Rapid plasmid library screening using RecA-coated biotinylated probes.
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.
Rigas
B
B
Welcher
A A
AA
Ward
D C
DC
Weissman
S M
SM
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D001360:Avidin; D001710:Biotin; D002846:Chromatography, Affinity; D004247:DNA / Q000378:metabolism; D004274:DNA, Recombinant; D004277:DNA, Single-Stranded / Q000378:metabolism; D005818:Genetic Engineering / Q000379:methods*; D009693:Nucleic Acid Hybridization; D010957:Plasmids; D011938:Rec A Recombinases
D004274:DNA, Recombinant; D004277:DNA, Single-Stranded; D001360:Avidin; C022571:adenosine 5'-O-(3-thiotriphosphate); D001710:Biotin; D000255:Adenosine Triphosphate; D004247:DNA; D011938:Rec A Recombinases