BRAF mutation detection and identification by cycling temperature capillary electrophoresis.
BRAF mutations are found in many human tumors, namely melanomas ( approximately 70%) and colon carcinomas ( approximately 15%). This paper presents a method for identification of exon 15 BRAF mutations by denaturant capillary electrophoresis (CE), an analysis method that is sensitive, cost-effective (involving only polymerase chain reaction (PCR) and electrophoresis) and capable of high-throughput screening. In total, we found 21 (70%) out of 30 melanoma cell lines with BRAF mutations in exon 15: two of which were the p.Val600Asp (c.1799-800TG>AT) mutation, one cell line contained the p.Val600Arg (c.1798-99GT>AG) mutation, and 18 cell lines contained the p.Val600Glu (c.1799T>A) mutation. Of the nine cell lines that did not contain a BRAF mutation, five contained an NRAS mutation at exon 2, and no mutations were detected in NRAS exon 1. There was no overlap of NRAS and BRAF mutations in the same cell line. In addition, we looked at 221 colon biopsy samples and identified one further BRAF mutation, the p.Asp594Gly (c.1781A>G) mutation, in seven samples. The p.Val600Glu mutation was identified in 11 of the colon biopsy samples. Using the four mutations of BRAF exon 15, we then constructed a denaturing CE standard capable of distinguishing between each of the mutations; therefore, sequencing does not need to be performed to confirm the mutation. In conclusion, this sensitive, cost-effective mutation assay for BRAF (and RAS) will provide the opportunity to detect and determine mutations without the need to purify samples for sequencing. Future large-scale studies will provide the clinical usefulness of such mutations.
Hinselwood
David C
DC
Department of Surgical Oncology, The Norwegian Radium Hospital, Oslo, Norway.
Abrahamsen
Torveig W
TW
Ekstrøm
Per O
PO
D045744:Cell Line, Tumor; D015179:Colorectal Neoplasms / Q000235:genetics; D004252:DNA Mutational Analysis / Q000379:methods*; D004259:DNA-Directed DNA Polymerase; D019075:Electrophoresis, Capillary / Q000379:methods*; D005091:Exons; D006801:Humans; D008545:Melanoma / Q000235:genetics; D009154:Mutation; D009369:Neoplasms / Q000235:genetics*; D009691:Nucleic Acid Denaturation; D016133:Polymerase Chain Reaction; D048493:Proto-Oncogene Proteins B-raf / Q000235:genetics*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013696:Temperature
C482119:BRAF protein, human; D048493:Proto-Oncogene Proteins B-raf; C072146:Pfu DNA polymerase; D004259:DNA-Directed DNA Polymerase