Enhancement of in vivo targeted nucleotide exchange by nonspecific carrier DNA

• Published in: Methods in molecular biology (Clifton, N.J.) Vol. 262; p. 209
• Main Authors: Maguire, Katie K, Kmiec, Eric B
• Format: Journal Article
• Language: English
• Published: United States 2004
• Subjects: Base Sequence; Chromosomes, Fungal - drug effects; Chromosomes, Fungal - genetics; Gene Targeting - methods; Molecular Sequence Data; Oligodeoxyribonucleotides - chemistry; Oligodeoxyribonucleotides - pharmacology; Plasmids - genetics; Polymerase Chain Reaction - methods; Restriction Mapping - methods; Saccharomyces cerevisiae - genetics; Saccharomyces cerevisiae - growth & development
• ISSN: 1064-3745
• DOI: 10.1385/1-59259-761-0:209

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.