Efficient construction of long DNA duplexes with internal non-Watson-Crick motifs and modifications.
Efficient construction of long DNA duplexes with internal non-Watson-Crick motifs and modifications.
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DOI：
10.1093/nar/29.2.e6
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发表时间：
2001-01
影响因子：
14.9
通讯作者：
X. Zheng;P. Bevilacqua
中科院分区：
文献类型：
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作者：
X. Zheng;P. Bevilacqua
我们开发了一种半合成方法来制备包含内部化学修饰和/或非Watson-Crick结构基序的长片段DNA(>；100bp)，该方法依赖于无夹板、无细胞的DNA连接和通过非PCR热循环回收副产物。中间含有多接头的双链DNA PCR片段被两个限制性内切酶消化，并在中间引入一个含有修饰或非Watson-Crick基序的小插入物(约20bp)。不正确的产品通过使用适当的限制性内切酶进行消化，回收为起始原料，而正确的产品则抵抗消化，因为它不包含这些限制位点。这种半合成方法比DNA夹板介导的连接方法有几个优点，包括步骤更少，产量显著更高(总产量约60%)和易用性。这种方法有许多潜在的应用，包括在DNA中引入荧光团和交联剂等修饰，大规模控制DNA的形状，以及研究非序列特异性的核酸-蛋白质相互作用。
We have developed a semi-synthetic approach for preparing long stretches of DNA (>100 bp) containing internal chemical modifications and/or non-Watson-Crick structural motifs which relies on splint-free, cell-free DNA ligations and recycling of side-products by non-PCR thermal cycling. A double-stranded DNA PCR fragment containing a polylinker in its middle is digested with two restriction enzymes and a small insert ( approximately 20 bp) containing the modification or non-Watson-Crick motif of interest is introduced into the middle. Incorrect products are recycled to starting materials by digestion with appropriate restriction enzymes, while the correct product is resistant to digestion since it does not contain these restriction sites. This semi-synthetic approach offers several advantages over DNA splint-mediated ligations, including fewer steps, substantially higher yields ( approximately 60% overall yield) and ease of use. This method has numerous potential applications, including the introduction of modifications such as fluorophores and cross-linking agents into DNA, controlling the shape of DNA on a large scale and the study of non-sequence-specific nucleic acid-protein interactions.