Open AccessApplications of SSCP and HMA for polymorphic analysis of horse MHC-I alleles
Author(s) -
Wang Xiang,
Jian Ma,
Xuefeng Wang,
Yongju Zhao,
Jianhua Zhou
Publication year - 2009
Publication title -
yichuan
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.125
H-Index - 16
ISSN - 0253-9772
DOI - 10.3724/sp.j.1005.2008.01635
Subject(s) - single strand conformation polymorphism , microbiology and biotechnology , dna , biology , genetics , gel electrophoresis , polyacrylamide gel electrophoresis , major histocompatibility complex , restriction fragment length polymorphism , horse , plasmid , polymerase chain reaction , mhc class i , allele , genetic analysis , gene , biochemistry , paleontology , enzyme
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.