RAPID SHOTGUN CLONING UTILIZING THE 2 BASE RECOGNITION ENDONUCLEASE CVIJI

被引:32
作者
FITZGERALD, MC
SKOWRON, P
VANETTEN, JL
SMITH, LM
MEAD, DA
机构
[1] UNIV WISCONSIN, DEPT CHEM, MADISON, WI 53706 USA
[2] CHIMERX, MADISON, WI 53704 USA
[3] UNIV NEBRASKA, DEPT PLANT PATHOL, LINCOLN, NE 68583 USA
关键词
D O I
10.1093/nar/20.14.3753
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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-mu-g instead of 2 - 5-mu-g), 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).
引用
收藏
页码:3753 / 3762
页数:10
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