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Nucleic Acids Research, 1992, Vol. 20, No. 14 3753-3762
© 1992

MOLECULAR BIOLOGY

Rapid shotgeun cloning utillizing the two base recongition endonuclease CviJI

Michael C. Fitzgerald, Peter Skowron1, James L. Van Etten2, Lloyd M. Smith and David A. Maed*

Chemistry Department, University of Wisconsin Madison, WI 53706 1CHIMERx, Madison, WI 53704 2Department of Plant Pathology, University of NEbraska Licoln, NE 548583, USA

*To whom correspondence should be addressed

Received February 27, 1992. Revised June 23, 1992. Accepted June 23, 1992.

A new approach has been developed for the rapid fragmentation and fractionation of DNA Into a size suitable for shotgun cloning and sequencing. The restriction endonuclease CviJI normally cleaves the recognition sequence PuGCPy between the G and C to leave blunt ends. Atypical reaction conditions which alter the specificity of this enzyme (CviJI**) yield a quasi-random distribution of DNA fragments from the small molecule pUC19 (2686 base pairs). To quantitatively evaluate the randomness of this fragmentation strategy, a CviJI** digest of pUC19 was size fractionated by a rapid gel filtration method and directly llgated, without end repair, to a lacZ minus M13 cloning vector. Sequence analysis of 76 clones showed that CviJI** restricts PyGCPy and PuGCPu, in addition to PuGCPy sites, and that new sequence data is accumulated at a rate consistent with random fragmentation. Advantages of this approach compared to sonicatlon and agarose gel fractionation Include: smaller amounts of DNA are required (0.2–0.5 µg instead of 2 – 5 µg), fewer steps are involved (no preligation, end repair, chemical extraction, or agarose gel electrophoresls and elutlon are needed), and higher cloning efficiencies are obtained (CviJI** digested and column fractionated DNA transforms 3 – 16 times more efficiently than sonicated, end-repaired, and agarose fractionated DNA).


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