SSCP和HMA方法在马MHC-I类分子基因多态性研究中的应用

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

文章使用SSCP和HMA两种基于聚丙烯酰胺凝胶电泳的方法对马MHC-I类分子基因多态性进行了分析。在应用SSCP法进行分析时,尽管经过实验条件优化,仍未得到对MHC-I基因理想的分离效果,提示该方法对分离多态性较高的基因有一定局限性。在对HMA法用参考标准DNA对影响DNA分子构象的温度和变性剂浓度等实验条件进行优化后,获得了对马MHC-I类分子基因较好的分离效果。6、7、8、9和10号马的样本在相对应泳道上分别出现了6、5、6、5和7个条带。从凝胶中进行DNA条带回收后克隆测序的结果表明,这一方法可以有效地分离高度多态性的MHC-I类分子基因。

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 gener- ating 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 run- ning 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 prim- ers, 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.