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KMID : 0613820100200050789
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Journal of Life Science
2010 Volume.20 No. 5 p.789 ~ p.793
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Development of Simultaneous YAC Manipulation-Amplification (SYMA) system by Chromosome Splitting Technique Harboring Copy Number Amplification System
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Kim Yeon-Hee
Nam Soo-Wan
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Abstract
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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 ㎎/㎖ sulfanilamide and 50 ㎍/㎖ methotrexate (S3/M50) as inducing substances.
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KEYWORD
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Yeast artificial chromosome(YAC), PCR-mediated chromosome splitting(PCS), copy number amplification, GAL1 promoter, thymidine kinase(TK) gene
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