Putney S D, Benkovic S J, Schimmel P R
Proc Natl Acad Sci U S A. 1981 Dec;78(12):7350-4. doi: 10.1073/pnas.78.12.7350.
2'-Deoxyadenosine 5'-O-(1-thiotriphosphate) (dATP[alpha S]) was introduced into the 3' ends of DNA restriction fragments with Escherichia coli DNA polymerase I to give phosphorothioate internucleotide linkages. Such "capped" 3' ends were found to be resistant to exonuclease III digestion. Moreover, the resistance to digestion is great enough that, under conditions used by us, just one strand of a double helix is digested by exonuclease III when a cap is placed at only one end; when digestion is carried to completion, this results in production of intact single strands. When digestion with exonuclease III is limited and is followed by S1 nuclease treatment, double-stranded DNA fragments asymmetrically shortened from just one side are produced. In this was thousands of nucleotides can be selectively removed from one end of a restriction fragment. In vitro introduction of phosphorothioate linkages into one end of a linearized replicative plasmid, followed by exonuclease III and S1 nuclease treatments, gives rise to truncated forms that, upon circularization by blunt-end ligation, transform E. coli and replicate in vivo.
用大肠杆菌DNA聚合酶I将2'-脱氧腺苷5'-O-(1-硫代三磷酸)(dATP[αS])引入DNA限制性片段的3'末端，以形成硫代磷酸酯核苷酸间连接。发现这种“加帽”的3'末端对外切核酸酶III的消化具有抗性。此外，这种抗性足够强，以至于在我们使用的条件下，当仅在一端加上帽时，双链螺旋的仅一条链会被外切核酸酶III消化；当消化完成时，这会导致完整单链的产生。当外切核酸酶III的消化受到限制并随后进行S1核酸酶处理时，会产生仅从一侧不对称缩短的双链DNA片段。通过这种方法，可以从限制性片段的一端选择性地去除数千个核苷酸。在线性化的复制质粒的一端体外引入硫代磷酸酯连接，随后进行外切核酸酶III和S1核酸酶处理，会产生截短形式，通过平端连接环化后，可转化大肠杆菌并在体内复制。
