Open AccessRapid shotgeun cloning utillizing the two base recongition endonucleaseCviJI
Author(s) -
Michael C. Fitzgerald,
Piotr M. Skowron,
James L. Van Etten,
Lloyd M. Smith,
David A. Maed
Publication year - 1992
Publication title -
nucleic acids research
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 9.008
H-Index - 537
eISSN - 1362-4954
pISSN - 0305-1048
DOI - 10.1093/nar/20.14.3753
Subject(s) - biology , puc19 , restriction enzyme , microbiology and biotechnology , dna , agarose , sticky and blunt ends , endonuclease , molecular cloning , cloning (programming) , dna fragmentation , genetics , plasmid , peptide sequence , gene , computer science , programming language , apoptosis , programmed cell death
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 ligated, 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 sonication and agarose gel fractionation include: smaller amounts of DNA are required (0.2-0.5 micrograms instead of 2-5 micrograms), fewer steps are involved (no preligation, end repair, chemical extraction, or agarose gel electrophoresis and elution 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).