遗传 ›› 2008, Vol. 30 ›› Issue (12): 1635-1639.doi: 10.3724/SP.J.1005.2008.01635

• 技术与方法 • 上一篇    下一篇

SSCP和HMA方法在马MHC-I类分子基因多态性研究中的应用

项伟1, 2; 马建2, 3; 王雪峰2, 4; 赵玉军1; 周建华2

  

  1. 1. 沈阳农业大学畜牧兽医学院, 沈阳 110161;
    2. 中国农业科学院哈尔滨兽医研究所 兽医生物技术国家重点实验室, 哈尔滨 15001;
    3. 东北林业大学野生动物资源学院, 哈尔滨 150040;
    4. 内蒙古农业大学动物科学与医学学院, 呼和浩特 010018

  • 收稿日期:2008-03-12 修回日期:2008-04-21 出版日期:2008-12-10 发布日期:2008-12-10
  • 通讯作者: 周建华

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

XIANG Wei1, 2; MA Jian2; WANG Xue-Feng2, 4; ZHAO Yu-Jun1; ZHOU Jian-Hua 2

  

  1. 1. Institutes of Animal Husbandry and Veterinary Medicine, Shenyang Agricultural University, Shenyang 100161, China;
    2. National Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin 150001, China;
    3. College of Wildlife Resources, Northeast Forestry University, Harbin 150040, China;
    4. College of Animal Science and Veterinary Medicine, Inner Mongolia Agricultural University, Hohhot 010018, China

  • Received:2008-03-12 Revised:2008-04-21 Online:2008-12-10 Published:2008-12-10
  • Contact: ZHOU Jian-Hua

摘要:

摘要: 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。

关键词: SSCP, HMA, 基因多态性, MHC-I类分子基因

Abstract:

Abstract: 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.