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用PCR快速筛选递次截短的DNA克隆 |
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胡培蓉 余龙 张民 范玉新 王小柯 赵寿元
【摘要】 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。
【关键词】 多聚酶链反应 快速筛选 DNA递次截短分析
RAPID SELECTION OF THE GRADUALLY SHORTENED DNA CLONES BY USING
PCRAMPLIFICATION Hu Peirong,Yu Long*,Zhang Min,Fan Yuxin, Wang Xiaoke, Zhao
Shouyuan.*Genetic Institute of Fudan University,Shanghai,200433
【Abstract】 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 GEM 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[1] [2] 下一页
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