Applications of SSCP and HMA for polymorphic analysis of horse MHC-I alleles

In this article, we report the analysis of genetic polymorphisms of horse MHC-I molecules by SSCP and HMA, which are methods based on the technique of polyacrylamide gel electrophoresis (PAGE). Our results showed that SSCP was not a suitable method for the analysis of genetic polymorphisms of horse MHC-I molecules due to the failure in generating satisfied separation of DNA fragments, even if experimental conditions were optimized. However, the HMA method produced clearly separated DNA fragments of horse MHC-I molecules, after the experimental conditions, such as the running temperature and the concentration of detergent, were optimized by using a reference plasmid. PCR-amplified samples from horses No. 6, No. 7, No. 8, No. 9 and No. 10 generated 6, 5, 6, 5, and 7 bands, respectively, in corresponding lanes of the polyacrylamide gel. DNA fragments in each band cut from the gel were amplified by PCR using a second pair of primers, and were cloned for sequencing. Alignment analysis of these sequences revealed that HMA was a proper method to efficiently analyze the polymorphisms of MHC-I molecule genes.