Aguiar Tatiana Q, Oliveira Carla, Domingues Lucília
CEB-Centre of Biological Engineering, University of Minho, Campus de Gualtar, 4710-057, Braga, Portugal.
Methods Mol Biol. 2017;1620:101-112. doi: 10.1007/978-1-4939-7060-5_6.
The polymerase chain reaction (PCR) is the technique of choice used to obtain DNA for cloning, because it rapidly provides high amounts of desired DNA fragments and allows the easy introduction of extremities adequate for enzyme restriction or homologous recombination, and of artificial, native, or modified sequence elements for specific applications. In this context, the use of megaprimer-based PCR strategies allows the versatile and fast assembly and amplification of tailor-made DNA sequences readily available for cloning.In this chapter, we describe the design and use of a megaprimer-based PCR protocol to construct customized fusion genes ready for cloning into commercial expression plasmids by restriction digestion and ligation.
聚合酶链反应（PCR）是用于获取用于克隆的DNA的首选技术，因为它能快速提供大量所需的DNA片段，并便于引入适合酶切或同源重组的末端，以及用于特定应用的人工、天然或修饰的序列元件。在这种情况下，基于大引物的PCR策略的使用允许对定制的DNA序列进行通用且快速的组装和扩增，这些序列可随时用于克隆。在本章中，我们描述了一种基于大引物的PCR方案的设计和使用，以构建定制的融合基因，这些基因可通过限制性消化和连接直接克隆到商业表达质粒中。
