摘要
为了解Dystrophin基因缺失断裂点和连接片段的序列特点 ,以分析Dystrophin基因缺失的分子机制 ,利用巢式反向PCR克隆了 1名 5 1号外显子缺失DMD(DuchenneMuscularDystrophy ,DMD)患者的缺失连接片段 ,通过测序 ,确定 5′和 3′断裂点及连接片段的序列。对 5′、3′断裂点和连接片段进行重复序列、TOPOI、TOPOII酶切位点等分析。结果共测得 5 0号内含子 16 14bp ,确定该患者Dystrophin基因的 5′断裂点位于THE1(Transposon likeHumanElement,THE)内 ,3′断裂点位于L2序列内。连接片段有 3bp的连接同源序列cta ,局部无小的缺失、插入和碱基置换。本研究首次在 5 0号内含子内发现一THE1序列 ,再次发现Dystrophin基因的缺失断裂点位于THE1结构内。反向PCR操作简单、耗时短 ,可以推扩应用于缺失连接片段的克隆 ;THE1可能与部分Dystrophin基因的缺失有关 ;Dystrophin基因缺失大多与同源重组无关 ,非同源末端连接可能参与了Dystrophin基因缺失的形成。
To study the mechanism of Dystrophin gene deletion, we obtained the deletion junction fragment of exon 51 by inverse PCR and analyzed the sequence characteristic of breakpoints and deletion juction fragment. After the full sequence of intron 51 was finished, the rough site of breakpoint in intron 51 of a DMD patient with exon 51 deletion was detected by PCR with 9 pairs of primers spaced every 3～5kb in intron 51. Then the junction fragment was amplified by nested inverse PCR. After sequencing the junction fragment, the 3′ breakpoint and partial sequences of intron 50 were determined by comparing with the normal sequences in intron 51. The primer was designated to sequence intron 50 according to the sequence of junction fragment, and then the 5′ breakpoint was determined. A total of 1?614bp in intron 50 was sequenced. The 5′ and 3′ breakpoints were located in the THE 1 internal seqeucne (Transposon like Human Element, THE) and L2 sequence respectively. There are 3bp junctional homology and no errors near the junction point. This is the second report that the deletion breakpoint located directly in THE 1 sequence studied at the DNA level. We here firstly reported that there is a THE 1 sequence in intron 50. THE 1 and non homologue end joining repair mechanism may be associated with the Dystrophin gene deletion.
基金
国家自然科学基金 (No.3 9870 80 4)
广东省自然科学基金 (No .970 0 610 0 10 3 2 )
卫生部临床重点项目基金 (No.970 40 2 2 9)
CMB基金 (No .98-677)~~
