Activating mutations of the BRAF and KRAS genes cause constitutive stimulation 
of an important cell-signaling pathway promoting tumorigenesis, and are 
increasingly recognized as determinants of response to targeted cancer 
therapies. The V600E mutation accounts for most of the BRAF mutations in cancer, 
and KRAS mutations are predominantly encoded by nucleotide substitutions within 
codons 12 and 13. We designed novel pyrosequencing assays for the detection of 
the common "hotspot" mutations in these genes, which demonstrated analytical 
sensitivities of <or=10% in titrations of mutant cell lines. The KRAS 
pyrosequencing assay has the ability to simultaneously identify all potential 
nucleotide changes within the mutation cluster at codons 12 and 13, with a 
sequence output in the sense direction to facilitate results interpretation. 
These assays were used to determine the mutation status in a prospective series 
of 1198 sporadic colorectal cancers. The BRAF V600E mutation was detected in 
13.2% of the colorectal cancers. The frequency of KRAS mutations in our cohort 
was 32.4%, with G>A transitions at position 2 of codons 12 and 13 being most 
prevalent. Both assays proved highly sensitive and specific when applied to 
clinical specimens, and were applicable to both fresh-frozen and formalin-fixed 
paraffin-embedded archival tissues. These assays would serve as a suitable 
platform for large-scale mutation detection in cancer specimens where the 
facility for pyrosequencing is available.