SSCP在寡核苷酸适配子ssDNA多克隆筛选中的应用

目的利用单链构象多态性技术弥补SELEX(systematic evolution of ligands by exponential en-richment,SELEX)实验在寡核苷酸适配子克隆、测序环节中的不足,减少SELEX实验的环节。方法利用单链构象多态性技术(singel-strand conformation polymorphism,SSCP)将多轮SELEX筛选的198个寡核苷酸适配子克隆在测序前进行SSCP筛查。结果对198个克隆的SSCP电泳条带位置分析,挑选出58个克隆测序。SSCP筛查得到有效克隆的检出率为20.69%,是不用SSCP筛查检出率6.07%的3.4倍;如果去除组间误差,有效克隆的检出率44.44%是不用SSCP筛查检出率的7.32倍。结论通过对198个克隆的20批次SSCP电泳筛查,并经58个克隆测序后的实验结果证明:同一SSCP电泳胶片上电泳条带泳动位置相同的克隆片段,基因序列相同;电泳条带泳动位置不同的片段,基因序列不同。根据SSCP电泳条带泳动位置,挑选有代表性的克隆测序,避免对多克隆盲目测序,为多克隆测序前的筛查探索一条新的途径。

Application in screening multiclone of ssDNA olig- aptamer with SSCP

Objective To compensate the shortage of SELEX（systematic evolution of ligands by exponential enrichment, SELEX）in olig-aptamer clone and sequencing, and avoid missing any precious positive clone of olig-aptamer. Methods SSCP（singel-strand conformation polymorphism, SSCP）was used to screen 198 olig-aptamers multiclones selected by several rounds of SELEX. Results After analyzing the band position of 198 multiclones in SSCP electrophoresis, 58 clones was selected to sequence. The isolation rate of positive clone with SSCP was 20.69% (12/58), which was 3.4 times that of positive clone without SSCP. If interclass error of SSCP was eliminated, the isolation rate of positive clone with SSCP was 44.44% (12/27), which was 7.32 times that of positive clone without SSCP. Conclusions The experiment result indicates that in one SSCP gel, same position bands have the same sequence while different position bands have distinct sequences, which indicates that SSCP electrophoresis band can be used to choose typical clones for sequencing. SSCP electrophoresis provides a new method to screen positive multi-clones before sequencing.