FastCloning: a highly simplified, purification-free, sequence- and ligation-independent PCR cloning method.
FastCloning: a highly simplified, purification-free, sequence- and ligation-independent PCR cloning method.
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DOI：
10.1186/1472-6750-11-92
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发表时间：
2011-10-12
影响因子：
3.5
通讯作者：
Chang Y
中科院分区：
文献类型：
--
作者：
Li C;Wen A;Shen B;Lu J;Huang Y;Chang Y
虽然有各种各样的方法和昂贵的试剂盒，但分子克隆可能是一个耗时且令人沮丧的过程。在这里，我们报告了一种高度简化，可靠，高效的基于PCR的克隆技术，将任何DNA片段插入到质粒载体或载体中的基因（cDNA）的任何所需位置。使用这种方法，使用高保真DNA聚合酶分别PCR扩增载体和插入片段，仅用18个循环。扩增的插入片段具有与扩增的载体的末端重叠约16个碱基的末端。在Dpn I消化扩增的载体和插入片段的混合物以消除PCR反应中使用的DNA模板后，将混合物直接转化到感受态E. coli细胞，以获得所需的克隆。该技术与其他克隆方法相比具有许多优点。首先，它不需要PCR产物或线性化载体的凝胶纯化。第二，不需要任何克隆试剂盒或专门的克隆酶。此外，随着PCR循环次数的减少，它也减少了随机突变的机会。此外，该方法是高效和可重复的。最后，由于这种克隆方法也是序列独立的，我们证明了它可以用于嵌合体的构建，插入，和多个突变跨越一段DNA高达120 bp。我们的FastCloning技术为分子克隆、嵌合体构建、任何感兴趣的DNA序列插入以及cDNA短片段中的多个突变提供了一种非常简单、有效、可靠和通用的工具。
Although a variety of methods and expensive kits are available, molecular cloning can be a time-consuming and frustrating process. Here we report a highly simplified, reliable, and efficient PCR-based cloning technique to insert any DNA fragment into a plasmid vector or into a gene (cDNA) in a vector at any desired position. With this method, the vector and insert are PCR amplified separately, with only 18 cycles, using a high fidelity DNA polymerase. The amplified insert has the ends with ~16-base overlapping with the ends of the amplified vector. After DpnI digestion of the mixture of the amplified vector and insert to eliminate the DNA templates used in PCR reactions, the mixture is directly transformed into competent E. coli cells to obtain the desired clones. This technique has many advantages over other cloning methods. First, it does not need gel purification of the PCR product or linearized vector. Second, there is no need of any cloning kit or specialized enzyme for cloning. Furthermore, with reduced number of PCR cycles, it also decreases the chance of random mutations. In addition, this method is highly effective and reproducible. Finally, since this cloning method is also sequence independent, we demonstrated that it can be used for chimera construction, insertion, and multiple mutations spanning a stretch of DNA up to 120 bp. Our FastCloning technique provides a very simple, effective, reliable, and versatile tool for molecular cloning, chimera construction, insertion of any DNA sequences of interest and also for multiple mutations in a short stretch of a cDNA.
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影响因子：
14.9
作者：
ASLANIDIS, C;DEJONG, PJ
通讯作者：
DEJONG, PJ
影响因子：
16.2
作者：
LIMAN, ER;TYTGAT, J;HESS, P
通讯作者：
HESS, P
使用切口 DNA 核酸内切酶的不依赖于连接的克隆方法
影响因子：
2.7
作者：
Yang, Jie;Zhang, Zhihong;Luo, Qingming
通讯作者：
Luo, Qingming
影响因子：
2.7
作者：
Bryksin AV;Matsumura I
通讯作者：
Matsumura I