应用限制性显示PCR技术构建细菌poly(A)化mRNA的cDNA文库

针对细菌mRNA poly(A)化位点的高度多态性,利用oligo(dT)与poly(A)特异结合的特性,以oligo(dT)一纤维素纯化mRNA,并以oligo(dT)18为引物逆转录合成cDNA,用限制性内切酶消化cDNA,所得的限制性内切酶片段与通用接头相连,通过10个选择性引物组合进行选择性PCR,使各片段得以扩增并分布于10个亚组中,并进行克隆,成功地克隆了100多个基因片段,已对其中40个进行了测序分析,探讨了限制性显示PCR技术在细菌poly(A)化mRNA cDNA库构建中的应用价值。

Apply restriction display-PCR to constructing cDNA library ofmRNA with poly(A) tracts in bacteria

Due to great diversity of polyadenylation and short poly(A)tracts of mRNA in bacteria,it seldom suc-ceeds in constructing cDNA library in bacteria.There is a new technique Restriction Display-PCR(RD-PCR)to con-struct cDNA library of bacteria.The technique involves four steps:(1)The mRNAs with poly(A)tracts were enriched by oligo(dT)-cellulose chromatography?Double strands cDNA were synthesized subsequently;(2)Restriction of the cDNA and ligation of oligonucleotide adapter;(3)Selective amplification of sets of restriction fragments;(4)The PCR products were then cloned into T-vectors.PCR amplification of restriction fragments is achieved by using the adapter and restriction site sequence as target sites for primer annealing.The selective amplification is achieved by the use of primers that extend into the restriction fragments,amplifying only those fragments in which the primer extensions match the nucleotides flanking the restriction sites.More than100gene fragments have been cloned,and40gene fragments have been sequenced.Our research demonstrates that RD-PCR is an effective technique of constructing cDNA library in bacteria.It can largely decrease the redundancy of cDNA library.