Purification of circular DNA using benzoylated naphthoylated DEAE-cellulose.
Un-nicked circular DNA can be separated from protein, RNA, and other DNA in a simple three-step protocol consisting of exonuclease III digestion, extraction with benzoylated naphthoylated DEAE-cellulose (BND cellulose) in 1 M NaCl, and alcohol precipitation of the remaining supercoiled DNA. Exonuclease III treatment introduces single-stranded regions into contaminating linear and nicked circular DNA. This DNA, together with most RNA and protein, is adsorbed onto BND cellulose leaving form I DNA in solution. The protocol can be used to purify analytical as well as preparative amounts of supercoiled DNA. This procedure is a substitute for cesium chloride-ethidium bromide gradient ultracentrifugation and gives a comparable yield of pure form I DNA. Other classes of DNA can be isolated by changing the pretreatment step. Selective digestion of linear DNA with lambda exonuclease permits the isolation of both nicked circular and supercoiled DNA while brief heat-induced or alkali-induced denaturation leads to the recovery of rapidly reannealing DNA. In large-scale purifications, the basic protocol is usually preceded by one or more BND cellulose extractions in 1 M NaCl to remove contaminants absorbing UV or inhibiting exonuclease III.
D002482:Cellulose / Q000031:analogs & derivatives; D002848:Chromatography, DEAE-Cellulose / Q000379:methods*; D004270:DNA, Circular / Q000302:isolation & purification*; D004277:DNA, Single-Stranded / Q000378:metabolism; D005090:Exodeoxyribonucleases; D009691:Nucleic Acid Denaturation; D012965:Sodium Chloride; D014764:Viral Proteins
D004270:DNA, Circular; D004277:DNA, Single-Stranded; D014764:Viral Proteins; D012965:Sodium Chloride; D002482:Cellulose; C023583:benzoylated cellulose; D005090:Exodeoxyribonucleases; C020133:exodeoxyribonuclease III; C043586:exo protein, Bacteriophage lambda