Synthesis of fusion genes for cloning by megaprimer-based PCR

Abstract

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.