Enhancement of In Vivo Targeted Nucleotide Exchange by Nonspecific Carrier DNA

Targeted nucleotide exchange (TNE) is a process in which an oligonucleotide bearing sequence complementarity aligns with the sequence of a target gene and directs the alteration of a single base. This technique can be used to repair a point mutation or mediate site-specific mutagenesis. A critical factor in the development of this approach centers around the elevation and stabilization of the frequencies with which these events occur. Here we describe a protocol for increasing the frequency of TNE in the true yeast, Saccharomyces cerevisiae , through the use of nonspecific, carrier oligonucleotides. These molecules, when added to the reaction, increase the TNE frequency up to 25-fold in some cases, perhaps by providing a molecular trap to bind factors, which may inactivate the specific targeting oligos.