BRCA1/2 Variant Identification Protocol

Genomic DNA was obtained from blood using the DNeasy Blood & Tissue Kit (QiaGen) according to the manufacturer’s instructions. BRCA1 and BRCA2 coding regions and their intron–exon boundaries were amplified using PCR primers complementary to flanking intron sequences. Primers were designed by primer 3 software [7 (link)] and then evaluated by single nucleotide polymorphism (SNP) check software [8 ] to test for the presence of SNPs in their length, especially at the 3′ end. Sequencing reactions were performed by using an ABI Prism Big Dye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems). Sequenced PCR products were purified using CentriSeptfiltration columns (Applied Biosystems) following the manufacturer’s instructions. Sequencing was carried out using an ABI 3130 genetic analyser (Applied Biosystems). Visual inspection of base calling was used to evaluate the quality of DNA sequencing. NCBI reference sequences (RefSeq) NM_007294.3 and NM_000059.3 were used for the annotation of BRCA1 and BRCA2 variants, respectively. These RefSeq transcripts are included in the Locus Reference Genomic (LRG) data LRG_292-BRCA1 and LRG_293-BRCA2. Bi-directional sequencing review was performed using Mutation Surveyor Software (v.5.0.0, Soft Genetics, State College, PA). BRCA1/2 variant data were submitted to the Clinical Variation Database (ClinVar) [9 (link)].