利用ATP依赖的脱氧核糖核酸酶从全细胞DNA中分离环状DNA分子。
Isolation of circular DNA molecules from whole cellular DNA by use of ATP-dependent deoxyribonuclease.
作者信息
Mukai T, Matsubara K, Takagi Y
出版信息
Proc Natl Acad Sci U S A. 1973 Oct;70(10):2884-7. doi: 10.1073/pnas.70.10.2884.
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.
本文描述了一种从细菌细胞中分离出闭环和开环双链形式质粒DNA的技术，该技术利用了从藤黄微球菌中纯化得到的依赖ATP的脱氧核糖核酸酶。这种脱氧核糖核酸酶仅作用于线性DNA分子，可降解以线性形式提取的所有细菌染色体DNA。环状质粒DNA则保持完整，随后通过蔗糖梯度沉降进行分离。与以往分析质粒DNA的技术不同，该技术不仅可用于分离闭环DNA，还可用于分离开环DNA。通过该技术，几种质粒，如来自噬菌体（λdv1和λdv21）、一种产大肠杆菌素因子（Col E2）、一种性因子（F(8)' gal）以及大肠杆菌15中的“微型环”，其开环和闭环形式均能与染色体DNA良好分离。
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