Centre for Enzyme Innovation, University of Portsmouth, Portsmouth, UK.
Methods Mol Biol. 2023;2633:45-53. doi: 10.1007/978-1-0716-3004-4_4.
The Gibson Assembly is a popular method for molecular cloning which has been developed specifically to join several fragments together in a specific order, without the constraint of restriction enzyme sites. This method is based on the assembly of overlapping fragments, generally produced by PCR, and then combining them using three enzymes: a 5' exonuclease, a DNA polymerase, and a DNA ligase, in an isothermal reaction. Here, we describe this method, including the design of primers for the generation of the overlapping fragments and the assembly; to this end, we provide an example involving joining two fragments in a single plasmid.
Gibson 组装是一种流行的分子克隆方法，专门用于以特定顺序连接几个片段，而不受限制酶位点的限制。该方法基于重叠片段的组装，通常通过 PCR 产生，然后使用三种酶将它们结合在一起：5'核酸外切酶、DNA 聚合酶和 DNA 连接酶，在等温反应中。在这里，我们描述了这种方法，包括用于生成重叠片段和组装的引物设计；为此，我们提供了一个涉及在单个质粒中连接两个片段的示例。
