SELEX技术中ssDNA文库PCR扩增条件的优化  

Optimization of PCR Reaction System for Random Single-strand DNA Pool in SELEX Technology

作　　者：曹立亭[1] 许李丽[1] 万向[1] 王秋菊[1] 马跃[1] 

机构地区：[1]西南大学荣昌校区动物医学系,重庆荣昌402460

出　　处：《安徽农业科学》2012年第6期3241-3242,3297,共3页Journal of Anhui Agricultural Sciences

基　　金：中央高校基本科研业务费专项资金资助项目(XDJK2011C026);西南大学博士基金(09BSR04)

摘　　要：[目的]对SELEX技术中ssDNA文库的PCR扩增条件进行优化。[方法]采用L16(45)正交试验设计在4个水平上对影响单链随机DNA文库PCR反应体系的Mg2+浓度、dNTP浓度、TaqDNA聚合酶含量、引物浓度和随机单链DNA文库量5个重要因素进行了优化,同时对PCR反应的退火温度和循环次数进行了优化,确立最优反应体系和扩增程序。[结果]20μlPCR反应体系及反应程序中各因素优化组合为:10×Buffer2.0μl,随机ssDNA文库0.5ng,Mg2+2.5mmol/L,dNTP Mixture0.25mmol/L,上下游引物各0.6μmol/L,Taq DNA聚合酶1.5U;退火温度为68℃,最佳循环数为12。此反应系统下,随机ssDNA文库PCR扩增谱带清晰、稳定、特异性高。[结论]为SELEX技术中筛选到特异性更强的适配子奠定了基础。[Objective] This study aimed to optimize the PCR amplification conditions for random ssDNA pool in SELEX technology. [Method] L16（45） orthogonal experimental design was adopted for optimization of five important factors that affecting PCR reaction system for random single-stranded DNA pool including Mg2＋ concentration, dNTP concentration, amount of Taq DNA polymerase, primer concentration and amount of random single-stranded DNA pool at four levels. Meanwhile, the annealing temperature and number of PCR reaction cycles were optimized to establish the optimal reaction system and PCR procedure. [Result] The optimal combination of PCR reaction system for random ssDNA pool was obtained, with a total system volume of 20 μl containing 2.0 μl of 10×Buffer, 0.5 ng of random ssDNA pool, 2.5 mmol/L of Mg2＋, 0.25 mmol/L of dNTP Mixture, 0.6 μmol/L of upstream and downstream primers and 1.5 U of Taq DNA polymerase; the optimal annealing temperature was 68 ℃ and the optimal number of cycles was 12. Under the above conditions, clear and stable bands with high specificity for random ssDNA pool were amplified. [Conclusion] This study had laid the foundation for selection of aptamers with higher specificity in SELEX technology.

关 键 词：随机单链DNA文库 正交试验设计 PCR 体系优化 

分 类 号：Q93-33[生物学—微生物学]