A restriction-free method for gene reconstitution using two single-primer PCRs in parallel to generate compatible cohesive ends.
A restriction-free method for gene reconstitution using two single-primer PCRs in parallel to generate compatible cohesive ends.
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DOI：
10.1186/s12896-017-0346-5
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发表时间：
2017-03-17
影响因子：
3.5
通讯作者：
Lin Y
中科院分区：
文献类型：
--
作者：
Zeng F;Hao Z;Li P;Meng Y;Dong J;Lin Y
无限制(RF)克隆是一种基于聚合酶链式反应(PCR)的方法，用于创建定制的DNA质粒，允许将任何序列插入到任何所需位置的任何质粒载体中，而不依赖于限制性内切酶和/或连接。在这里，我们描述了一种简单而快速的方法，通过改进的RF克隆进行基因重组。首先用常规的聚合酶链式反应扩增双链插入片段和受体。然后将扩增的片段作为模板，进行两个单独的线性扩增反应，其中包含正向或反向引物，以产生两个单链反向互补对应物，它们可以相互退火。通过连接反应封闭具有5‘和3’粘结端的退火插入物和受体。使用这种方法，我们构建了46个包含长达20kb的插入片段的构建体。经菌落聚合酶链式反应和插入片段测序证实，平均克隆效率高于85%。我们的方法提供了一种替代的克隆方法，能够高效地将至少20kb的任何DNA片段插入到质粒中。这种新方法不需要限制酶切位点，也不需要改变质粒或感兴趣的基因，也不需要额外的治疗。该方法设计简单，操作简便，适用于高通量克隆和结构基因组学研究。本文的在线版本(doi：10.1186/s12896-0170346-5)包含补充材料，授权用户可以使用。
Restriction-free (RF) cloning, a PCR-based method for the creation of custom DNA plasmids, allows for the insertion of any sequence into any plasmid vector at any desired position, independent of restriction sites and/or ligation. Here, we describe a simple and fast method for performing gene reconstitution by modified RF cloning. Double-stranded inserts and acceptors were first amplified by regular PCR. The amplified fragments were then used as the templates in two separate linear amplification reactions containing either forward or reverse primer to generate two single-strand reverse-complement counterparts, which could anneal to each other. The annealed inserts and acceptors with 5’ and 3’ cohesive ends were sealed by ligation reaction. Using this method, we made 46 constructs containing insertions of up to 20 kb. The average cloning efficiency was higher than 85%, as confirmed by colony PCR and sequencing of the inserts. Our method provides an alternative cloning method capable of inserting any DNA fragment of up to at least 20 kb into a plasmid, with high efficiency. This new method does not require restriction sites or alterations of the plasmid or the gene of interest, or additional treatments. The simplicity of both primer design and the procedure itself makes the method suitable for high-throughput cloning and structural genomics. The online version of this article (doi:10.1186/s12896-017-0346-5) contains supplementary material, which is available to authorized users.