PCR Restriction Fragment Length Polymorphism (PCR-RFLP) analysis

A pair of PCR primers (5′-GGGATATTACAGGCTCTCAGCAATG-3′/5′-TTAGGCTGCAAGCGGAAGGCAAAG-3′) were designed for the PCR amplification of the region surrounding the DraI site on position 22,416,901 of chromosome 4. Tks Gflex DNA Polymerase (TaKaRa) with the following PCR routine: 94°C 1 min; (98°C 10 sec, 55°C 15 sec, 68°C 30 sec) × 35 cycles; 68°C 5 min, was used to amplify the PCR fragment. Five μl of the PCR product was digested by 0.2 μl of DraI (15 U/μl, TaKaRa) with incubation at 37°C for two hours. PCR products were electrophoresed on a 1% agarose gel.