Zhong Min, Pan Su-yue, Lu Bing-xun, Jiang Li, Li Wei
Department of Neurology, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China.
Nan Fang Yi Ke Da Xue Xue Bao. 2006 Jun;26(6):757-9.
To study the mechanisms of dystrophin gene deletion by cloning and sequencing the junction fragment of dystrophin gene with exons 3 to 5 deletion.
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.
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 the deletion-junction fragment.
Repeat sequence and strong topoisomerase II cleavage site around the breakpoint may predispose double-strand DNA breaks and recombination, which, in addition to the nonhomologous end-joining mechanism, may contribute as important factors to the gene deletion.
通过克隆和测序缺失外显子3至5的肌营养不良蛋白基因的连接片段，研究肌营养不良蛋白基因缺失的机制。
采用聚合酶链反应（PCR）验证一名杜氏肌营养不良症患者的肌营养不良蛋白基因外显子3至5缺失情况。运用基于PCR的基因组步移法定位内含子2和5中的断点，通过PCR方法使用正向和反向引物直接扩增缺失连接片段，引物与内含子2和5中尽可能靠近断点的DNA序列退火。将缺失连接片段的测序结果与正常内含子序列进行比较。
获得了一个包含连接片段的2113 bp序列。5'断点位于内含子2的SINE/Alu元件中，3'断点位于靠近TTTAAA序列的独特序列中。断点与一个强拓扑异构酶II切割位点相关。一个26 bp的片段插入到断点处，并在缺失连接片段周围形成了3个13 bp的重复序列（GGCTTATATTTAA）。
断点周围的重复序列和强拓扑异构酶II切割位点可能易导致双链DNA断裂和重组，除了非同源末端连接机制外，可能作为重要因素促成基因缺失。
