A high-throughput protocol for mutation scanning of the BRCA1 and BRCA2 genes
1 Molecular Pathology Research and Development Laboratory, Department of Pathology, Peter MacCallum Cancer Centre, Locked Bag 1, A'Beckett St, Melbourne, Victoria, 8006, Australia
2 Department of Pathology, The University of Melbourne, Parkville, Victoria, 3010, Australia
3 Familial Cancer Centre, Peter MacCallum Cancer Centre, Locked Bag 1, A'Beckett St, Melbourne, Victoria, 8006, Australia
4 School of Biomedical Sciences, University of Newcastle, New South Wales, 2308, Australia
BMC Cancer 2011, 11:265 doi:10.1186/1471-2407-11-265Published: 24 June 2011
Detection of mutations by DNA sequencing can be facilitated by scanning methods to identify amplicons which may have mutations. Current scanning methods used for the detection of germline sequence variants are laborious as they require post-PCR manipulation. High resolution melting (HRM) is a cost-effective rapid screening strategy, which readily detects heterozygous variants by melting curve analysis of PCR products. It is well suited to screening genes such as BRCA1 and BRCA2 as germline pathogenic mutations in these genes are always heterozygous.
Assays for the analysis of all coding regions and intron-exon boundaries of BRCA1 and BRCA2 were designed, and optimised. A final set of 94 assays which ran under identical amplification conditions were chosen for BRCA1 (36) and BRCA2 (58). Significant attention was placed on primer design to enable reproducible detection of mutations within the amplicon while minimising unnecessary detection of polymorphisms. Deoxyinosine residues were incorporated into primers that overlay intronic polymorphisms. Multiple 384 well plates were used to facilitate high throughput.
169 BRCA1 and 239 BRCA2 known sequence variants were used to test the amplicons. We also performed an extensive blinded validation of the protocol with 384 separate patient DNAs. All heterozygous variants were detected with the optimised assays.
This is the first HRM approach to screen the entire coding region of the BRCA1 and BRCA2 genes using one set of reaction conditions in a multi plate 384 well format using specifically designed primers. The parallel screening of a relatively large number of samples enables better detection of sequence variants. HRM has the advantages of decreasing the necessary sequencing by more than 90%. This markedly reduced cost of sequencing will result in BRCA1 and BRCA2 mutation testing becoming accessible to individuals who currently do not undergo mutation testing because of the significant costs involved.