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The Utility of TAR Vectors Used for Selective Gene Isolation by TAR Cloning.  

박정은 (동아대학교 자연과학대학 생물학과)
이윤주 (동아대학교 자연과학대학 생물학과)
정윤희 (동아대학교 자연과학대학 생물학과)
김재우 (동아대학교병원 임상병리실)
김승일 (기초과학지원연구소 프로테옴 분석팀)
김수현 (기초과학지원연구소 프로테옴 분석팀)
박인호 (동아대학교 자연과학대학 생물학과)
선우양일 (동아대학교 자연과학대학 생물학과)
임선희 (동아대학교 자연과학대학 생물학과)
Publication Information
Microbiology and Biotechnology Letters / v.31, no.4, 2003 , pp. 322-328 More about this Journal
Abstract
The Transformation-Associated Recombination (TAR) cloning technique allows selective isolation of chromosomal regions and genes from complex genomes. The procedure requires knowledge of relatively small genomic sequences that reside adjacent to the chromosomal region of interest. This technique involves homologous recombination during yeast spheroplast transformation between genomic DNA and a TAR vector that has 5'and 3' gene targeting sequences. In this study, we examined the minimum size of specific hooks required for a single-copy gene isolation and compared the utility of different TAR vectors, radial and unique vectors, by cloning the same single-copy gene. The efficiency of TAR cloning of the hHPRT gene was same using hooks varying from 750 to 63 bp. The number of transformants decreased approximately 20-fold when the TAR vector contained two unique hooks versus using a radial vector, but the percentage of positive recombinants increased over 2-fold when a unique TAR vector was used. Therefore, we suggest that the two-unique TAR vector is suitable for general TAR cloning given its high selectivity, and the radial TAR vector is more suitable when genomic DNA is in limited quantity, for example, DNA isolated from pathological specimens. Moreover, we confirm the minimal length of a unique sequence in a TAR vector is approximately 60 bp for a single-copy gene isolation.
Keywords
TAR (Transformation-Associated Recombination) cloning; TAR vector; hHPRT gene isolation; yeast;
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