克隆测序方法检测三核苷酸重复次数的可靠性  

The reliability of cloning-sequencing to detect the number of trinueleotide repeats

作　　者：郑巧娟[1] 甘世锐[1] 王柠[1] 吴志英[2] 

机构地区：[1]福建医科大学附属第一医院神经内科,福州350005 [2]复旦大学附属华山医院神经内科

出　　处：《中华神经科杂志》2010年第9期659-663,共5页Chinese Journal of Neurology

基　　金：福建省高校创新团队培育计划（FMU-RT002）;复旦大学特聘教授华山医院配套基金资助项目

摘　　要：目的探讨克隆测序技术用于检测三核苷酸（CAG）重复次数的可靠性。方法对1例临床确诊的遗传性脊髓小脑性共济失调1型（SCA1）患者，PCR法扩增ATXN1基因的CAG重复次数，8％变性聚丙烯酰胺凝胶电泳（DPAGE）分离PCR产物以协助确诊。2．5％琼脂糖凝胶电泳分离PCR产物的大小片段，割胶回收大小片段测序。另外将割胶回收的大片段进行TA克隆测序。结果DPAGE提示大片段存在CAG的异常扩增，故该患者可确诊为SCA1。PCR产物大小片段直接测序结果为：小片段为26次CAG重复，大片段为47次重复。而将大片段克隆测序后，则有50、47、46、41、32、28、27、26、25及24次共10种不同的CAG重复次数，而且出现了CCG、CGG、CTG、CAA、TAT等序列变异现象。结论克隆测序检测CAG重复次数会造成重复次数变异，且存在各种碱基变异。因此不宜单用TA克隆测序检测CAG重复次数及筛查碱基变异，需联合应用多种方法以提高结果的可靠性。Objective Cloning-sequencing is a common method to detect the number of trinucleotide repeats. The aim of the present study is to discuss its reliability. Methods One clinically diagnosed SCA1 patient was recruited in the study. The numbers of CAG repeats in ATXN1 gene were estimated via polymerase chain reaction （PCR） and denaturing polyacrylamide gel electrophoresis （DPAGE）. To verify accuracy of CAG numbers estimated, the PCR products were electrophoresed on a 2. 5% agarose gel and separated bands were excised for direct sequencing. Also, the longer separated band underwent cloning-sequencing using a TA cloning kit. Results The patient was identified as SCA1 by DPAGE. After direct sequencing, the numbers of CAG repeats were 26 and 47 in the shorter and longer bands, respectively. However, after cloning-sequencing of the longer band, there are 10 different numbers of CAG repeats, including 50, 47, 46, 41,32, 28, 27, 26, 25 and 24. Furthermore, there are other kinds of trinucleotide repeats, such as CCG, CGG, CTG, CAA and TAT scattered among the CAG repeats. Conclusions It is not reliable to identify the number of trinucleotide repeats by cloning-sequencing alone. To improve the reliability, it is better to combine cloning-sequencing with other methods.

关 键 词：脊髓小脑共济失调 三核苷酸重复 神经组织蛋白质类 核蛋白质类 克隆分子 可重复性 结果 

分 类 号：R440[医药卫生—诊断学]