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Microbiology and Biotechnology Letters

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Microbiol. Biotechnol. Lett. 2003; 31(4): 322-328

TAR Cloning에 의한 선별적 유전자 분리에 사용되는 TAR Vectors의 유용성에 관한 연구

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

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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