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

Abstract

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. © 1990 Oxford University Press.