通过序列特异性R环形成介导的DNA双链断裂。

Double stranded scission of DNA directed through sequence-specific R-loop formation.

作者信息

Landgraf R, Chen C H, Sigman D S

机构信息

Department of Biological Chemistry, School of Medicine, University of California-Los Angeles 90024-1570, USA.

出版信息

Nucleic Acids Res. 1995 Sep 11;23(17):3524-30. doi: 10.1093/nar/23.17.3524.

DOI:10.1093/nar/23.17.3524

PMID:7567465

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC307233/

Abstract

R-loop formation with short (100 nt) RNAs provides a highly flexible and stringent method to achieve sequence-specific separation of target DNA at any given sequence. After stabilization of R-loops with glyoxal and removal of the RNA through RNase treatment the remaining single-stranded DNA bubble provides a highly favorable substrate for attenuated micrococcal nuclease. We investigated this method for sequence-specific scission of double-stranded DNA and achieved quantitative scission of 3-5 kb plasmids. The applicability to larger size DNA is demonstrated through specific excision of the intervening segment between two R-loops from a P1 plasmid of approximately 120 kb.

摘要

与短（100个核苷酸）RNA形成R环提供了一种高度灵活且严格的方法，可在任何给定序列处实现靶DNA的序列特异性分离。在用乙二醛稳定R环并通过核糖核酸酶处理去除RNA后，剩余的单链DNA气泡为弱化的微球菌核酸酶提供了非常有利的底物。我们研究了这种用于双链DNA序列特异性切割的方法，并实现了3-5 kb质粒的定量切割。通过从约120 kb的P1质粒中特异性切除两个R环之间的间隔片段，证明了该方法对更大尺寸DNA的适用性。