论文部分内容阅读
家族性高胆固醇血症(Familial Hypercholesterolemia,FH)是冠心病的一种很重要的危险因素,早期发现FH患者,进行预防和治疗,就能减少心肌梗死的危险性。但是只根据临床表现不足以作出准确诊断(尤其是杂合子患者),所以,对其病因即低密度脂蛋白受体(Low Density Lipoprotein Receptor,LDL-R)基因突变的研究非常重要。本研究将PCR-SSCP与银染技术相结合,建立稳定的PCR-SSCP检测条件,以期对LDL-R基因的全部18个外显子进行点突变的筛查。 方法:从外周静脉血中提取基因组DNA。参照文献共设计了19对引物,分别扩增LDL-R基因的18个外显子。其中外显子4片段较长。因此设计了2对引物,分别扩增外显子4的5′和3′部分,以提高SSCP的敏感性,SSCP分析:将6μl扩增产物与2倍体积的变性液混合,95C变性5～10min,冰中骤冷。用夹心式垂直电泳槽进行8%非变性聚丙烯酰胺凝胶(交联度49:1)电泳,4C 180V电泳3h左右。凝胶的硝酸银染色:固定
Familial Hypercholesterolemia (FH) is a very important risk factor of coronary heart disease. Early detection of FH patients can reduce the risk of myocardial infarction for prevention and treatment. However, the diagnosis of low density lipoprotein receptor (LDL-R) gene mutation is very important because it is not enough to make accurate diagnosis based on the clinical manifestations (especially heterozygous patients). In this study, PCR-SSCP combined with silver staining technology to establish a stable PCR-SSCP detection conditions, in order to point mutation of all 18 exons of LDL-R gene screening. Methods: Genomic DNA was extracted from peripheral venous blood. A total of 19 pairs of primers were designed by reference to amplify 18 exons of LDL-R gene respectively. Exon 4 fragments of which longer. Therefore, two pairs of primers were designed to amplify the 5 ’and 3’ portions of exon 4, respectively, to improve SSCP sensitivity. SSCP analysis: 6 μl of amplification product was mixed with 2 volumes of denaturing solution, 10min, cold in the ice. 8% non-denatured polyacrylamide gels (49: 1 degree of crosslinking) were electrophoresed on a sandwich vertical electrophoresis cell and electrophoresed at 4C 180V for about 3 hours. Silver nitrate gel staining: fixed