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Nucleic Acids Research, 2003, Vol. 31, No. 6 e29
© 2003 Oxford University Press

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

Sun-Hee Leem1,2, Vladimir N. Noskov1, Jung-Eun Park1,2, Seung Il Kim3, Vladimir Larionov1 and Natalay Kouprina1

1 Laboratory of Biosystems and Cancer, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA, 2 Department of Biology, Dong-A University, Pusan 604-714, Korea and 3 Proteome Analysis Team, Korea Basic Science Institute, Daejon 305-806, Korea

*To whom correspondence should be addressed at Laboratory of Biosystems and Cancer, National Cancer Institute, NIH, Building 37, Room 5032, Bethesda, MD 20892-4471, USA. Tel: +1 301 496 7941; Fax: +1 301 496 0332; Email: kouprinn{at}mail.nih.gov

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 ~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 at the ends of the genomic DNA fragment. We demonstrate that the yield of gene-positive clones increases ~20-fold after endonuclease digestion of genomic DNA, which caused double strand breaks near the targeted sequences. These findings have led to a greatly improved protocol.


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