Rapid shotgun cloning utilizing the two base recognition endonuclease 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 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).
Fitzgerald
M C
MC
Chemistry Department, University of Wisconsin, Madison 53706.
Skowron
P
P
Van Etten
J L
JL
Smith
L M
LM
Mead
D A
DA
D010582:Bacteriophage lambda / Q000235:genetics; D001483:Base Sequence; D002850:Chromatography, Gel; D003001:Cloning, Molecular / Q000379:methods*; D004262:DNA Restriction Enzymes / Q000378:metabolism*; D004279:DNA, Viral / Q000235:genetics / Q000378:metabolism*; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism*; D008969:Molecular Sequence Data; D015183:Restriction Mapping; D013010:Sonication
D004279:DNA, Viral; D004262:DNA Restriction Enzymes; C075913:endodeoxyribonuclease CviJI; D015252:Deoxyribonucleases, Type II Site-Specific