Rapid shotgun cloning utilizing the two base recognition endonucelase CviJI

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 Cv/JI 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 (Cv/JI**) 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 Cv/JI * * 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 Cv/JI* * 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 pg instead of 2- 5 /*g), fewer steps are involved (no pre-ligation, end repair, chemical extraction, or agarose gel electrophoresls and elutlon are needed), and higher cloning efficiencies are obtained (Cv/JI * * digested and column fractionated DNA transforms 3- 1 6 times more efficiently than sonicated, end-repaired, and agarose fractionated DNA)