Double stranded scission of DNA directed through sequence-specific R-loop formation.
Double stranded scission of DNA directed through sequence-specific R-loop formation.
复制标题
通过序列特异性 R 环形成引导 DNA 双链断裂。
DOI：
10.1093/nar/23.17.3524
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发表时间：
1995
影响因子：
14.9
通讯作者：
Sigman,DS
中科院分区：
文献类型：
--
作者：
Landgraf,R;Chen,CH;Sigman,DS
用短（100 nt）RNA形成R环提供了一种高度灵活和严格的方法，以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RNase处理去除RNA后，剩余的单链DNA泡为减毒的微球菌核酸酶提供了非常有利的底物。我们研究了该方法用于双链DNA的序列特异性切割，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环之间的插入片段，证明了对较大尺寸DNA的适用性。
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 ∼120 kb.