Selective isolation of genomic loci from complex genomes by transformation-associated recombination cloning in the yeast Saccharomyces cerevisiae
Selective isolation of genomic loci from complex genomes by transformation-associated recombination cloning in the yeast Saccharomyces cerevisiae
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DOI：
10.1038/nprot.2008.5
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发表时间：
2008-01-01
期刊：
影响因子：
14.8
通讯作者：
Larionov, Vladimir
中科院分区：
文献类型：
--
作者：
Kouprina, Natalay;Larionov, Vladimir
在这里，我们描述了一个协议的任何基因组片段或感兴趣的基因的大小高达250 kb的复杂基因组作为一个环状酵母人工染色体（YAC）的选择性分离。该方法基于酵母酿酒酵母中基因组DNA和线性化TAR克隆载体之间的转化相关重组（TAR），所述线性化TAR克隆载体含有与感兴趣区域同源的靶向序列。载体和共转化的哺乳动物DNA中的同源序列之间的分离导致能够在酵母中繁殖、分离和被选择的YAC的建立。基因阳性克隆的产率从1%到5%不等。一旦构建了TAR载体并制备了基因组DNA，整个过程需要2周才能完成。TAR克隆方法在功能和比较基因组学、长距离单体型分析和染色体重排（包括拷贝数变异）的表征中具有广泛的应用。
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.