[The selective isolation of cosmid clones by homologous recombination in Escherichia coli--a cosmid clone containing t complex linkage DNA sequence of mouse was isolated].
A procedure for the selective isolation of specific cosmid clones by homologous recombination between cosmid clones of genomic library and a probe DNA sequence cloned in a plasmid in vivo has been developed. The cosmid library was constructed in a rec- host cell strain and packaged into phage particles in vivo. The rec+ host cells containing a DNA sequence used as selection probe cloned in the pUC plasmid were infected by packaged cosmid phage particles. There is no homology between cosmid and the plasmid vectors. After a period of 1-3 hr. for the recombination to take place, the probe plasmids were integrated into cosmid, in which the DNA sequence are homologous with the probe, by homologous recombination. The cosmids are then packaged in vivo and transferred into a rec- cell strain. The specific cosmid clones were selected by double antibiotic resistance carried by both vectors. The probe plasmid can be excised by lambda excision enzyme by using superinfection with red+ phage. After packaging in vivo, these cosmid revertants can be identified on Xgal plate. A cosmid clone containing the t complex linkage DNA sequence of mouse was selected by using the procedure above with a probe derived from microdissected metaphase chromosome.
Chai
J H
JH
Institute of Genetics, Fudan University, Shanghai.
D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D003360:Cosmids; D004269:DNA, Bacterial / Q000302:isolation & purification*; D004274:DNA, Recombinant / Q000302:isolation & purification*; D004926:Escherichia coli / Q000235:genetics*; D005796:Genes; D008040:Genetic Linkage; D051379:Mice; D008815:Mice, Inbred Strains; D011995:Recombination, Genetic
D004269:DNA, Bacterial; D004274:DNA, Recombinant