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使用RecA包被的生物素化探针进行快速质粒文库筛选。

Rapid plasmid library screening using RecA-coated biotinylated probes.

作者信息

Rigas B, Welcher A A, Ward D C, Weissman S M

出版信息

Proc Natl Acad Sci U S A. 1986 Dec;83(24):9591-5. doi: 10.1073/pnas.83.24.9591.

Abstract

A method for the rapid physical isolation of recombinant plasmids of interest from a mixture of plasmids such as a plasmid cDNA library is presented. This method utilizes the ability of RecA protein to form stable complexes between linear single-stranded and circular double-stranded DNA molecules sharing sequence homology, and procedures allowing isolation of biotinylated nucleic acid. Biotinylated linear DNA probes coated with RecA have been used to screen reconstituted plasmid libraries consisting of two plasmid species, one homologous and the other heterologous to the probe. When the link between biotin and the nucleotide base could be cleaved by reducing agents, the complex was purified by streptavidin-agarose chromatography and the recovered plasmid was propagated in Escherichia coli. When the link was not cleavable the complex was bound to avidin in solution and purified by cupric iminodiacetic acid-agarose chromatography. The complex was then dissociated and the plasmids were propagated in E. coli. With either protocol, homologous plasmid recovery was between 10% and 20%, and enrichment was between 10(4)- and 10(5)-fold. Potential applications and extensions of this method, such as plasmid, cosmid, and phage library screening and facilitation of physical mapping of macroregions of mammalian genomes are presented and discussed.

摘要

本文介绍了一种从质粒混合物(如质粒cDNA文库)中快速物理分离目标重组质粒的方法。该方法利用RecA蛋白在具有序列同源性的线性单链和环状双链DNA分子之间形成稳定复合物的能力,以及允许分离生物素化核酸的程序。涂有RecA的生物素化线性DNA探针已用于筛选由两种质粒组成的重组质粒文库,其中一种与探针同源,另一种与探针异源。当生物素与核苷酸碱基之间的连接可被还原剂切割时,通过链霉亲和素-琼脂糖色谱法纯化复合物,并将回收的质粒在大肠杆菌中繁殖。当连接不可切割时,复合物在溶液中与抗生物素蛋白结合,并通过亚氨基二乙酸铜-琼脂糖色谱法纯化。然后使复合物解离,并将质粒在大肠杆菌中繁殖。无论采用哪种方案,同源质粒的回收率在10%至20%之间,富集倍数在10⁴至10⁵倍之间。本文还介绍并讨论了该方法的潜在应用和扩展,如质粒、黏粒和噬菌体文库筛选以及促进哺乳动物基因组大区域的物理图谱绘制。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/8018/387186/3b40669165fc/pnas00328-0328-a.jpg

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