Optimum conditions for selective isolation of genes from complex genomes by transformation-associated recombination cloning.

ABSTRACT: Transformation-associated recombination (TAR) cloning in yeast is used to isolate a desired chromosomal region or gene from a complex genome without construction of a genomic library. The technique involves homologous recombination during yeast spheroplast transformation between genomic DNA and a TAR vector containing short 5' and 3' gene-specific targeting hooks. Efficient gene capture requires a high yield of transformants, and we demonstrate here that the transformant yield increases approximately 10-fold when the genomic DNA is sheared to 100-200 kb before being presented to the spheroplasts. Here we determine the most effective concentration of genomic DNA, and also show that the targeted sequences recombine much more efficiently with the vector's targeting hooks when they are located

SUBMITTER: Leem SH 

PROVIDER: S-EPMC152883 | biostudies-literature | 2003 Mar

REPOSITORIES: biostudies-literature