Rescue of end fragments of yeast artificial chromosomes by homologous recombination in yeast.
Yeast artificial chromosomes (YACs) provide a powerful tool for the isolation and mapping of large regions of mammalian chromosomes. We developed a rapid and efficient method for the isolation of DNA fragments representing the extreme ends of YAC clones by the insertion of a rescue plasmid into the YAC vector by homologous recombination. Two rescue vectors were constructed containing a yeast LYS2 selectable gene, a bacterial origin of replication, an antibiotic resistance gene, a polylinker containing multiple restriction sites, and a fragment homologous to one arm of the pYAC4 vector. The 'end-cloning' procedure involves transformation of the rescue vector into yeast cells carrying a YAC clone, followed by preparation of yeast DNA and transformation into bacterial cells. The resulting plasmids carry end-specific DNA fragments up to 20 kb in length, which are suitable for use as hybridization probes, as templates for direct DNA sequencing, and as probes for mapping by fluorescence in situ hybridization. These vectors are suitable for the rescue of end-clones from any YAC constructed using a pYAC-derived vector. We demonstrate the utility of these plasmids by rescuing YAC-end fragments from a human YAC library.
Hermanson
G G
GG
Molecular Genetics Laboratory, Salk Institute for Biological Studies, La Jolla, CA 92037.
Hoekstra
M F
MF
McElligott
D L
DL
Evans
G A
GA
D001483:Base Sequence; D015825:Chromosomes, Fungal; D015723:Gene Library; D005822:Genetic Vectors; D015894:Genome, Human; D006801:Humans; D008969:Molecular Sequence Data; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011995:Recombination, Genetic; D014170:Transformation, Genetic