Rapid shotgeun cloning utillizing the two base recongition endonuclease CviJI

Michael C. Fitzgerald, Peter Skowron, James L. Van Etten, Lloyd M. Smith, David A. Maed

Research output: Contribution to journalArticle

32 Scopus citations

Abstract

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).

Original languageEnglish (US)
Pages (from-to)3753-3762
Number of pages10
JournalNucleic acids research
Volume20
Issue number14
DOIs
StatePublished - Jul 25 1992

ASJC Scopus subject areas

  • Genetics

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