rapid generation of long tandem dna repeat arrays by homologous recombination in yeast to study their function in mammalian genomes快速代长串联dna重复阵列通过同源重组酵母在哺乳动物基因组研究它们的功能.pdfVIP

Noskov et al. Biological Procedures Online 2011, 13:8 /content/13/1/8 Biological Procedures Online METHODOLOGY Open Access Rapid generation of long tandem DNA repeat arrays by homologous recombination in yeast to study their function in mammalian genomes * Vladimir N Noskov, Nicholas CO Lee, Vladimir Larionov and Natalay Kouprina Abstract We describe here a method to rapidly convert any desirable DNA fragment, as small as 100 bp, into long tandem DNA arrays up to 140 kb in size that are inserted into a microbe vector. This method includes rolling-circle phi29 amplification (RCA) of the sequence in vitro and assembly of the RCA products in vivo by homologous recombination in the yeast Saccharomyces cerevisiae. The method was successfully used for a functional analysis of centromeric and pericentromeric repeats and construction of new vehicles for gene delivery to mammalian cells. The method may have general application in elucidating the role of tandem repeats in chromosome organization and dynamics. Each cycle of the protocol takes ~ two weeks to complete. Introduction different tandem repeated DNA sequences. Although Tandem repeats, also referred to as satellite DNA, repre- centromeric DNA repeat sequences are thought to be sent a major class of repetitive DNA, whose function in structurally and/or functionally important for forming a the shaping of the human genome is only beginning to functional kinetochore, they are poorly conserved be explored. These repetiti