High resolution melting as an alternative method to genotype diacylglycerol O-acyltransferase 1 (DGAT1) K232A polymorphism in cattle

Abstract

PCR-RFLP analysis is a common method for genotyping the DGAT1 K232A polymorphism in cattle. Our purpose was to develop a high resolution melting (HRM) assay in order to genotype the polymorphic alleles. Firstly, the PCR-RFLP method was used and the 411 bp products including the DGAT1 polymorphism were digested by CfrI enzyme. Direct sequencing was performed to confirm genotypes of the K232A polymorphism for 30 samples that presented different PCR-RFLP patterns. It was determined according to sequencing results that partial enzyme digestion had occurred for some samples. A 130 bp fragment including the polymorphism was amplified for real time PCR. Then, the HRM analysis was carried out using two fluorescent dyes, SYBR Green I and EvaGreen<sup>TM</sup>. Although the HRM genotyping using SYBR Green I was contradicted by the sequencing results, three correct melting curves were obtained for the K232A polymorphism when EvaGreen<sup>TM</sup> was used. There were no false genotypes and all genotypes were in agreement with their sequencing results. The difference in the T<sub>m</sub> between the two homozygous groups was about 0.5°C and the AA genotypes showed a higher T<sub>m</sub> than the KK genotypes. The heterozygous genotypes showed a different pattern. Similar results were obtained from different concentrations of EvaGreen<sup>TM</sup> in the reactions. All 206 DNA samples were genotyped using this fluorescent dye with estimated allele frequencies of 0.66 and 0.34 for the A and K alleles, respectively. Our study showed that HRM analysis will be applicable for genotyping the DGAT1 K232A polymorphism in large populations of dairy cattle.