A rapid and sensitive approach to mutation detection using real-time polymerase chain reaction and melting curve analyses, using BRCA1 as an example

Background: The detection of 2 recurrent mutations in the BRCA1 gene (Ashke nazi Jewish and Dutch populations) was studied with real-time polymerase ch ain reaction (PCR) and melting curve analyses. Methods: PCR products were designed around the 185delAG in exon 2 and the s ingle-base substitution 2841G-->T in exon 11. Hybridization probe sets were designed for both PCR products, with each probe overlapping the specific m utation. The exon 11 probe set also covered another mutation, the 2814insA. The 3' end of the 5' probe was labeled with fluorescein isothiocyanate and the 5' end of the 3' probe with LightCycler Red 640 (Roche Diagnostics, In dianapolis, IN). Results: The 185del and 2841G mutations were easily detected with the hybri dization probes, resulting in dual peaks for heterozygotes in melting curve analyses. The differences in melting characteristics of the heteroduplexes in heterozygotes were not detectable with SYBR Green I. Conclusion: For known mutations, melting curve analyses using hybridization -specific probes provide a sensitive, rapid, and efficient approach to muta tion detection.