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Updated: Jul 5, 2026
Employing Digital Droplet PCR to Detect BRAF V600E Mutations in Formalin-fixed Paraffin-embedded Reference Standard Cell Lines
Published on: October 8, 2015
Detection of BRAF V600E mutation by pyrosequencing
Yi Hui Tan1, Yanqun Liu, Kong Weng Eu
1Oncology Research Institute, National University of Singapore, Singapore.
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.
