Methylase-limited partial NotI cleavage for physical mapping of genomic DNA.

AUTOR(ES)

Hanish, J

RESUMO

Partial cleavage of DNA with the restriction endonuclease NotI (5'...GC/GGCCGC...3') is an important 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 methylase-limited partial DNA cleavage is shown to be superior to standard techniques on bacterial genomic DNA. Abbreviations: bp, base-pair; kb, one thousand base-pairs; Mb, one million base-pairs; Tris, Tris(hydroxy-methyl)aminomethane; EDTA, (ethylenedinitrilo)tetraacetic; beta-ME, beta-mercaptoethanol; PMSF, phenyl methyl-sulfonyl fluoride; PEG, polyethyleneglycol (MW = 8000); 3H, tritium; SAM, S-adenosylmethionine; KGB, potassium glutamate buffer; DTT, dithiothreitol; IPTG, isopropyl-beta-D-thiogalactopyranoside; BSA, bovine serum albumin.

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