplify rare mutant DNA molecules. A blocker primer having a non-extendable 3'-end and a 3'-penultimate mismatch relative to the wild-type DNA sequence, but a double 3'-terminal mismatch relative to the mutant DNA sequence is included in ACB-PCR to selectively repress amplification from abundant wild-type molecules. Consequently, ACB-PCR can quantify the level of a single base pair substitution mutation in a DNA population when present at a mutant:wild-type ratio of 1×10-5 or greater. Quantification of rare mutant alleles is achieved by parallel analysis of unknown samples and mutant fraction (MF) standards (defined mixtures of mutant and wild-type DNA sequences). The ability to quantify specific mutations with known association to cancer has several important applications in evaluating the carcinogenic potential of chemical exposures in rodent models. Further, the measurement of cancer-driver mutant subpopulations is important for precision cancer treatment (selecting the most appropriate targeted therapy and predicting the development of therapeutic resistance). This chapter provides a step-by-step description of the ACB-PCR methodology as it has been used to measure human PIK3CA codon 1047, CAT→CGT (H1047R) mutation.