dna sequencing sequencher heterozygous mutations

All heterozygous variants were detected with the optimised assays.Ĭonclusions: 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. We also performed an extensive blinded validation of the protocol with 384 separate patient DNAs. Results: 169 BRCA1 and 239 BRCA2 known sequence variants were used to test the amplicons. Multiple 384 well plates were used to facilitate high throughput. Deoxyinosine residues were incorporated into primers that overlay intronic polymorphisms. Significant attention was placed on primer design to enable reproducible detection of mutations within the amplicon while minimising unnecessary detection of polymorphisms. A final set of 94 assays which ran under identical amplification conditions were chosen for BRCA1 (36) and BRCA2 (58). Methods: Assays for the analysis of all coding regions and intron-exon boundaries of BRCA1 and BRCA2 were designed, and optimised. It is well suited to screening genes such as BRCA1 and BRCA2 as germline pathogenic mutations in these genes are always heterozygous. High resolution melting (HRM) is a cost-effective rapid screening strategy, which readily detects heterozygous variants by melting curve analysis of PCR products. Current scanning methods used for the detection of germline sequence variants are laborious as they require post-PCR manipulation. Heather L Hondow, Stephen B Fox ', Gillian Mitchell, Rodney J Scott, Victoria Beshay, Stephen Q Wong, kConFab Investigators and Alexander Dobrovic1,2*īackground: Detection of mutations by DNA sequencing can be facilitated by scanning methods to identify amplicons which may have mutations. A high-throughput protocol for mutation scanning of the BRCA1 and BRCA2 genes

Dna sequencing sequencher heterozygous mutations