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[19] Rapid and efficient site-specific mutagenesis without phenotypic selection

1987; Academic Press; Linguagem: Inglês

10.1016/0076-6879(87)54085-x

1557-7988

Thomas A. Kunkel, John D. Roberts, Richard A. Zakour,

DNA Repair Mechanisms

This chapter discusses the use of standard and well-established procedures of rapid and efficient site-specific mutagenesis without phenotypic selection in conjunction with an unusual template. This method takes advantage of a strong biological selection against the original deoxyribo nucleic acid (DNA) template, which is preferentially destroyed on transfection. The use of this special template can be combined with many of the previously described in vitro mutagenesis methods. This combination permits flexibility in the choice of mutagenesis techniques and makes possible the highly efficient recovery of mutants. The basis of this method is the performance of site-directed mutagenesis, using a DNA template that contains a small number of uracil residues in place of thymine. This procedure may also prove to be useful for investigating the mutagenic potential of specific DNA adducts located at defined positions in genes and for the studies of mutational specificity of in vitro DNA synthesis. The applications of these techniques for engineering DNA sequences and the proteins for which they code are limited only by the need and the imagination of the investigator.