High-resolution melting analysis for detection of a single-nucleotide polymorphism and the genotype of the myostatin gene in warmblood horses.

The research article is a study on the development of a high-resolution melting analysis (HRM) for detecting a specific polymorphism in the myostatin gene (MSTN) present in warmblood horses. The efficiency of the HRM analysis is evaluated against standard PCR methods.

Objective and Sample Collection

• The main objective of the research was to create a HRM assay which could effectively detect the g.66493737C>T polymorphism in the MSTN gene in warmblood horses. The frequency of three pre-defined genotypes related to this polymorphism was also studied.
• The sample basis for this study was blood samples collected from 23 different horses.

Procedures and Protocol Development

• DNA from the collected blood samples was extracted and then analyzed through standard PCR methods as well as the developed HRM assay to determine the MSTN genotype.
• The assay included three protocols namely standard protocol, a method involving a high-salt solution added prior to the first melting cycle, and a technique where an unlabeled probe was added to the reaction mixture before analysis. The protocols were designed and then compared.
• The HRM protocol resulting in the most consistent melting curves was chosen. Results thus obtained were compared with those achieved by sequencing.

Results and Outcome

• The HRM protocol involving the unlabeled probe added to the reaction mixture had the most consistent melting curves.
• The genotypes of the g.66493737C>T polymorphism for 22 horses were decided (16 by HRM analysis and 20 by sequencing), where 14 horses had the T/T genotype, 7 had T/C and 1 had C/C genotype.
• The genotype determined by HRM analysis corresponded with the ones revealed by sequencing for 14 out of 16 horses.
• The frequency of T and C alleles was 79.5% and 20.5% respectively.

Conclusions

• HRM analysis was found to be a faster and more economical alternative for genotyping than PCR.
• The genotyping results could potentially be used as predictors for athletic performance in horses.