High resolution melting analysis on temperature allows the genotype determination of indicators related to beef

L. E. López Rojas, Laura Patiño Cadavid, Juliana María Martínez Garro, Silvana Durán Ortiz, Lina Johana Correa Agudelo, Santiago González Escudero, Albeiro López Herrera, José Julián Echeverri Zuluaga

Abstract


Some SNPs within μ-calpain (CAPN1), calpastatin (CAST) and leptin (LEP) genes have been associated to beef tenderness and, in order to optimize the processes of genetic improvement in cattle rearing farms, it is necessary to apply fast, reliable and accessible methodologies to producers, for determining the genotype of these polymorphisms. Although there are several methodologies, the HRM is stated as a promising alternative that meets these conditions. Therefore, the PCR-HRM was standardized and validated for genotyping the SNPs CAPN4751, CAPN316, CAST2959, CAST282, E2FB and E2JW of 50 animals from Bos taurus and Bos indicus breeds, using PCR-RFLP as gold standard test. There was an agreement among the results obtained by PCRHRM, compared to PCR-RFLP, and high sensitivity, specificity and appropriate negative and positive predictive values for PCRHRM. Similarly, repeatability and reproducibility were verified after obtaining homogeneity in the intra-run and inter-run results. Although the results show that the PCR-HRM is an efficient technique, and allows to obtain reliable results in a short time, these can be affected by the extraction method, quality and quantity of DNA, among other undetermined factors that can generate false positives and atypical dissociation profiles. Therefore, these variables must be controlled and the PCR should be monitored to identify unusual dissociation curves, caused by excess or deficiency in the amplification.

Keywords: quality, calpastatin, leptin, μ-calpain, HRM.

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