降落聚合酶链反应技术在低密度脂蛋白受体基因点突变研究中的应用

Application of TOUCH DOWN PCR technique in the research of detecting gene point mutation in LDL-R gene

目的 建立降落 (TOUCHDOWN)聚合酶链反应 (PCR)方法 ,快速检测家族性高胆固醇血症患者低密度脂蛋白受体 (LDL R)基因点突变。方法 设计LDL R基因 2 1对引物 ,根据TOUCHDOWN原理设计包括启动子和 18个外显子特异性片段在内的PCR程序 ,通过试验选择PCR最佳反应条件 ,在同一程序中分别对 2 1个片段进行扩增 ,琼脂糖凝胶电泳检测扩增产物 ,纯化后DNA产物测序分析该方法的特异性。结果 编设退火温度范围从 6 8℃降至 5 4℃的PCR程序 ,优化PCR扩增条件 ,电泳检测采用同一程序同时分别扩增的LDL R基因 2 1个片段条带清晰 ,测序证实了此组片段的特异性。结论 成功建立降落PCR方法 ,提示此法为LDL R基因突变筛查提供了快速可靠的手段。

Objective To establish a method for detecting point mutations of low density lipoprotein receptor(LDL-R) gene in patients with Familial hypercholesterolemia(FH)by TOUCH DOWN PCR technique. Methods To design 21 pairs of primer of LDL-R gene which included 18 specific extrons and promoter. Twenty-one fragments were amplified simultaneously through designing an optimized procedure of decreasing annealing temperature in a certain scale and choosing optimal PCR reaction condition. Amplified products were tested by agarose gel electrophoresis and analyzed by sequence analysis. Results TOUCH DOWN PCR procedure whose annealing temperature covering from 68℃ to 54℃ optimized PCR amplifying condition. The twenty-one aimed fragments of LDL-R gene could be amplified simultaneously by a single procedure, electrophoresis band was clear and specificity of these fragments had been proved by sequence analyze, satisfying gene type was obtained. Conclusion Successfully establishing TOUCH DOWN PCR technique provided an quick and reliable way to detect LDL-R gene mutation.