Protocol abstract
Nature Protocols 3, - 371 - 377 (2008)
Published online: 14 February 2008 | doi:10.1038/nprot.2008.5
Subject Categories: Genetic modification | Isolation, purification and separation | Microbiology and virology | Nucleic acid based molecular biology
Selective isolation of genomic loci from complex genomes by transformation-associated recombination cloning in the yeast Saccharomyces cerevisiae
Natalay Kouprina1 & Vladimir Larionov1
Abstract
Here, we describe a protocol for the selective isolation of any genomic fragment or gene of interest up to 250 kb in size from complex genomes as a circular yeast artificial chromosome (YAC). The method is based on transformation-associated recombination (TAR) in the yeast Saccharomyces cerevisiae between genomic DNA and a linearized TAR cloning vector containing targeting sequences homologous to a region of interest. Recombination between the vector and homologous sequences in the co-transformed mammalian DNA results in the establishment of a YAC that is able to propagate, segregate and be selected for in yeast. Yield of gene-positive clones varies from 1% to 5%. The entire procedure takes 2 weeks to complete once the TAR vector is constructed and genomic DNA is prepared. The TAR cloning method has a broad application in functional and comparative genomics, long-range haplotyping and characterization of chromosomal rearrangements, including copy number variations.
- Laboratory of Molecular Pharmacology, National Cancer Institute, National Institutes of Health, 9000 Rockville Pike, Bethesda, Maryland 20892, USA.
Correspondence to: Natalay Kouprina1 e-mail: kouprinn@mail.nih.gov
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A-Z product listing
- 100 mm
15 mm sterile disposable Petri plates(Fisher Scientific Ltd.) - 14 mM beta-mercaptoethanol(Sigma Chemical Co. Ltd.)
- 250-ml baffled Erlenmeyer flask, sterile(Fisher Scientific Ltd.)
- Adenine hemisulfate(Sigma Chemical Co. Ltd.)
- Bacto agar(Fisher Scientific Ltd.)
- Bacto peptone(Fisher Scientific Ltd.)