[5] Terminal transferase: Use in the tailing of DNA and for in Vitro mutagenesis

Terminal deoxynucleotidyltransferase catalyzes the addition of deoxynucleotides to the 3´ termini of DNA. In 1962, this enzyme was recognized as being different from DNA polymerase, and the name was changed to terminal transferase. By use of a tailing method, which adds homopolymer deoxynucleotide tails to denatured DNA, any double-stranded DNA fragment can be joined to a cloning vehicle. Because each DNA fragment carries the same type of tail, it cannot hybridize with another molecule of the same species. Thus, after cloning, each transformant should represent the desired recombinant DNA. These advantages make this technique particularly useful for the construction of recombinant DNA molecules for cloning. This chapter presents an improved procedure for the efficient addition of homopolymer tails to DNA, and also describes a new procedure for the addition of a single nucleotide to the 3´ end of a DNA as a method for in vitro mutagenesis, which can prove to be useful in investigating the control and expression of genes.