一种构建全长cDNA文库的方法

建立了一种以LD-PCR为基础的cDNA文库的快速构建方法.以人胎盘组织为材料获得总RNA,利用引物F1、R2在逆转录酶M-MLV的作用下合成cDNA第1链,进而利用引物F3、R4在DNA聚合酶的作用下通过LD-PCR方法合成cDNA第2链;双链cDNA经SfiⅠ酶切,通过T4 DNA连接酶连接到经相同酶切的JG45质粒载体后构建成cDNA文库,并对文库的容量、重组率以及多样性进行了分析.结果表明,通过该方法构建的cDNA文库容量约为7.01×105 个/μgds-cDNA,重组率为96%;对随机提取的100个克隆质粒的插入序列进行分析,共获得80个不同的cDNA序列,且未见重复序列.该法构建的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,可用于大规模的基因分析.

Study on Full Length cDNA Library Construction

A simple method of full length cDNA library construction based on long-distance PCR was established.Total RNA from the human placenta tissues were isolated,and the first cDNA was synthesized through RT-PCR with the help of M-MLV reverse enzyme.Then double strand cDNA(ds-cDNA) with the restriction sites of SfiⅠ was synthesized through LD-PCR(Long-distance-PCR) and ligated into the JG45 vector with the restriction sites of Sfi I.Recombinant vectors were transformed into E.coli JM109 and then amplified.Then qualities of the cDNA library were analyzed.The average capacities of the cDNA library was about 7.01×105 clones per μg ds-cDNA with recombinant rate of 96%.Among the 80 detected randomly selected cDNA clones,no repetitive sequence was found.cDNA library constructed with this method was suitable for functional gene analysis.