Synthesis of fusion genes for cloning by megaprimer-based PCR

Tatiana Q. Aguiar, Carla Oliveira*, Lucília Domingues

*Corresponding author for this work

Research output: Chapter in Book/Report/Conference proceedingChapterpeer-review

3 Citations (Scopus)

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.
Original languageEnglish
Title of host publicationMethods in molecular biology
Place of PublicationNew York
PublisherHumana Press Inc.
Pages101-112
Number of pages12
ISBN (Electronic)9781493970605
DOIs
Publication statusPublished - 2017
Externally publishedYes

Publication series

NameMethods in Molecular Biology
Volume1620
ISSN (Print)1064-3745

Keywords

  • Expression plasmids
  • Fusion genes
  • Megaprimers
  • Molecular cloning
  • Restriction enzymes
  • Two-step PCR

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