Genomic Reconstruction by Serial Mitotic Recombination of Yeast Artificial Chromosomes

Abstract

DNA cloned in yeast artificial chromosomes (YACs) is a valuable resource for functional experiments in cell culture as well as whole animal systems. Where the size or chimerism of a YAC clone are limiting factors it may be desirable to generate recombinant YAC clones. One such approach is based on mitotic recombination, and we describe the development of a methodology that allows multiple recombination cycles for serial reconstruction of overlapping YACs. This approach employs retrofitting with standard plasmid vectors, transfer of YACs to a common haploid host by Kar1 mating, and selection for recombination with 5-fluoro-orotic acid.

Global Survey of Research and Capabilities in Genetically Engineered Organisms That Could be Used in Biological Warfare or Bioterrorism

“…In addition to standard in vitro cloning techniques or chemical synthesis de novo, there is a variety of either alternative or complementary approaches which, due to their efficiency and simplicity, may be used to produce GEOs. These approaches involving in vivo recombination of desired sequences or properties include: DNA shuffling or molecular breeding [Stemmer W.P.C., 1994;Soong et al, 2000], recombineering [Court et al, 2002;Thomason et al, 2007], and genomic reconstruction in yeast artificial chromosomes [Markie et al, 2006]. The reported total times involved in some of these methods range from approximately a week to 10 days.…”