Transformation-Associated Recombination (TAR) Cloning

Description:

Transformation-Associated Recombination (TAR) cloning in yeast is a unique method for selective isolation of large chromosomal fragments or entire genes from complex genomes without the time-consuming step of library construction.¹ The technique involves homologous recombination during yeast spheroplast transformation between genomic DNA and a TAR vector that has short (approximately 60bp) 5’ and 3’ gene targeting sequences (hooks). Further, because up to 15% sequence divergence does not prevent recombination in yeast, TAR cloning is highly efficient for isolation of gene homologs and synthenic regions. Using this technology, chromosomal regions up to 250kb can be rescued in yeast as circular YACs within 3-5 working days..

NIH researchers Drs. Larionov, Kouprina and Resnick have championed the use of this technology and TAR cloning has been used to efficiently isolate haplotypes, gene families⁴ as well as genomic regions which are not present in existing BAC libraries. Known mutations and new modifications, including point mutations, deletions and insertions, can easily be introduced into DNA fragments hundreds of kilobases in size without introducing any unwanted alterations. The modified DNAs can then be tested functionally in mammalian cells and transgenic mice. TAR has also been used for structural biology studies, long-range haplotyping, evolutionary studies, centromere analysis and analysis of other regions which cannot be cloned by a routine technique based on in vitro ligation.⁵ In particular, construction of human artificial chromosome vectors and the combining of a HAC vector with a gene of interest can be effectively performed using the TAR methodology. Human genes isolated by TAR for expression in HACs include HPRT (60kb), BRCA1 (84kb), BRCA2 (90kb), PTEN (120kb), hTERT (60kb), KA11 (200kb), ASPM (70kb), SPANX-C (83kb) among others. TAR is a flexible and efficient means for employing in vivo recombination in yeast in order to clone entire genomic loci which can then be used for structural and functional analysis and for expression in HAC vectors for a variety of uses including for potential use in gene therapy.

The TAR cloning portfolio, including methods of use and vectors, is available for licensing and will be of direct use to those using a functional genomics approach in their work.