与人遗传病相关(CTG)n·(CAG)n重复序列的分子克隆

Molecular Cloning on(CTG)_n·(CAG)_n Repeat Sequences Associated with Human Genetic Diseases

为了得到（CTG）n·（CAG）n重复序列,设计了一个可获得具有单个G/C悬端的重复序列的克隆策略.化学合成重复序列及两侧接头序列,两侧接头有EcoR Ⅰ、BamH Ⅰ和Ple Ⅰ酶切位点,EcoR Ⅰ、BamHⅠ位点用于将重复序列插入载体,Ple Ⅰ酶切位点可方便地将重复序列从重组质粒中取出.使用该方案,可获得长度在200～1000bp的重复序列片段.本工作为进一步研究和人遗传病相关的重复序列的结构特性及该类遗传疾病的分子机理奠定基础.

The generation of long uninterrupted DNA repeats is important for the study of repeat instability associated with several human genetic diseases. To clone （CTG）n·（CAG）n repeat sequences,a cloning vector is designed from which the pure repeat fragments containing a G/C overhang can be generated for further ligation. There exist EcoR Ⅰ, BamH Ⅰ and Ple Ⅰ sites in chemical synthesized repeat sequences and adaptors. The sites of EcoR Ⅰ and BamH Ⅰ are used to insert repeats sequences into vector. Ple Ⅰ sites can be used to take out repeats sequences from recombinant plasmid easily. （CTG）n·（CAG）n DNA molecules longer than 800bp were generated using this approach. This approach also worked for other repeats sequences. This work was the basis of the further investigation on structural feature of repeat sequences and molecular mechanism of this kind of genetic diseases.