基于RhD蛋白Aptamer筛选技术的随机ssDNA次级文库制备条件的优化

Optimization of aptamer screening technology for RhD protein to generate subprime ss-DNA library

目的优化筛选RhD蛋白DNA-aptamer的ssDNA次级文库制备条件。方法对影响dsDNA扩增的主要参数(退火温度、循环数、模板用量、引物用量等)进行优化,在确定dsDNA扩增条件已优化的基础上,进而优化上、下游引物用量比例及扩增循环数,确定不对称PCR制备ssDNA的最佳条件。结果不对称PCR扩增的最佳条件为:上游引物终浓度为1 pmol/μL,上、下游引物浓度比例为20∶1,退火温度为59℃,循环数为30—35。结论制备条件优化后,经过不对称PCR扩增可获得浓度高、纯度好的ssDNA次级文库。

Objective To optimize the DNA-aptamer screening for RhD protein to generate good quality subprime ssD- NA library. Methods The principal parameters affecting the dsDNA amplification, such as the annealing temperature, cycle numbers, dosage of template and primers, were optimized. Through determining the optimal parameters for ssDNA amnpli- fication, the usage of sense and antisense primers were optimized, and the numbers of cycle were expanded. Results The optimal conditions for generating a subprime ssDNA library were as followed： sense primer amount at 10 pmol/μL, a primer ratio （sense primer： antisense primer） at 20：1, an annealing temperature at 59℃, and approximately 30 to 35 PCR cycles. Conclusion The generation of ssDNA by this method can produce a ssDNA library with high concentration and purity.