A novel method for producing partial restriction digestion of DNA fragments by PCR with 5-methyl-CTP
A novel method for producing partial restriction digestion of DNA fragments by PCR with 5-methyl-CTP
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DOI：
10.1093/nar/25.20.4169
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发表时间：
1997-10-15
影响因子：
14.9
通讯作者：
Saffer, JD
中科院分区：
文献类型：
--
作者：
Wong, KK;Markillie, LM;Saffer, JD
DNA片段的部分消化是进行亚克隆分析和生成限制图的标准程序。我们开发了一种新的方法，可以对任何可以通过PCR扩增的DNA片段进行部分酶切。该方法包括将5-甲基-dCTP掺入到聚合酶链式反应产物中，以保护大多数限制位点。因此，用5-甲基-dCTP敏感的酶完全消化修饰的PCR产物将产生一系列的限制片段，相当于对未甲基化的PCR产物进行的部分限制性内切酶消化反应。这种方法减少了用传统方法生产部分消化的DNA片段所需的时间和材料。此外，在反应中使用荧光素标记的引物，我们能够使用荧光成像仪或抗荧光素-AP抗体来检测酶消化产生的荧光素标记的末端片段，从而确定限制性内切酶图谱。
Partial digestion of DNA fragments is a standard procedure for subcloning analysis and for generating restriction maps. We have developed a novel method to generate a partial digestion for any DNA fragment that can be amplified by PCR. The method involves the incorporation of 5-methyl-dCTP into the PCR product to protect most of the restriction sites. As a result, complete digestion of the modified PCR products with a 5-methyl-dCTP-sensitive enzyme will produce an array of restriction fragments equivalent to a partial restriction enzyme digestion reaction done on unmethylated PCR products. This method reduces the time and material needed to produce partially-digested DNA fragments by traditional methods. Furthermore, using fluorescein-labeled primers in the reaction, we were able to detect the fluorescein-labeled end fragments resulting from the enzyme digestion using a fluorimager or anti-fluorescein-AP antibody and thus determine the restriction maps.