激光诱导荧光毛细管电泳法优化随机寡核苷酸文库聚合酶链反应扩增的条件

Optimization of Polymerase Chains Reaction Amplification for ssDeoxyribonucleic Acid Library Using Capillary Electrophoresis with Laser-induced Fluorescence Detection

寡核苷酸文库的聚合酶链反应(PCR)扩增是指数富集系统配基进化技术(SELEX)筛选适配子技术中的重要步骤。本研究采用毛细管电泳技术结合激光诱导荧光检测器(CE-LIF),通过对双链产物、PCR副产物以及剩余引物的考察,研究了寡核苷酸文库PCR扩增时的影响因素,并对其进行优化。结果表明,随机寡核苷酸文库的扩增与传统均一模板的扩增显著不同,其在扩增十几个循环时产物就达到最大值;此外,初始模板数、退火温度、DNA聚合酶浓度等条件也有所不同。因此,在进行SELEX筛选之前必须对文库PCR条件进行详细优化。本研究中N39文库优化后的PCR扩增条件为:初始模板的量为105个分子,DNA聚合酶浓度0.05U/μL,退火温度70℃,18个循环。本研究为利用SELEX技术有效筛选适配子,降低筛选的假阳性及提高特异性提供了参考依据。

For random DNA libraries, the amplification of polymerase chains reaction（PCR） is very important to select aptamers by systematic evolution of ligand exponential enrichment（SELEX）. The influencing factors of PCR amplification were investigated and optimized according to the changes of dsDNA product, ss-dsDNA by-products and primers using capillary electrophoresis with laser-induced fluorescence detection（CE-LIF）. The results suggested that there were prominent differences between PCR amplification of homogeneous DNA templates and that of random DNA libraries. For random DNA library, the yield of products reached its maximum at the eighteenth cycle. Other influence factors including the number of initial templates, anneal temperature of PCR, concentration of DNA polymerase were also investigated. To ensure efficiency of SELEX, it is necessary to optimize the PCR amplification conditions of random DNA libraries. In this study, for the N39 DNA libraries, the optimized conditions were as follows： number of initial templates was 105 molecules, concentration of DNA polymerase was 0.05 U/μL, and annealing temperature of PCR was 70 ℃, the optimization PCR cycles was 18. This study provided important references for the selection of high efficient aptamers by SELEX method.