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首页> 外文期刊>Nucleic acids research >Inhibition of restriction endonuclease Nel I cleavage by phosphorothioate groups and its application to oligonucleotide-directed mutagenesis
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Inhibition of restriction endonuclease Nel I cleavage by phosphorothioate groups and its application to oligonucleotide-directed mutagenesis

机译：限制性内切核酸酶的抑制NEL I通过硫代磷酸酯基团切割及其在寡核苷酸的脱核诱变中的应用

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摘要

M13 RF IV DNA where phosphorothioate groups are incorporated at restriction endonuclease Nci I recognition sites in the (?) strand is efficiently nicked by the action of this enzyme. Incubation of such nicked DNA with exonuclease III produces gapped DNA. The gap can be filled by reaction with deoxynuclease side triphosphates and DNA polymerase I. When this sequence of reactions is performed with DNA containing a mismatch oligonucleotide primer in the (?)strand mutational frequencies of 70–90 % can be obtained upon transformation. The general nature of this methodology has been further shown to be applicable to other restriction enzymes such as Hind II, Pst I and Fsp I. The mutational frequency obtained using these enymes is between 40–80% mainly because of less efficient nicking and gapping. Studies on inhibition of Nci I cleavage show that in addition to a phosphorothioate group at the position of cleavage an additional group in the 5'-neighbouring position is necessary for complete inhibition.
机译：M13 RF IV DNA，其中硫代脲基团在限制性内切核酸酶中掺入NCI I识别位点（α）链中的识别位点通过该酶的作用有效地卷绕。用外切核酸酶III孵育这种切屑DNA III产生螺纹DNA。通过与脱氧核酸酶侧三磷酸和DNA聚合酶I反应填充间隙。当通过含有含有错配寡核苷酸引物的DNA进行这种反应序列，在转化时可以获得70-90％的脱模寡核苷酸引物的DNA。该方法的一般性质进一步示出可应用于其他限制酶，如后II，PST I和FSP I.使用这些酶获得的突变频率主要是由于效率较低的切口和间隙介于40-80％之间。关于NCI的抑制的研究表明，除了乳化磷酸硫代磷酸盐基团之外，还需要在5'相邻位置中的另外的基团进行完全抑制。

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