A quantitative allele-specific PCR test for the BRAF V600E mutation using a single heterozygous control plasmid for...

We describe a novel method for mutant allele quantitation using allele-specific PCR. The method uses a heterozygous plasmid containing one wild-type and one mutant sequence as a calibrator that is run at a single concentration, with each quantitative allele-specific PCR run. PCR data from both calibrator alleles, together with predetermined PCR efficiencies, are used to quantitate the mutant allele burden in unknown specimens. We demonstrate the utility of this method by using it to calculate BRAF V600E allele frequencies in cases of hairy-cell leukemia and show that it generates data that are comparable to those obtained via allele quantitation using conventional standard curves over a wide range of allelic ratios. This method is not subject to errors that may be introduced in traditional standard curves as the result of variations in pippetting or errors in the calculation of the absolute copy numbers of standards. Furthermore, it simplifies the workflow in the clinical laboratory and would provide significant advantages for efforts to standardize clinical quantitative PCR testing.