Genetic Screening for Crystallin Mutations

Genomic DNA was extracted from 500 μL of peripheral blood using a TIANamp Blood DNA Midi Kit (Tiangen, Beijing, China). After genomic polymerase chain reaction (PCR) performed, we sequenced the coding exons and their flanking intronic sequences of six crystallin family genes CRYAA, CRYBA1, CRYBB1, CRYBB2, CRYGC and CRYGD, for pathogenic mutations in the proband. The primers and reaction conditions used in PCR have been described in earlier reports [16 (link)–21 (link)]. To more clearly show the mutation c.451_452insGACT in CRYGD, the obtained fragment was cloned into pMD-18T vector (TaKaRa, Dalian, China) and sequenced. To confirm the mutation, we performed genomic polymerase chain reaction (PCR) using primers CRYGD forward (5’-TACGAGCTGTCCAACTACCGAG-3’) and reverse (5’-GAGAAATCTATGACTCTCCTCAG-3’) in the family and 103 unrelated individuals. The PCR fragments were separated by 8% polyacrylamide gel electrophoresis. The analysis of amino acid conservation around the mutation site was carried out by CLC DNA Workbench.