Comparison of three PCR-based methods to detect a Piedmontese cattle point mutation in the Myostatin gene

Abstract

Despite many recent advances in single nucleotide polymorphism (SNP) genotyping technologies, there is still the need for methodologies with a reasonable throughput. In this study, we compared three PCR-based methods for SNP detection: (1) a conventional PCR-based allele detection system with fluorescent genotyping technology, (2) a SNaPshot methodology by single nucleotide primer extension and, (3) a real-time PCR-based method by allele-specific minor groove-binder probes. These three methodologies were used to analyze 104 meat samples of a particular Italian cattle breed known for producing excellent quality meat and for a characteristic increased development of muscle mass, caused by a point mutation (C313Y) in the Myostatin gene. The analysis revealed 98 samples to be homozygous (mh/mh) and five to be heterozygous (mh/1) for the mutation whereas one sample resulted to be homozygous for the wild type (1/1). The results obtained with the three different assays were consistent. Overall, all three methodologies proved to be efficient for allelic discrimination studies; however, real-time PCR was faster and allowed to genotype up to 96 samples in a single step, minimizing the number of steps required for samples manipulation.

A Regression-Based Approach to Selection Mapping

“…Animals with this phenotype have a significantly greater percentage of muscle than those with the wild-type genotype, and thus produce more meat at slaughter (Wiener et al 2009), suggesting that this characteristic has been strongly selected in these breeds due to its positive association with meat production capacity. The Piedmontese breed carries at high frequency a nonsynonymous mutation in myostatin (Kambadur et al 1997;McPherron and Lee 1997;Pozzi 2009), rather than the 11-bp deletion found in the Belgian Blue, Asturiana de los Valles, and South Devon breeds.…”

Biosensors for Point Mutation Detection

“…PCR based methods, such as PCR based allele detection system (AS-PCR) and fluorescent gene detection technology, allele specific small groove binding probe real-time PCR detection (RT-PCR), PCR restriction fragment length polymorphism (RFLP), polymerase chain reaction single strand conformation polymorphism (PCR-SSCP) and heteroduplex mobility analysis (HMA) belong among the most commonly used point mutation detection methods ( Callens and De Clercq, 1999 ; Pozzi et al, 2009 ; Riahi et al, 2015 ; Prykhozhij et al, 2018 ).…”