Geisel J, Walz T, Bodis M, Nauck M, Oette K, Herrmann W
Klinisch-Chemisches Zentrallabor, Universitätskliniken des Saarlandes, Homburg, Germany.
J Chromatogr B Biomed Sci Appl. 1999 Mar 19;724(2):239-47. doi: 10.1016/s0378-4347(98)00581-7.
We describe here a new method to screen for unknown mutations in the low density lipoprotein (LDL) receptor gene by the use of capillary electrophoresis in single-strand conformation polymorphism (SSCP) analysis. To analyze the promoter and all 18 exons, 20 different amplification reactions were necessary. For each polymerase chain reaction (PCR), the forward and reverse primers were 5' fluorescent-labelled with FAM and HEX, respectively. To test the accuracy of the newly developed method, 61 genetic variants distributed in 16 exons were analyzed. Under identical electrophoresis conditions (13 kV, 30 degrees C, 30 min), 59 mutations were detected by a distinct abnormal SSCP pattern. The two remaining mutations showed only slight abnormalities, which could be amplified by increasing the electrophoresis temperature. The high accuracy, the degree of automation and the speed of analysis make fluorescence-based SSCP analysis with capillary electrophoresis ideal for rapid mutation screening and the technique is well-suited for clinical applications.
我们在此描述一种通过在单链构象多态性（SSCP）分析中使用毛细管电泳来筛选低密度脂蛋白（LDL）受体基因中未知突变的新方法。为了分析启动子和所有18个外显子，需要进行20种不同的扩增反应。对于每个聚合酶链反应（PCR），正向和反向引物分别用FAM和HEX进行5'荧光标记。为了测试新开发方法的准确性，分析了分布在16个外显子中的61个基因变体。在相同的电泳条件下（13 kV，30℃，30分钟），通过独特的异常SSCP模式检测到59个突变。其余两个突变仅显示轻微异常，可通过提高电泳温度进行扩增。基于荧光的毛细管电泳SSCP分析的高准确性自动化程度和分析速度使其成为快速突变筛选的理想选择，该技术非常适合临床应用。
