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Nat Protoc. 2008;3(3):371-7. doi: 10.1038/nprot.2008.5.

Selective isolation of genomic loci from complex genomes by transformation-associated recombination cloning in the yeast Saccharomyces cerevisiae.

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  • 1Laboratory of Molecular Pharmacology, National Cancer Institute, National Institutes of Health, 9000 Rockville Pike, Bethesda, Maryland 20892, USA. kouprinn@mail.nih.gov

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.

PMID:
18323808
[PubMed - indexed for MEDLINE]
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