摘要
目的为了在ｃＤＮＡ文库杂交筛选目的基因过程中，当复筛的杂交信号对应于１个多克隆融合噬菌斑时，能够快速、准确地分离出特异克隆的插入片段。方法依据定量ＰＣＲ扩增的原理，利用初始模板量的不同，通过两次ＰＣＲ反应，鉴定和分离出所需ｃＤＮＡ片段。结果利用这一方法，在β－１，４－半乳糖苷转移酶ｃＤＮＡ的“步移”复筛中，从一个双克隆融合斑中，得到了特异性克隆的插入片段，经测序证实为β－１，４－半乳糖苷转移酶的５′ｃＤＮＡ序列，长１．９ｋｂ，从而完成了全长ｃＤＮＡ的克隆，节省了进行再次复筛的时间和材料。结论该方法简便、快捷，可以显著地节省时间和实验材料，在新基因克隆中有一定的实用价值。
Objective To isolate quickly and exactly the
specific inserted fragment from fused phage clones which were obtained from cDNA library by
hybridization. Methods According to the amplification principle of quantitative polymerase chain
reaction (PCR) and based on the difference of original template quantity, target cDNA fragment
was isolated and identified by two PCRs. Results A positive clone with specific cDNA fragment
of HumGTH1 gene was obtained from a twophage fused clone by using this method, and the
inserted fragment was verified to be the 5cDNA sequence of HumGTH1,1.9kb in length. So
another hybridization screening is not necessary.Conclusion The method presented is effective
and rapid in gene cloning and can greatly save time and materials.
出处
《中华医学遗传学杂志》
EI
CAS
CSCD
北大核心
1999年第3期185-187,共3页
Chinese Journal of Medical Genetics
基金
国家863高技术项目
关键词
杂交筛选
融合噬菌斑
基因克隆
定量PCR
uantitative
polymerase chain reactionHybridization screeningFused phage clonesGene cloning
