ACB-PCR Quantification of Somatic Oncomutation
Allele-specific competitive blocker-polymerase chain reaction (ACB-PCR) is a sensitive approach for the selective amplification of an allele. Using the ACB-PCR technique, hotspot point mutations in oncogenes and tumor-suppressor genes (oncomutations) 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 with 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 the abundant wild-type molecules. Consequently, ACB-PCR is capable of quantifying the level of a single basepair substitution mutation in a DNA population when present at a mutant:wild type ratio of 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, including evaluating the carcinogenic potential of chemical exposures in rodent models and in the diagnosis and treatment of cancer. This chapter provides a step-by-step description of the ACB-PCR methodology as it has been used to measure human KRAS codon 12 GGT to GAT mutation.
Key wordsAllele-specific competitive blocker-polymerase chain reaction (ACB-PCR) Oncomutation Point mutation Allele-specific PCR Mutation detection Base substitution KRAS
The authors thank Drs. Robert Heflich and Wei Ding for their critical review of this book chapter. The contents of this book chapter neither necessarily reflect the views or the policies of the US FDA nor does the mention of trade names or commercial products constitute endorsement for use.