Application of methylase-limited partial NotI cleavage for a long-range restriction map of the human ABL locus.
The use of partial restriction digests for mapping complex genomes by pulsed-field gel electrophoresis has been limited by the difficulty of consistently obtaining these digests in agarose, which is a necessary matrix for high-molecular-weight DNA. Enzyme cleavage in agarose is faster then diffusion for most of the enzymes which cleave infrequently. We have developed a method for the production of partial digests in agarose for the endonuclease NotI (5' . . . GC/GGCCGC . . . 3') which circumvents the diffusion problem by using the blocking methylase M. BspRI (5' . . . GGmCC . . . 3'), which competes for the same sites. Using various ratios of the methylase and endonuclease results in partial digests in any size range desired. We report the successful application of this technique to the production of NotI partial digests of human genomic DNA for the mapping of the ABL locus of human chromosome 9.
Hanish
J
J
Department of Biochemistry and Molecular Biology, University of Chicago, Illinois 60637.
Rebelsky
M
M
McClelland
M
M
Westbrook
C
C
D001483:Base Sequence; D001667:Binding, Competitive; D002899:Chromosomes, Human, Pair 9; D004273:DNA, Neoplasm / Q000235:genetics / Q000378:metabolism; D015257:DNA-Cytosine Methylases / Q000494:pharmacology*; D015252:Deoxyribonucleases, Type II Site-Specific / Q000037:antagonists & inhibitors; D004587:Electrophoresis, Agar Gel; D016313:Genes, abl; D005819:Genetic Markers; D006801:Humans; D007938:Leukemia / Q000473:pathology; D008745:Methylation; D010677:Philadelphia Chromosome; D015183:Restriction Mapping; D013379:Substrate Specificity; D014407:Tumor Cells, Cultured
D004273:DNA, Neoplasm; D005819:Genetic Markers; D015257:DNA-Cytosine Methylases; C027823:Cytosine 5-methyltransferase; D015252:Deoxyribonucleases, Type II Site-Specific; C038565:GCGGCCGC-specific type II deoxyribonucleases