Gaubatz J W, Flores S C
Department of Biochemistry, University of South Alabama, College of Medicine, Mobile 36688.
Anal Biochem. 1990 Feb 1;184(2):305-10. doi: 10.1016/0003-2697(90)90685-3.
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.
本文描述了一种分离真核细胞染色体外环状（ecc）DNA的方法。使用核酸外切酶III对DNA的线性和开环形式进行制备性消化；然后通过浮力密度梯度沉降对所得的抗核酸外切酶分子进行表征，发现其基本上是共价闭合环状DNA。将核酸外切酶方法的效率与超速离心技术进行了比较，发现其产量高于通过两个或更多平衡密度梯度获得的产量。通过从小鼠肝脏、大脑、心脏和肾脏组织中纯化eccDNA来确定核酸外切酶III技术的实用性。结果表明，eccDNA含量存在组织相关差异。
