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

作　　者：张印则[1] 周丹[1] 吴凡[1] 苏宇清[1] 梁延连[1] 李大成[1] 徐华[2] 周华友 

机构地区：[1]深圳市血液中心,广东深圳518035 [2]陕西省血液中心 [3]广州中医药大学第二附属医院输血科

出　　处：《中国输血杂志》2014年第12期1293-1296,共4页Chinese Journal of Blood Transfusion

基　　金：深圳市科技计划项目(医疗卫生类)重点项目(201201015)

摘　　要：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.

关 键 词：RhD蛋白 Aptamer筛选技术 次级文库 核酸适配体 单链DNA 

分 类 号：R457.11[医药卫生—治疗学] Q503[医药卫生—临床医学]