Extraction of DNA and genotyping of SNPs
Genomic DNA obtained was extracted from the peripheral blood of all of
the subjects with the QIAamp DNA Blood Mini kit (Qiagen, Valencia, CA,
USA). The targeted sequence was amplified by the PCR using appropriate
primers(Yu, Liu, Bai, Kijlstra, & Yang, 2014).Genotyping of the nine
SNPs was performed using the polymerase chain reaction restriction
fragment length polymorphism (PCR-RFLP) assay. A total of 7 μL PCR
product was digested with 3 U of restriction enzyme according to the
manufacturer’s instructions, involving Pdi-I, DraIII, Rsal, Eco471,
BshNI, HindIII, TscAI, Ndel, and BstNI (ThermoFisher Scientific,
Waltham, MA, USA) overnight (Table S1). Digestion products were
separated on 4% agarose gels added with goldview I nucleic acid dye
(Beijing Solarbio Science & Technology Co., Ltd., Beijing, China). To
confirm the PCR-RFLP genotyping results, we randomly selected a part of
the samples to reperform genotyping using direct sequencing.