The mechanism of gap repair in yeast in the basis for development of a facile method for in vivo cloning of large fragments of genomic DNA. A yeast-bacteria shuttle vector, pCLASPER, linearized to expose interchangeable site-specific recombinogenic ends, can target homologous sequences in co-transformed genomic DNA. The genomic region delimited by the recombinogenic ends is recovered in pCLASPER via the high frequency of yeast homologous recombination. Recombinants are identified by acquisition of a yeast selectable marker. The cloned products can be transferred to E. Coli for large-scale growth and plasmid DNA extraction by standard alkaline lysis methods. Methods will be devised and adapted for optimal co-transformation efficiency of linearized vector and uncloned genomic DNA in S. cerevisiae, the length of homology necessary for target-specific cloning will be determined, and sources for generating targeting sequence will be tested. Targeted in vivo cloning provides a method for selective recovery of a single, specific genomic fragment from one or many individuals of a population, without library construction and of a size larger than amplified by long-range PCR. Applications include sequence gap closure , mapping and subcloning, haplotyping, population sampling mutational analysis, gene funtion, and phylogientic comparisons.
Thesaurus Terms:biotechnology, gene targeting, genome, molecular cloning, transfection vector bacterial genetics, cell transformation, fungal genetics, method development, nucleic acid sequence, transposon /insertion element Escherichia coli, Saccharomyces cerevisiae, polymerase chain reaction, site directed mutagenesisNational Institute of Child and Human Development (NICHD)
