Selective isolation of genomic loci from complex genomes by transformation-associated recombination cloning in the yeast Saccharomyces cerevisiae.
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.
Kouprina
Natalay
N
Laboratory of Molecular Pharmacology, National Cancer Institute, National Institutes of Health, 9000 Rockville Pike, Bethesda, Maryland 20892, USA. kouprinn@mail.nih.gov
Larionov
Vladimir
V
D000818:Animals; D018244:Chromosomes, Artificial, Yeast / Q000235:genetics; D003001:Cloning, Molecular / Q000379:methods*; D004270:DNA, Circular / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics; D016678:Genome / Q000235:genetics*; D023281:Genomics / Q000379:methods*; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D014170:Transformation, Genetic
D004270:DNA, Circular