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Nucleic Acids Research, 2002, Vol. 30, No. 2 e8
© 2002 Oxford University Press

A genetic system for direct selection of gene-positive clones during recombinational cloning in yeast

Vladimir Noskov, Natalay Kouprina, Sun-Hee Leem1, Maxim Koriabine, J. Carl Barrett and Vladimir Larionov*

Laboratory of Biosystems and Cancer, National Cancer Institute, NIH, Building 37, Room 5032, Bethesda, MD 20892-4264, USA and 1Department of Biology, Dong-A University, Pusan 604-714, Korea

Transformation-associated recombination (TAR) is a cloning technique that allows specific chromosomal regions or genes to be isolated directly from genomic DNA without prior construction of a genomic library. This technique involves homologous recombination during spheroplast transformation between genomic DNA and a TAR vector that has 5' and 3' gene targeting sequences (hooks). Typically, TAR cloning produces positive YAC recombinants at a frequency of ~0.5%; the positive clones are identified by PCR or colony hybridization. This paper describes a novel TAR cloning procedure that selects positive clones by positive and negative genetic selection. This system utilizes a TAR vector with two targeting hooks, HIS3 as a positive selectable marker, URA3 as a negative selectable marker and a gene-specific sequence called a loop sequence. The loop sequence lies distal to a targeting hook sequence in the chromosomal target, but proximal to the targeting hook and URA3 in the TAR vector. When this vector recombines with chromosomal DNA at the gene-specific targeting hook, the recombinant YAC product carries two copies of the loop sequence, therefore, the URA3 negative selectable marker becomes mitotically unstable and is lost at high frequency by direct repeat recombination involving the loop sequence. Positive clones are identified by selecting against URA3. This method produces positive YAC recombinants at a frequency of ~40%. This novel TAR cloning method provides a powerful tool for structural and functional analysis of complex genomes.

* To whom correspondence should be addressed. Tel: +1 301 496 7941; Fax: +1 301 480 2772; Email: larionov{at}mail.nih.gov


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