Dystrophin基因3～5号外显子缺失连接片段的克隆和 - 南方医科大学.DOC

Dystrophin基因3～5号外显子缺失连接片段的克隆和测序 作者：钟敏1；潘速跃1；陆兵勋1；姜立2；李伟1　　(南方医科大学1南方医院神经内科，2分子生物学研究所，广东??广州??510515) 摘要：目的 通过对抗肌萎缩蛋白(dystrophin)基因3～5号外显子缺失后连接片段的克隆和测序分析，探讨dystrophin基因缺失的发生机制。方法 先以外显子PCR反应检测证实1例杜氏肌营养不良症患者3～5号外显子缺失，然后在2、5号内含子上用PCR步移方法寻找断裂位点，最后用靠近断裂位点处设计的引物，以PCR法直接扩增dystrophin基因的缺失连接片段并测序，测序结果和正常内含子序列作对比分析。结果 获得2113 bp PCR产物，本例基因缺失片段长约49 000 bp。5’端断裂点位于2号内含子短散在元件Alu序列内，3’ 端断裂点在单一序列，附近有TTTAAA序列。断裂点附近有较强的拓扑异构酶Ⅱ酶切位点。连接片段插入了26 bp序列并在断裂点周围形成3个13 bp的短序列重复(GGCTTATATTTAA)连接断裂点两端。结论 推测重复序列、断裂点附近较强的拓扑异构酶Ⅱ酶切位点关联易引起基因的断裂重组，加上非同源末端连接修复机制等综合因素可能是导致基因缺失的重要原因。 关键词： 肌营养不良症；连接片段；基因缺失 中图分类号：Q78??文献标识码： A??文章编号：1673-4254(2006)06-0757-03 Cloning and sequencing of the junction fragment of dystrophin gene with exons 3 to 5 deletionZHONG Min1； PAN Su-yue1； LU Bing-xun1； JIANG Li2； LI Wei11Department of Neurology, Nanfang Hospital;??2Institute of Molecular Biology, Southern Medical University, Guangzhou 510515, ChinaAbstract: Objective To study the mechanisms of dystrophin gene deletion by cloning and sequencing the junction fragment of dystrophin gene with exons 3 to 5 deletion. Methods PCR was performed to verify dystrophin gene exons 3 to 5 deletion in a patient with Duchenne muscular dystrophy. A PCR-based genome-walking method was used to localize the breakpoint in introns 2 and 5, and the deletion-junction fragment was directly amplified by PCR approach with forward and reverse primers annealing to a DNA sequence as close as possible to the breakpoint in the introns 2 and 5. The sequencing result of the deletion-junction fragment was compared with the normal intron sequences. Results A sequence of 2113 bp containing the junction fragment was obtained. The 5’ breakpoint was located in SINE/Alu element of intron 2, and the 3’ breakpoint was located in the unique sequence near the sequence TTTAAA. The breakpoints were associated with a strong topoisomerase Ⅱ cleavage site. A 26-bp fragment was inserted into the breakpoint and formed 3 duplications (GGCTTATATTTAA) of 13 bp around th