Differential cloning of genomic DNA: cloning of DNA with an altered primary structure by in-gel competitive reassociation.
A procedure was developed for cloning (anonymous) DNA sequences whose primary structures differ between two DNA samples. The procedure is based upon in-gel competitive DNA reassociation after electrophoresis of a mixture of restriction enzyme-digested target DNA (from which clones are to be isolated) and a large excess of unclonable reference DNA (competitor DNA). Inclusion of polyethylene glycol in the reassociation buffer greatly improved the in-gel reassociation efficiency, which was critical for the practical use of the procedure. Using this technique, we obtained several clones from rat brain (target) DNA, which may have been derived from tissue (brain)-specific altered DNA structures. The details of this procedure and its possible applications are discussed.
Yokota
H
H
Institute of Applied Microbiology, University of Tokyo, Japan.
Oishi
M
M
D000818:Animals; D015139:Blotting, Southern; D001921:Brain / Q000378:metabolism; D003001:Cloning, Molecular / Q000379:methods*; D004247:DNA / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D015246:Deoxyribonuclease EcoRI; D005260:Female; D005796:Genes; D007700:Kinetics; D008099:Liver / Q000378:metabolism; D011092:Polyethylene Glycols / Q000494:pharmacology; D051381:Rats; D011919:Rats, Inbred Strains
D011092:Polyethylene Glycols; D004247:DNA; D015246:Deoxyribonuclease EcoRI