Detection of BRAF V600E mutation in colorectal cancer: comparison of automatic sequencing and real-time chemistry methodology

J Mol Diagn. 2006 Nov;8(5):540-3. doi: 10.2353/jmoldx.2006.060070.


Mutation V600E of BRAF, a kinase-encoding gene from the RAS/RAF/MAPK pathway, in colorectal carcinoma (CRC) suggests a sporadic origin of the disease, providing an exclusion criterion for hereditary nonpolyposis colorectal cancer. Here we describe detection of this mutation by real-time chemistry TaqMan MGB probes, confirmed by direct DNA sequencing as the gold standard. DNA was extracted from paraffin-embedded tissue from 112 tumors obtained from the EPICOLON study. Seventy-two tumors were CRC with defective DNA mismatch repair (MMR; microsatellite instability and/or loss of protein expression by immunohistochemical analysis), and 40 were proficient MMR controls. BRAF mutation was detected in 20/72 (27.8%) CRC with defective MMR and in 3/40 (7.5%) proficient MMR controls (P = 0.011). BRAF mutation was detected in 19/51 (37.3%) tumors with loss of MLH1 expression and in none of the tumors with loss of MSH2 expression (0/13). BRAF mutation was not found in cases with germline mutation of MLH1 (4/112) or MSH2 (3/112) genes. The sensitivity and specificity of our real-time chemistry were both 100% for detecting the V600E mutation. Because real-time chemistry methodology has advantages in cost, time, and labor, we consider it a valuable alternative to automatic direct sequencing, particularly for serial measurements.

Publication types

  • Comparative Study
  • Evaluation Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Colorectal Neoplasms / diagnosis*
  • Colorectal Neoplasms / genetics
  • Colorectal Neoplasms, Hereditary Nonpolyposis / economics*
  • Colorectal Neoplasms, Hereditary Nonpolyposis / genetics
  • DNA Mutational Analysis / economics*
  • Humans
  • Mutation
  • Proto-Oncogene Proteins B-raf / genetics*
  • Reverse Transcriptase Polymerase Chain Reaction / economics*
  • Sequence Analysis, DNA / economics*


  • Proto-Oncogene Proteins B-raf