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Nucleic Acids Research, 1990, Vol. 18, No. 11 3287-3291
© 1990

GENOME STRUCTURE AND MAPPING

Methylase-limited partial NotI cleavage for physical maping of genomic DNA

John Hanish and Michael McClelland1

Department of Biochemistry and Molecular Biology, TheUniversity of Chicago 920 East 58th Stret, Chicago, IL 60637 1California Institute of Biological Research 11099 North Torey Pines Road. La Jolla., CA 92037, USA

Received February 16, 1990. Accepted May 7, 1990.

Partial cleavage of DNA with the restriction endonuclease Not (5 prime;...GC/GGCCGC...3') is animportant technique for genomic mapping. However, partial genomic cleavage with this enzyme is impaired by the agarose matrix In which the DNA must be suspended. To solve this problem we have purified the blocking methylase M. BspRI (5'...GGmCC...3') for competition digests with Noti. The resulting methylaselimited partial DNA cleavage Is shown to be superior to standard techniques on bacterial genomic DNA. Abbreviations: bp, base-pain kb, one thousand basepairs; Mb, one million base-pairs; Tris, Tris(hydroxymethyl)aminomethane; EDTA, (ethylenedinitrilo)tetraacetlc; sol;3-ME, /3-mercaptoethanol; PMSF, phenyl methyl-sulfonyl fluoride; PEG, polyethyleneglycol (MW= 8000); H, tritium; SAM, S- denosylmethionlne; GB, potassium glutamate buffer; DTT, drthiothreltol; IPTG, isopropyl-0-D-thiogalactopyranoside; BSA, bovine serum albumin.


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