Sanger Sequencing for Mutation Screening

Primer pairs for individual exons were designed using the Primer3 program. The primer sequences and amplification conditions are available upon request. The PCR amplifications were performed in 10 μl reactions with 20 ng genomic DNA as described previously [41,42]. The PCR primers for each exon were used for bidirectional sequencing with the BigDye Terminator Ready Reaction Mix, in accordance with the manufacturer’s instructions (Thermo Fisher Scientific, Waltham, MA).

Sequencing products were dissolved in 10 μl of Formamide (Applied Biosystems) and resolved on an ABI PRISM 3100 Genetic Analyzer (Applied Biosystems). Sequencing results were assembled with ABI PRISM sequencing analysis software, version 3.7, and analyzed with SeqScape software (Applied Biosystems).