Detection of BRAF V600E mutation by pyrosequencing

Introduction: Detection of the V600E hotspot mutation in BRAF oncogene is extremely useful for the screening of hereditary non-polyposis colorectal cancer (Lynch's syndrome) and for the prediction of sensitivity to MEK inhibitors. Here we describe a method for detecting this mutation based upon pyrosequencing technology. Methods: The efficiency of pyrosequencing for detecting BRAF V600E mutations was compared with the conventional dideoxy sequencing method in 12 tumour cell lines and in 108 colorectal tumours. Results: The results from pyrosequencing were 100% concordant with those from dideoxy sequencing. This method was capable of detecting BRAF V600E mutations at a much lower ratio of mutant to wild-type alleles (1:50) than dideoxy sequencing (1:5) while being considerably faster and less expensive. Conclusions: Pyrosequencing offers a specific, sensitive, rapid and cost-effective alternative to dideoxy sequencing for the detection of BRAF V600E mutations in clinical tumour specimens.