Detection of BRAF V600E mutation by pyrosequencing

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Abstract
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. 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. 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. Pyrosequencing offers a specific, sensitive, rapid and cost-effective alternative to dideoxy sequencing for the detection of BRAF V600E mutations in clinical tumour specimens.