聚合酶链反应-单链构象多态性银染后胶回收在基因改变检测中的应用

Application of polyacrylamide gel reclamation after polymerase chain reaction-single strand conformation polymorphism-argentation in the detection of gene alteration

目的:探讨聚合酶链反应-单链构象多态性银染后胶回收的方法在基因改变检测中的应用。方法:实验于2004-09/2005-10在南方医院心内科实验室完成。样本来源:于2004-09/2005-08选择南方医院心内科收治的冠状动脉粥样硬化性心脏病(简称冠心病)患者123例,均符合WHO冠心病诊断标准,并经心电图、超声心动图、CT确诊。患者均知情同意。聚合酶链反应-单链构象多态性银染方法检测冠心病患者三磷酸腺苷结合盒转运体A1基因改变,对1例怀疑存在基因改变样本的聚丙烯酰胺凝胶采用银染后单链胶回收,以胶回收后的单链聚合酶链反应产物为模板重复聚合酶链反应后进行测序。结果:在对三磷酸腺苷结合盒转运体A1基因Exon7进行单链构象多态性检测时,发现一样本泳带异常,对其样本进行银染后胶回收,结合限制性内切酶酶切,证实此样本存在碱基改变,从而验证了非变性聚丙烯酰胺凝胶银染后胶回收的准确性和可行性。结论:经聚合酶链反应-单链构象多态性银染后的非变性聚丙烯酰胺凝胶胶也可进行胶回收。聚合酶链反应-单链构象多态性银染后胶回收在基因改变检测中可以增强单链构象多态性基因突变检测的准确性,由于其无毒性、成本低及操作简单等优点,更易被实验人员所接受。

AIM： To investigate the application of polyacrylamide gel （PAGE） reclamation after polymerase chain reaction （PCR） -single-strand conformation polymorphism （SSCP）-argentation in the detection of gene alteration, METHODS ： The experiment was conducted in the laboratory of Department of Cardiology, Nanfang Hospital from September 2004 to October 2005. The samples were derived from 123 patients that were diagnosed by coronary artery disease from the Department of Cardiology of Nangfang Hospital between September 2004 and October 2005. They were all in accordance with the diagnostic criteria of WHO for coronary artery disease and diagnosed through electrocardiogram, echocardiogram and CT. All the patients were informed and agreed to the experiment. The genetic alteration of ABCA1 gene in patients with coronary artery disease was detected with PCR-SSCP-argentation. Recovery of PAGE, repeated PCR and sequencing were performed in one case with suspect of gene alteration. RESULTS： When detecting Exon7 of ABCA1 gene with SSCP, a sample band with exited abnormal was found, which was detected with the method of PAGE reclamation after PCR-SSCP-argentation and digested with restriction fragment length polymorphism to demoristrate that there was basic radical alteration in this case and non-denaturing PAGE with argentation could be reclaimed successfully, CONCLUSION： The non-denaturing PAGE with argentation could be reclaimed after PCR-SSCP-argentation. The method of PAGE reclamation after PCR-SSCP-argentation could enhance the accuracy in detecting the gene alteration with SSCP; moreover, this method is more acceptable by experimenter as its avirulent, lower cost and simple manipulation.