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An asymmetric PCR-based, reliable and rapid single-tube native DNA engineering strategy

机译：基于非对称PCR的可靠，快速的单管天然DNA工程策略

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Background Widely used restriction-dependent cloning methods are labour-intensive and time-consuming, while several types of ligase-independent cloning approaches have inherent limitations. A rapid and reliable method of cloning native DNA sequences into desired plasmids are highly desired. Results This paper introduces ABI-REC, a novel strategy combining asymmetric bridge PCR with intramolecular homologous recombination in bacteria for native DNA cloning. ABI-REC was developed to precisely clone inserts into defined location in a directional manner within recipient plasmids. It featured an asymmetric 3-primer PCR performed in a single tube that could robustly amplify a chimeric insert-plasmid DNA sequence with homologous arms at both ends. Intramolecular homologous recombination occurred to the chimera when it was transformed into E.coli and produced the desired recombinant plasmids with high efficiency and fidelity. It is rapid, and does not involve any operational nucleotides. We proved the reliability of ABI-REC using a double-resistance reporter assay, and investigated the effects of homology and insert length upon its efficiency. We found that 15?bp homology was sufficient to initiate recombination, while 25?bp homology had the highest cloning efficiency. Inserts up to 4?kb in size could be cloned by this method. The utility and advantages of ABI-REC were demonstrated through a series of pig myostatin (MSTN) promoter and terminator reporter plasmids, whose transcriptional activity was assessed in mammalian cells. We finally used ABI-REC to construct a pig MSTN promoter-terminator cassette reporter and showed that it could work coordinately to express EGFP . Conclusions ABI-REC has the following advantages: (i) rapid and highly efficient; (ii) native DNA cloning without introduction of extra bases; (iii) restriction-free; (iv) easy positioning of directional and site-specific recombination owing to formulated primer design. ABI-REC is a novel approach to DNA engineering and gene functional analysis.
机译：背景技术广泛使用的依赖限制性的克隆方法是劳动密集型且费时的，而几种不依赖连接酶的克隆方法具有固有的局限性。迫切需要一种快速可靠的将天然DNA序列克隆到所需质粒中的方法。结果本文介绍了ABI-REC，这是一种将不对称桥式PCR与细菌内分子内同源重组相结合的新策略，用于天然DNA克隆。开发了ABI-REC，可将其以定向方式精确地克隆到受体质粒内的指定位置。它的特点是在单个试管中进行不对称3引物PCR，可以牢固地扩增两端均具有同源臂的嵌合插入质粒DNA序列。当嵌合体被转化为大肠杆菌并以高效率和保真度产生所需的重组质粒时，嵌合体发生了分子内同源重组。它是快速的，并且不涉及任何可操作的核苷酸。我们使用双重抗性报告基因检测法证明了ABI-REC的可靠性，并研究了同源性和插入长度对其效率的影响。我们发现15 bp的同源性足以启动重组，而25 bp的同源性具有最高的克隆效率。用这种方法可以克隆最大4？kb的插入片段。通过一系列猪肌肉生长抑制素（MSTN）启动子和终止子报道质粒证明了ABI-REC的实用性和优势，并在哺乳动物细胞中评估了它们的转录活性。我们最终使用ABI-REC构建了猪MSTN启动子-终止子盒式报道基因，并表明它可以协同表达EGFP。结论ABI-REC具有以下优点：（i）快速且高效; （ii）不引入额外碱基的天然DNA克隆; （iii）无限制; （iv）由于配制的引物设计，容易定位定向和位点特异性的重组。 ABI-REC是一种用于DNA工程和基因功能分析的新颖方法。

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