M13 forward or reverse primers

The purified PCR products were ligated in pGEM-T Easy vector according to standard protocols (Promega, Madison, WI, USA). Procedures for transformation and plating of E. coli DH5 strain were described elsewhere [30 ]. Individual colonies (134) were randomly chosen to grow in liquid medium for plasmid DNA extraction using Concert Rapid Plasmid Miniprep System (Life Tech) to be subsequently analyzed by Southern-blot hybridization using salivary gland DNA of T. pubescens as a probe. Sequencing reactions of both strands of plasmid DNAs were performed with Big Dye Terminator Cycle Sequencing Ready Reaction as recommended by the manufacturer (Applied Biosystems, Foster City, CA, USA) using M13 Forward or Reverse primers (Life Tech) and subsequently run in the ABI PRISM 310 Genetic Analyzer (Applied Biosystems). GenBank searches were done with BLAST [31 (link)].

PCR was performed using genomic DNA as a template and primer pairs flanking the deleterious variant sites. PCR products were purified using the QIAquick PCR Purification Kit (QIAGEN) according to the manufacturer's instructions. Sequencing was performed with BigDye® Terminator v3.1 using M13 forward or reverse primers (Life Technologies). PCR products were purified and subsequently analyzed by the 3500 Genetic Analyzer (Applied Biosystems, Tokyo, Japan). GenBank sequences of human BRCA1 (accession number: NP_009225.1) and BRCA2 (accession number: NP_000050.2) were referred to at the NCBI Reference Sequence Database. Primer sequences are provided in the Table S1.