祝军,汪丽,张景海
ZHU Jun,Wang Li,ZHANG Jing-hai(School of Life Science and Biopharmaceutics

• 1:沈阳药科大学生命科学与生物制药学院
• 2:沈阳药科大学生命科学与生物制药学院
• 3:沈阳药科大学生命科学与生物制药学院 辽宁沈阳110016
• 4:辽宁沈阳110016

摘要(Abstract)：

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。
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 oligaptamers 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.

关键词(KeyWords)： 单链构象多态性技术(SSCP);指数级富集的配体系统进化(SELEX);寡核苷酸适配子;多克隆;测序
SSCP;SELEX;oligo-aptamers;multiclone;sequencing

Abstract:

Keywords:

基金项目(Foundation):

作者(Author): 祝军,汪丽,张景海
ZHU Jun,Wang Li,ZHANG Jing-hai(School of Life Science and Biopharmaceutics

参考文献(References)：

• [1]戚豫,黄丽英.DNA单链构象多态性的原理[J].北京医科大学学报,1997,29(1):81-82.
• [2]欧阳建华,黄建安.PCR-SSCP技术的研究进展[J].上海畜牧兽医通讯,2002,4:11-12.
• [3]姜适良,李宁,赵兴波,等.影响PCR-SSCP的因素分析[J].农业生物技术学报,2000,8(3):245-247.
• [4]ROBERTSON D L,JOYCE G F.Selectionin vitroofan RNA enzyme that specifically cleaves single-strandedDNA[J].Nature,1990,344(6265):467-468.
• [5]TUERK C,GOLD L.Systematic evolution of ligands byexponential enrichment:RNA ligands to bacteriophageT4 DNA polymerase[J].Science,1990,249(4968):505-510.
• [6]BRODY E N,GOLD L.Aptamers as therapeutic and di-agnostic agents[J].J Biotechnol,2000,74(1):5-13.
• [7]ZHAO Shan-chuan,Siew Loon Ooi,YANG Fu-chai,etal.Three methods for identificstion of two positivecloned cDNA fragments in differential display[J].BioTechniques,1996,20:400-404.