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首页> 外文期刊>BMC Biotechnology >A restriction-free method for gene reconstitution using two single-primer PCRs in parallel to generate compatible cohesive ends
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A restriction-free method for gene reconstitution using two single-primer PCRs in parallel to generate compatible cohesive ends

机译：一种无限制的基因重组方法，使用两个单引物PCR并行产生相容的内聚末端

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BackgroundRestriction-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. ResultsDouble-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. ConclusionsOur 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.
机译：背景无限制（RF）克隆是一种基于PCR的创建定制DNA质粒的方法，可将任何序列插入任何质粒载体中的任何所需位置，而不受限制位点和/或连接的影响。在这里，我们描述了一种通过修改后的RF克隆执行基因重建的简单而快速的方法。结果首先通过常规PCR扩增双链插入物和受体。然后将扩增的片段用作包含正向或反向引物的两个单独的线性扩增反应中的模板，以生成两个可以彼此退火的单链反向互补互补物。通过连接反应密封末端为5'和3'的退火插入物和受体。使用这种方法，我们制成了46个含有最多20？kb插入片段的构建体。通过克隆PCR和插入物测序证实，平均克隆效率高于85％。结论我们的方法提供了另一种克隆方法，该方法能够高效地将至少20？kb的任何DNA片段插入质粒。这种新方法不需要限制性位点或质粒或目的基因的改变，也不需要其他处理。引物设计和程序本身的简单性使该方法适用于高通量克隆和结构基因组学。

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