Reliable fusion PCR mediated by GC-rich overlap sequences
Reliable fusion PCR mediated by GC-rich overlap sequences
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DOI：
10.1016/j.gene.2008.12.014
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发表时间：
2009-04-01
期刊：
影响因子：
3.5
通讯作者：
Akada, Rinji
中科院分区：
文献类型：
--
作者：
Cha-aim, Kamonchai;Fukunaga, Tomoaki;Akada, Rinji
重组DNA技术在很大程度上依赖于通过限制性内切酶消化和DNA连接构建大肠杆菌质粒。重叠延伸PCR是另一种构建重组DNA的简单技术，但不常用。这可能是由于通常从DNA片段中的真实序列设计的重叠退火后融合的效率低下。在我们目前的研究中，我们描述了新的重叠序列，可用于构建融合DNA片段，包括一步融合的三个片段在一个单一的PCR和框内融合的发展。新的聚G或C延伸显示出强烈的，也是特异性退火的互补序列的融合PCR。因此，这种DNA融合方法是一种简单而通用的重组DNA技术。(c)2008 Elsevier B.V.保留所有权利。
Recombinant DNA technology largely depends upon Escherichia coli plasmid construction via restriction enzyme digestion and DNA ligation. Overlap extension PCR is another simple technique for constructing recombinant DNA but is not commonly used. This is likely due to the inefficiency of fusion after the annealing of overlaps that are generally designed from authentic sequences in the DNA fragments. In our current study, we describe the development of novel overlap sequences that can be used for the construction of fusion DNA fragments, including the one-step fusion of three fragments in a single PCR and also for in-frame fusions. Novel poly G or C stretches showed strong and also specific annealing to the complementary sequences in the fusion PCR. This DNA fusion method is thus both a simple and versatile recombinant DNA technique. (c) 2008 Elsevier B.V. All rights reserved.