Real-time allele-specific amplification for sensitive detection of the BRAF mutation V600E.
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.
INSERM U539, Faculté de Médecine, 1 rue Gaston Veil, Nantes F-44035, France.
Anne
A
Jarry
Damien
D
Masson
Elisabeth
E
Cassagnau
Sigrid
S
Parois
Christian
C
Laboisse
Marc G
MG
Denis
D000483:Alleles; D045744:Cell Line, Tumor; D015179:Colorectal Neoplasms / Q000235:genetics; D006801:Humans; D009154:Mutation / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*; D048493:Proto-Oncogene Proteins B-raf / Q000235:genetics*; D012680:Sensitivity and Specificity; D013379:Substrate Specificity; D014633:Valine / Q000235:genetics*
D048493:Proto-Oncogene Proteins B-raf; D014633:Valine