Purification of small polydisperse circular DNA of eukaryotic cells by use of ATP-dependent deoxyribonuclease.
Small polydisperse circular (spc) DNAs of mouse thymocytes were purified by a procedure involving nitrocellulose column chromatography and the treatment of ATP-dependent DNase, which acts only upon linear DNA molecules. Nitrocellulose column chromatography prior to the enzyme treatment was essential because digestion of linear DNA duplexes by the enzyme was inhibited by the presence of concomitant single-stranded DNAs. Mitochondrial DNAs were eliminated by linearization with XhoI and digestion with ATP-dependent DNase. The size distribution of the purified spc DNA molecules ranged from 0.2 micron to more than 28 micron, with a mean length of 5.4 micron. Circular molecules of more than 0.4 micron long (or 1.2 kb) were free from the contamination of linear DNA fragments and pure enough to be cloned into plasmids.
D000818:Animals; D002845:Chromatography; D003101:Collodion; D004270:DNA, Circular / Q000302:isolation & purification*; D004272:DNA, Mitochondrial / Q000302:isolation & purification; D043211:Exodeoxyribonuclease V; D005090:Exodeoxyribonucleases; D051379:Mice; D013950:Thymus Gland / Q000032:analysis
D004270:DNA, Circular; D004272:DNA, Mitochondrial; D003101:Collodion; D005090:Exodeoxyribonucleases; D043211:Exodeoxyribonuclease V