BRCA1 and BRCA2 mutation screening using SmartCycler II high-resolution melt curve analysis.
BACKGROUND
Real-time polymerase chain reaction technologies have replaced many of the more labor-intense methods in the molecular diagnostics laboratory. Similarly, melt curve analysis can provide a rapid means of mutation screening.


OBJECTIVE
To determine if real-time polymerase chain reaction and melt curve analysis using the SmartCycler II could be used as a screening tool for 3 common mutations in BRCA1 and BRCA2.


METHODS
Real-time polymerase chain reaction amplification with SYBR Green I detection was performed on DNA from cell lines known to carry the 185delAG or 5382insC mutation in BRCA1 or the 6174delT mutation in BRCA2. The melting temperatures and the melt curves were analyzed for differences between wild-type DNA and cell lines that were heterozygous for each mutation.


RESULTS
Significant differences were present in the melt curves for each of the mutations compared with those of the wild-type sequences. The melt curve for the 185delAG mutation showed a separate peak at a lower temperature, which represented the melting temperature of the heteroduplex. For the 6174delT mutation, the melt curve had a shoulder at a lower temperature, while the melt curve for the 5382insC mutation was shifted to the left and was broader than that for the wild-type sequences.


CONCLUSIONS
High-resolution melt curve analysis is a quick, reliable method for identifying mutations due to small deletions or insertions. As a proof of principle, we used this assay to identify the 3 most common BRCA1 and BRCA2 mutations in the Ashkenazi Jewish population.
Dufresne
Scott D
SD
Department of Pathology, Dartmouth Medical School, Dartmouth-Hitchcock Medical Center, Lebanon, NH 03756-0001, USA.
Belloni
Dorothy R
DR
Wells
Wendy A
WA
Tsongalis
Gregory J
GJ
D045744:Cell Line, Tumor; D004252:DNA Mutational Analysis / Q000379:methods*; D017931:DNA Primers / Q000737:chemistry; D004273:DNA, Neoplasm / Q000235:genetics*; D005260:Female; D019398:Genes, BRCA1; D024522:Genes, BRCA2; D006580:Genetic Carrier Screening; D005820:Genetic Testing / Q000379:methods*; D006801:Humans; D007585:Jews / Q000235:genetics; D009691:Nucleic Acid Denaturation; D017354:Point Mutation; D020133:Reverse Transcriptase Polymerase Chain Reaction
D017931:DNA Primers; D004273:DNA, Neoplasm