Real-time allele-specific amplification for sensitive detection of the BRAF mutation V600E

Mol Cell Probes. 2004 Oct;18(5):349-52. doi: 10.1016/j.mcp.2004.05.004.


BRAF is a cytoplasmic serine/threonine kinase in the MAPK pathway that transduces signals from RAS family members to MEK1/2. Mutations in the BRAF gene have been described in the majority of cutaneous melanomas, papillary thyroid carcinoma and to a lesser extent in other cancers. The predominant mutation reported is a single transversion in exon 15 (T1799A). We designed a real-time allele-specific PCR to detect this mutation. This assay allowed us to detect this alteration in samples containing 2% of cells harboring this mutation, which is equivalent to 1% mutated DNA, assuming heterozygosity for the allele. Using this assay, we then tested 44 human primary colorectal tumors. We found the V600E mutation in four samples (9.1%). Analysis of DNA extracted from paraffin-embedded sections gave similar results, indicating that archived tissues can also be analyzed using this assay. The advantages of this method include: (1) rapidity; (2) sensitivity; (3) ease; (4) large throughput; (5) low cost and (6) the small quantities of DNA needed.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Alleles*
  • Cell Line, Tumor
  • Colorectal Neoplasms / genetics
  • Humans
  • Mutation / genetics*
  • Polymerase Chain Reaction / methods*
  • Proto-Oncogene Proteins B-raf / genetics*
  • Sensitivity and Specificity
  • Substrate Specificity
  • Valine / genetics*


  • Proto-Oncogene Proteins B-raf
  • Valine