Site-specific cleavage of DNA at 8- and 10-base-pair sequences.
Site-specific cleavage of DNA at 8- and 10-base-pair sequences.
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在 8 和 10 碱基对序列处对 DNA 进行位点特异性切割。
DOI：
10.1073/pnas.81.4.983
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发表时间：
1984
影响因子：
11.1
通讯作者：
M. Bittner
中科院分区：
文献类型：
--
作者：
Michael McClelland;L. Kessler;M. Bittner
描述了一种用于在8和10个碱基对长的特定位点切割DNA的方法。DNA首先用特异性甲基化酶处理，限制修饰酶M。Taq I，其将4-碱基序列T-C-G-A转化为T-C-G-mA，或类似的酶M。Cla I，其将6个碱基序列A-T-C-G-A-T转化为A-T-C-G-mA-T。然后用Dpn I切割DNA，Dpn I是一种识别序列G-mA-T-C的限制性内切酶。Dpn I的独特之处在于它只切割在其识别序列的两条链中腺嘌呤甲基化的DNA。在序列G-A-T-C的两条链中腺嘌呤没有甲基化的DNA中，Dpn I的切割仅发生在以下序列：Taq Ⅰ甲基化，5 ′ T-C-G-mA - T-C-G-mA 3 ′ 3 ′ mA-G-C-T-mA-G-C - T 5 ′; ClaI甲基化，5'A-T-C-G-mA - T-C-G-mA-T3' 3' T-mA-G-C - T-mA-G-C - T-A 5'。在这些甲基化酶/Dpn I产生的网站的具体切割和克隆实验。此外，我们描述了如何将上述技术扩展到产生多达12个碱基对的Dpn I切割位点。在含有等量随机分布的每个碱基的DNA中，8个和10个碱基对的识别序列平均分别约每65，000个和1，000，000个碱基对出现一次。潜在的应用，包括克隆载体的发展和染色体步移的快速方法，进行了讨论。
A method is described for cutting DNA at specific sites that are 8 and 10 base pairs long. The DNA is first treated with a specific methylase, either the restriction-modification enzyme M. Taq I, which converts the 4-base sequence T-C-G-A to T-C-G-mA, or the similar enzyme M. Cla I, which converts the 6-base sequence A-T-C-G-A-T to A-T-C-G-mA-T. The DNA is then cleaved with Dpn I, a restriction endonuclease that recognizes the sequence G-mA-T-C. Dpn I is unique in that it cuts only DNA that is methylated at adenine in both strands of its recognition sequence. In DNAs that are not otherwise methylated at adenine in both strands of the sequence G-A-T-C, cleavage by Dpn I occurs only at the following sequences: in the case of M. Taq I methylation, 5' T-C-G-mA - T-C-G-mA 3' 3' mA-G-C - T-mA-G-C - T 5'; in the case of M. Cla I methylation, 5' A - T-C-G-mA - T-C-G-mA-T 3' 3' T-mA-G-C - T-mA-G-C - T-A 5'. Specific cutting and cloning at these methylase/Dpn I-generated sites is shown experimentally. Further, we describe how the above technique can be extended to generate Dpn I cleavage sites of up to 12 base pairs. In DNA that contains equal amounts of each base distributed at random, 8- and 10-base-pair recognition sequences occur, on the average, approximately once every 65,000 and 1,000,000 base pairs, respectively. Potential applications, including the development of cloning vectors and a rapid method for chromosome walking, are discussed.