DOE Human Genome Program Contractor-Grantee Workshop VIII
February 27-March 2, 2000  Santa Fe, NM

109. Defining the Minimal Length of Sequence Homology Required for Selective Gene Isolation by TAR Cloning

Vladimir Noskov, Maxim Koriabine, Greg Solomon, Natalay Kouprina, J. Carl Barrett, Lisa Stubbs¹, and Vladimir Larionov

Laboratory of Molecular Genetics and Laboratory of Molecular Carcinogenesis, National Institute of Environmental Health Sciences, P.O. Box 12233, Research Triangle Park, NC 27709 and ¹Human Genome Center, Lawrence Livermore National Laboratory, Livermore, CA 95616

noskov@niehs.nih.gov

Using the recently developed TAR cloning technique, it is possible to directly isolate specific chromosomal regions and genes from complex genomes as linear or circular YACs. Over the last two years the new technique has been successfully applied for isolation of different genes and specific regions of human and mouse genomes. In this study we investigated the minimal length of sequence homology required for gene isolation by TAR cloning using the Tg.AC transgene as a model. The Tg.AC transgene unit consists of a zeta-globin promoter fused to the v-Ha-ras structural gene with a terminal simian virus 40 (SV40) polyadenylation signal sequence. We constructed a set of radial TAR cloning vectors containing the B1 repeat and different size SV40-specific hooks (from 800 bp to 20 bp). With a vector containing a 800 bp hook, cloning of Tg.AC transgene sequences from mouse genome was highly specific: one among fifty yeast transformants obtained contained a YAC with Tg.AC transgene. The same yield of positive clones was observed when length of homology was reduced to 60 bp. Therefore the minimal length of a unique sequence required for gene isolation is only 2 times larger than the minimal size of homology required for spontaneous mitotic recombination in yeast. This observation greatly facilitates selection of hooks for isolation of specific regions as well as construction of TAR vectors because the hooks can be synthesized as oligonucleotides instead of being isolated as genomic fragments.