The use of phosphorothioate-modified DNA in restriction enzyme reactions to prepare nicked DNA.
The use of phosphorothioate-modified DNA in restriction enzyme reactions to prepare nicked DNA.
复制标题
在限制酶反应中使用硫代磷酸酯修饰的 DNA 来制备带切口的 DNA。
DOI：
10.1093/nar/13.24.8749
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发表时间：
1985
影响因子：
14.9
通讯作者：
Fritz Eckstein
中科院分区：
文献类型：
--
作者：
John W. Taylor;W. Schmidt;Richard Cosstick;Andrzej Okruszek;Fritz Eckstein
M13 DNA 的 RF IV 形式是在体外酶促合成的，使用病毒 (+) 链作为模板，在新合成的 (-) 链中特定类型的每个碱基的 5' 侧包含硫代磷酸酯修饰的 Rp 构型的核苷酸间键。然后测试了 29 种限制性内切酶与适当修饰的 DNA 类型的反应，这些修饰的 DNA 类型具有恰好位于 (-) 链中这些酶的切割位点的硫代磷酸酯键。所测试的 17 种限制性酶中的 11 种具有 5 个或更多碱基的识别序列，可用于将硫代磷酸酯 DNA 完全转化为带切口的形式，方法是简单地用过量的酶（Ava I、Ava II、Ban II、Hind II、Nci I、Pst I 或 Pvu I）完成反应，或者在带切口的 DNA 线性化之前的适当时间停止反应（Bam HI、Bgl I、Eco RI 或 Hind）三）。只有对 (-) 链中的确切切割位点进行修饰才能阻止第一类酶的线性化。结果表明，硫代磷酸酯 M13 DNA 的限制性内切酶定向切口仅发生在 (+) 链中。
The RF IV form of M13 DNA was synthesized enzymatically in vitro, using the viral (+)strand as template, to contain phosphorothioate-modified internucleotidic linkages of the Rp configuration on the 5' side of every base of a particular type in the newly-synthesized (-)strand. Twenty nine restriction enzymes were then tested for their reactions with the appropriate modified DNA types having a phosphorothioate linkage placed exactly at the cleavage site(s) of these enzymes in the (-)strand. Eleven of the seventeen restriction enzymes tested that had recognition sequences of five bases or more could be used to convert the phosphorothioate DNA entirely into the nicked form, either by simply allowing the reaction to go to completion with excess enzyme (Ava I, Ava II, Ban II, Hind II, Nci I, Pst I or Pvu I) or by stopping the reaction at the appropriate time before the nicked DNA is linearized (Bam HI, Bgl I, Eco RI or Hind III). Only modification of the exact cleavage site in the (-)strand could block linearization by the first class of enzymes. The results presented imply that the restriction enzyme-directed nicking of phosphorothioate M13 DNA occurs exclusively in the (+)strand.