用PCR法直接快速筛查重组阳性克隆

A Rapid Screen for Recombinant Clones by Polymerase Chain Reaction

本研究目的是应用ＰＣＲ法快速筛查插入有苯丙氨酸脱氨酶（ＰＡＬ）ｃＤＮＡ重组阳性克隆。用于ＰＣＲ 扩增的引物是位于载体ｐＥＴ２３ｂ 启动子处的Ｔ７ 启动子引物和位于目的基因ＰＡＬｃ ＤＮＡ３′端终止密码ＴＡＡ 处的引物。以灭菌吸头挑一单菌落加入ＰＣＲ 体系扩增。结果：在筛查的３ 个克隆中，有２ 个阳性克隆，并且插入方向正确，经ＤＮＡ序列测定得到进一步证实。结果表明，以ＰＣＲ方法筛查重组阳性克隆，可以简便快速鉴定插入片段的大小和方向，不需提取质粒。

Objective: To develope a PCR technique for rapid screening of phenylalanine ammonia lyase(PAL) cDNA recombinant plasmid. Methods:PCR primers were designed so that one primer is complementary to the T 7promoter of pET23b vector, and another primer complementary to the 3′ end of PAL cDNA .The recombinant colonies were transferred by using sterile pipet tip into the PCR reaction mixture directly. After PCR amplification, the PCR products were subject to electrophoresis on 0.8% agrose gel.Results: 2 positive clones with a correct direction by using the screening method were detected. The positive clones were further confirmed by DNA sequencing. Conclusions: Recombinant vectors in bacterial cells can be analyzed directly with the use of PCR. This convenient and timesaving procedure eliminates the need for preparation and purification of the plasmid.