一种简单快速的cDNA文库构建方法

A Rapid and Simple Method of cDNA Library Construction

目的:建立一种以LDPCR(longdistancePCR)为基础的cDNA文库的快速构建方法。方法:以鲨鱼再生肝组织为材料获得总RNA,用偶联有Oligo(dT)的磁珠纯化mRNA后,利用含BamHⅠ酶切位点的Oligo(dT)16引物在逆转录酶MMLV的作用下合成cDNA第一链,进而利用末端转移酶在合成的cDNA第一链的3′末端引入polydC尾;以含BamHⅠ酶切位点的Oligo(dT)16和含HindⅢ酶切位点的Oligo(dG)10为引物,利用LDPCR合成双链cDNA,双链cDNA经BamHⅠ和HindⅢ双酶切,通过T4DNA连接酶连接到经相同双酶切的pUC19载体后构建成cDNA文库,对文库的容量、重组率以及多样性进行分析。结果:通过本方法构建的cDNA文库容量约为5.84×105个/μgdscDNA,重组率为96.7%;对随机提取的30个克隆质粒的插入序列进行分析,共获得25个不同的cDNA序列,且未见重复序列。结论:本法构建的cDNA质粒文库具有快速、简单的特点,构建的文库质量符合要求,可用于大规模的功能基因分析。

AIM:To establish a method of cDNA library construction based on long-distance RT-PCR.METHODS:Total RNA from the regenerated hepatic tissues were isolated,and mRNA were separated and purified through the magnetic substance combined with Oligo(dT).The poly-deoxythymidines and poly-deoxycytidines were introduced into the 5′ terminal and 3′terminal of first cDNA strand with the help of M-MLV reverse transcriptase and terminal transferase,respectively.Then double strand cDNA with the restriction site of BamH Ⅰ and Hind Ⅲ were synthesized through LD-PCR(Long-distance-PCR) and ligated into the pUC19 vector,recombinant vectors were transformed into E.coli DH5α and amplified,qualities of the cDNA library were analyzed.RESULTS:The average capacities of the cDNA library is about 5.84×10^(5)clones per μg ds-cDNA with recombant rate of 96.7%.Among the 25 detected randomly selected cDNA clones,none repeated inserted cDNA fragment were founded.CONCLUTION:cDNA library constructed with this method is suitable for functional genomic analyzing.