High-Resolution Melting Analysis

A pair of primers was designed to amplify the short genomic region (<300 bp) centered on the target PRR2 target SNP. Genotyping analysis was performed using a Rotor-Gene 6000 real-time PCR (Qiagen, Hilden, Germany). The reaction mixture was prepared in a total volume of 20 μL containing 80 ng of DNA, 0.3 μL of R Taq (Takara Bio), 2 μL of 10 × PCR buffer, 2 μL of 2.5 mM dNTPs, 0.6 μL of SYTO 9 (Thermo Fisher Scientific, Waltham, MA), and 0.5 μL of 10 pmol primers. Sequences of forward and reverse primers used in the study were as following: 5′-TTTGAAAGAGGAGAATGGTTCA-3′ (forward) and 5′-TGAGCTATGGGGACCAGAAG-3′ (reverse). The PCR conditions consisted of 95°C for 5 min; 55 cycles of 95°C for 20 s, 55°C for 20 s, and 72°C for 30 s; and 60°C for 1 min. For high-resolution melting (HRM) analysis, the temperature was increased 0.1°C per every minute from 65°C to 90°C. Melting curve and HRM-normalized graphs were analyzed using Rotor-Gene Q series software 2.1.0.