变性高效液相色谱法在FALS突变位点检测中的应用

Denaturing high performance liquid chromatography for detection of point mutation of familial ALS

目的检测一个肌萎缩侧索硬化症(amyotrophic lateral sclerosis,ALS)家系的铜、锌超氧化物歧化酶(Cu/Zn superoxide dismutase,SOD1)基因的突变位点,同时观察变性高效液相色谱法(denaturing high performance liquid chromatography,DHPLC)的实用价值。方法对PCR-SSCP检测阴性的外显子,应用DHPLC法及DNA直接测序技术,再次进行SOD1基因的突变位点检测。结果经DHPLC检测,家系成员Ⅲ1SOD1基因的第4外显子有突变峰,DNA直接测序证实存在杂合子,发生了GAA→GGA错义突变,使编码的氨基酸由谷氨酸变为甘氨酸。结论DHPLC技术与PCR-SSCP相比有更高的敏感性,可作为大样本筛查突变位点的一种便捷可靠手段。

Objective To identify the point mutation of Cu/Zn superoxide dismutase(SOD1) gene in an amyotrophic lateral sclerosis(ALS) family and observe the value of denaturing high performance liquid chromatography(DHPLC). Methods DHPLC and DNA sequencing were used to examine SOD1 gene of the ALS family which had not been found mutation by PCR-SSCP. Results DHPLC tests proved double peaks in one member(Ⅲ_1), Which indicated the possibility of mutation in SOD1 exon 4. DNA sequencing revealed that there was a heterozygote,with mutation of GAA to GGA in exon 4, and with a substitution of glutacid by glycine. Conclusion As compared with PCR-SSCP, DHPLC technique has proved to be a rapid and reliable method for screening mutation site in large samples.