CONTEXT: 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. DESIGN: 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.
CONTEXT: 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. DESIGN: 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.
Authors: Heather L Hondow; Stephen B Fox; Gillian Mitchell; Rodney J Scott; Victoria Beshay; Stephen Q Wong; Alexander Dobrovic Journal: BMC Cancer Date: 2011-06-24 Impact factor: 4.430