Isolation of circular DNA molecules from whole cellular DNA by use of ATP-dependent deoxyribonuclease.
A technique is described for isolation of plasmid DNA in closed and open circular double-stranded forms from bacterial cells, by use of ATP-dependent deoxyribonuclease purified from Micrococcus luteus. This DNase, acting only upon linear DNA molecules, degrades all bacterial chromosomal DNA extracted in the linear form. Circular plasmid DNAs are left intact, and are then separated by sedimentation through a sucrose gradient. Unlike previous techniques for analysis of plasmid DNA, this technique can be used to isolate not only closed circular DNA but also open circular DNA. Several plasmids, such as those from phage (lambdadv1 and lambdadv21), a colicinogenic factor (Col E2), a sex factor (F(8)' gal), and "minicircles" in Escherichia coli 15, in both the open and closed circular forms, were well separated from chromosomal DNA by this technique.
D000255:Adenosine Triphosphate; D002499:Centrifugation, Density Gradient; D003090:Coliphages; D004269:DNA, Bacterial / Q000032:analysis* / Q000096:biosynthesis; D004270:DNA, Circular / Q000302:isolation & purification*; D003851:Deoxyribonucleases; D004926:Escherichia coli / Q000032:analysis* / Q000166:cytology / Q000201:enzymology; D005092:Exonucleases; D006868:Hydrolysis; D002479:Inclusion Bodies / Q000032:analysis*; D007181:Inclusion Bodies, Viral; D008837:Micrococcus / Q000201:enzymology; D013936:Thymidine / Q000378:metabolism; D014316:Tritium
D004269:DNA, Bacterial; D004270:DNA, Circular; D014316:Tritium; D000255:Adenosine Triphosphate; D003851:Deoxyribonucleases; D005092:Exonucleases; D013936:Thymidine