用PCR快速筛选递次截短的DNA克隆

RAPID SELECTION OF THE GRADUALLY SHORTENED DNA CLONES BY USING PCR AMPLIFICATION

目的为了建立一种快速、简便而准确地筛选递次截短的ＤＮＡ克隆的技术，加速对基因全长ｃＤＮＡ或ＤＮＡ大片段的测序和鉴定。方法在载体多克隆位点两侧设计引物，直接ＰＣＲ扩增插入片段，以此为基础选择系列缺失亚克隆，并与常规酶切法作比较。结果根据ＰＣＲ产物大小排序，准确地鉴定出系列缺失亚克隆，并可将ＰＣＲ产物直接用于ＰＣＲ循环测序，比酶切法分辨率高，操作步骤简化，筛选时间短。结果所建立的ＰＣＲ快速筛选法不仅是一种行之有效的鉴定方法，而且可以显著地节省时间和实验材料。该方法也可适用于其它各种质粒载体。

Objective To establish a simple technique with which the gradually shortened DNA clones can be selected rapidly and accurately so that the sequencing and identification of the full length cDNA or longer DNA fragments can be performed as quick as possible.Methods Primers were designed according to the sequence flanking the multiple cloning site of pGEM vector and were used to amplify the inserted fragments in a series of deletion sub clones originated from a clone.with longer DNA insert fragment.These sub clones which contain the gradually shortened fragments can be selected directly.This method was compared with the routine method in which the restriction endonuclease was used to digest DNA samples.Results A series of deletion sub clones were identified accurately with this new method and the cycle sequencing could be performed on these PCR products as well.This method was characteristic of more accurate,more simple and less time consuming,compared with the routine method.Conclusion The method presented is very effective for rapid selection of the gradually shortened inserted fragments constructed in pGEM vector.It can greatly save time and materials,and the strategy in the report could also fit for selecting the clones containing gradually shortened fragments in other plasmid vectors.