克隆缺失连接片段——一种基于PCR技术的缺失型假肥大型肌营养不良症的携带者检测

目的 依据dystrophin基因缺失后断端重接可形成一段变异的DNA序列,提出一种利用缺失连接片段进行缺失型假肥大型肌营养不良症携带者检测的新方法.方法 实验以来自广东省肇庆地区的一个Becker型肌营养不良(Becket muscular dystrophy,BMD)家系为研究对象,其中2例确诊的男性BMD患者,3例待诊的女性携带者,1例待诊的人工流产绒毛.先证者经外显子PCR检测确定第3～5外显子缺失,随后采用PCR步移法在相应内含子设计引物定位断裂点的位点,最后利用靠近断裂点设计的引物直接对家系的6例基因组DNA进行缺失连接片段的PCR扩增和测序.结果 6例基因组DNA均扩增出阳性的产物片段且连接片段的测序序列完全一致,绒毛的性别诊断结果为女性,可以确诊本家系中的3个女性和流产绒毛均为缺失型BMD携带者.结论 作者成功地将整个家系患者和携带者的缺失连接片段进行克隆和测序分析,实现了利用缺失连接片段对缺失型假肥大型肌营养不良症携带者进行准确基因诊断的设想,同时对在产前诊断上的应用前景进行了探讨.

Cloning of deletion junctions: a method of PCR for detecting the deletional pseudohypertrophic muscular dystrophy carriers

Objective Dystrophin gene deletion junction is the tmique DNA sequence resulted from illegitimate recombination after the gene deletion.A novel accurate approach is presented here for the detection of deletional pseudohypertrophic muscular dystrophy carriers with the deletion junctions.Methods A Becker muscular dystrophy (BMD)family from Zhaoqing,Guangdong,China was used.Two males in the family were diagnosed as BMD patients,3 phenotypically normal females and 1 charionic villi sample of an artificial abortion were waiting for diagnosis.The index patient was identified as exons 3-5 deletion of the dystrophin gene.Then a PCR-based genome-walking method was used to locate the breakpoints in corresponding introns.Finally,deletion junctions of the 6 family members were amplified by PCR with primers adjacent to breakpoints and sequenced.Results The deletion junctions of all patients and carriers of the BMD family were cloned and sequenced.The 3 females and 1 ehorionic tissue were diagnosed as female carriers.Conclusion In this study researchers have successfully carried out accurate geno diagnosis of delefional pseudohypertrophic carriers by cloning and sequencing the deletion junctions,and explored the prospect of using deletion junctions in prenatal diagnosis of BMD.