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http://dx.doi.org/10.5352/JLS.2010.20.5.789

Development of Simultaneous YAC Manipulation-Amplification (SYMA) system by Chromosome Splitting Technique Harboring Copy Number Amplification System  

Kim, Yeon-Hee (Department of Biotechnology and Bioengineering, Dong-Eui University)
Nam, Soo-Wan (Department of Biotechnology and Bioengineering, Dong-Eui University)
Publication Information
Journal of Life Science / v.20, no.5, 2010 , pp. 789-793 More about this Journal
Abstract
Artificial chromosome manipulation and amplification of single-copy yeast artificial chromosome (YAC) are usually required in order to use YACs for applications such as physical mapping and functional analysis in eukaryotes. We designed and implemented a Simultaneous YAC Manipulation-Amplification (SYMA) system that combines the copy number amplification system of YAC with a convenient YAC manipulation system. To achieve the desired split and to amplify a YAC clone-harboring plant chromosome, a pBGTK plasmid containing a conditional centromere and thymidine kinase (TK) gene was constructed as a template to amplify the splitting fragment via PCR. By splitting, new 490-kb and 100-kb split YACs containing the elements for copy number amplification were simultaneously generated from a 590-kb YAC clone. The 100-kb split YAC was then successfully amplified 14.4-fold by adding 3 mg/ml sulfanilamide and $50\;{\mu}g/ml$ methotrexate (S3/M50) as inducing substances.
Keywords
Yeast artificial chromosome (YAC); PCR-mediated chromosome splitting (PCS); copy number amplification; GAL1 promoter; thymidine kinase (TK) gene;
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