指数富集配基系统进化技术筛选三阴乳腺癌适配子中聚合酶链反应扩增条件的优化及单链DNA的制备  

Selecting aptamers of triple negative breast cancer by systematic evaluation of ligands by exponential enrichment： addressing the ploymerase chain reaction and single-stranded DNA generation issues

作　　者：王芹芹[1] 胡慧[1] 崔军威[1] 韦伟[1] 

机构地区：[1]北京大学深圳医院乳腺外科,深圳518000

出　　处：《中华实验外科杂志》2014年第12期2732-2734,共3页Chinese Journal of Experimental Surgery

基　　金：深圳市科技局资助项目（201202015）

摘　　要：目的 优化三阴乳腺癌（TNBC）适配子筛选中单链DNA（ssDNA）文库扩增条件,探讨次级ssDNA最佳制备方法.方法 构建ssDNA文库,设置不同的模板量（1012、1011、1010、109、108、107、106、105条带）、循环数（9、12、15、18、21、24、27、30）、DNA聚合酶量（0.025、0.050、0.100、0.200、0.400 U/μl）进行聚合酶链反应（PCR）扩增;对比3种制备次级ssDNA的方法,取其中最佳方法进行筛选.结果 20μl体系最优扩增条件为：ssDNA模板量108个条带、扩增循环数为12、DNA聚合酶量为0.100 U/μl;不对称PCR法所得ssDNA量最多.结论 确立了ssDNA文库最优扩增条件及次级ssDNA最佳制备方法,为进一步筛选TNBC特异性适配子提供实验基础.Objective To optimize the polymerase chain reaction （PCR） amplification conditions of single-stranded DNA （ssDNA） pool and evaluate different methods for generation of ssDNA in selecting targeted aptamers of triple negative breast cancer （TNBC） by systematic evaluation of ligands by exponential enrichment （SELEX）.Methods ssDNA library was synthesized.The initial template concentration （1012,1011,1010,109,108,107,106 and 105 sequences）,amplification cycles （9,12,15,18,21,24,27 and 30） and DNA polymerase concentrations （0.025,0.050,0.100,0.200,and 0.400 U/μl） were set up for the amplification of random ssDNA.Efficiency of ssDNA generation was evaluated by comparing three most used methods.Results The optimal ssDNA template concentration was 108 sequences,cycle number was 12 and DNA polymerase concentration was 0.100 U/μl with a total system volume of 20 μl.Asymmetric PCR produced the highest amount of ssDNA.Conclusion The PCR amplification conditions of ssDNA library and the best method to generate ssDNA were established,which laid experimental foundations for the further selection of TNBC targeted aptamers.

关 键 词：单链DNA 适配子 三阴乳腺癌 

分 类 号：R737.9[医药卫生—肿瘤]