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Nucleic Acids Research, 1990, Vol. 18, No. 13 3863-3869
© 1990

MOLECULAR BIOLOGY

An F factor based cloning system for large DNA fragments

Fumie Hosoda1,2, Susumu Nishimura2, Hisao Uchida3 and Misao Ohki1,2

1Department of Immunology and Virology, Saitama Cancer Center Research Institute Ina-machi, Kitadachi-gun, Saitama 362 2Biology Division, National Cancer Center Research Institute Tsukiji 5–1–1, chuo-ku, Tokyo 104 3Department of Bioscience, Teikyo University Nagaoka-cho, Utsunomiya 320, Japan

Received March 28, 1990. Revised May 30, 1990. Accepted May 30, 1990.

An effective technique using an Escherichia coli plasmid system was developed to clone fragments of exogenous DNA of as large as 100 kilobase pairs. The characteristic features of this technique are the use of a low copy number (one to two) mini-F based plasmid vector and the introduction of artificial lambda cosR ends into the termini of DNA sources and then of the cosL ends into those of linearized vector molecules. This terminal modification greatly facilitated the formation of active large recombinant molecules, which was rarely achieved when the modification was omitted. The efficiency with which large recombinant clones can be generated is high enough to allow construction of a comprehensive library of higher organisms. All analyses of the plasmids recovered have revealed that the inserts were faithful replicas of the human DNAs used as sources.


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