Purification of eucaryotic extrachromosomal circular DNAs using exonuclease III.
A method for the isolation of eucaryotic extrachromosomal circular (ecc) DNA is described. Exonuclease III was used to preparatively digest linear and open circular forms of DNA; the resultant exonuclease-resistant molecules were then characterized by buoyant density gradient sedimentation and were found to be essentially covalently closed circular DNA. The efficiency of the exonuclease method was compared to ultracentrifugation techniques and was found to give yields greater than those obtained by two or more equilibrium density gradients. The utility of the exonuclease III technique was determined by purifying eccDNAs from mouse liver, brain, heart, and kidney tissues. The results showed that there are tissue-related differences in eccDNA content.
Gaubatz
J W
JW
Department of Biochemistry, University of South Alabama, College of Medicine, Mobile 36688.
Flores
S C
SC
D000818:Animals; D002499:Centrifugation, Density Gradient; D004269:DNA, Bacterial / Q000302:isolation & purification; D004270:DNA, Circular / Q000302:isolation & purification*; D005090:Exodeoxyribonucleases; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D009693:Nucleic Acid Hybridization; D009928:Organ Specificity; D010957:Plasmids
D004269:DNA, Bacterial; D004270:DNA, Circular; D005090:Exodeoxyribonucleases; C020133:exodeoxyribonuclease III