2 - Recombinant DNA cloning

Abstract

This chapter presents the background of recombinant DNA cloning and the procedures for cloning.The general steps of recombinant DNA cloning are the isolation of the DNA to be cloned, the isolation of the DNA to be used as a cloning vector, and the cutting of the two DNAs with a restriction endonuclease that makes sequence-specific cuts in the DNAs. The restriction endonucleases to be used in this cloning experiment generate a four-base-long single stranded region known as a “sticky end” or a “cohesive end.” Any fragment generated with this enzyme can base pair with any other fragment generated by the same enzyme. The restriction endonuclease subsequently is inactivated. Then the vector DNA and DNA to be cloned are mixed so that the complementary ends of the restriction endonuclease sites can base pair. That is, reannealing of the sticky ends is allowed to occur. These fragments are sealed together using T4 DNA ligase, and the DNA is introduced into a host cell where the cloning vector can replicate. This chapter describes an experiment in which the recombinant DNA is introduced into E. coli cells by transformation. The E. coli are subjected to a process that will select for the presence of