Defining the minimal length of sequence homology required for selective gene isolation by TAR cloning.
The transformation-associated recombination (TAR) cloning technique allows selective and accurate isolation of chromosomal regions and genes from complex genomes. The technique is based on in vivo recombination between genomic DNA and a linearized vector containing homologous sequences, or hooks, to the gene of interest. The recombination occurs during transformation of yeast spheroplasts that results in the generation of a yeast artificial chromosome (YAC) containing the gene of interest. To further enhance and refine the TAR cloning technology, we determined the minimal size of a specific hook required for gene isolation utilizing the Tg.AC mouse transgene as a targeted region. For this purpose a set of vectors containing a B1 repeat hook and a Tg.AC-specific hook of variable sizes (from 20 to 800 bp) was constructed and checked for efficiency of transgene isolation by a radial TAR cloning. When vectors with a specific hook that was >/=60 bp were utilized, approximately 2% of transformants contained circular YACs with the Tg.AC transgene sequences. Efficiency of cloning dramatically decreased when the TAR vector contained a hook of 40 bp or less. Thus, the minimal length of a unique sequence required for gene isolation by TAR is approximately 60 bp. No transgene-positive YAC clones were detected when an ARS element was incorporated into a vector, demonstrating that the absence of a yeast origin of replication in a vector is a prerequisite for efficient gene isolation by TAR cloning.
Laboratory of Molecular Genetics and Laboratory of Molecular Carcinogenesis, National Institute of Environmental Health Sciences, Research Triangle Park, NC 27709, USA.
V N
VN
Noskov
M
M
Koriabine
G
G
Solomon
M
M
Randolph
J C
JC
Barrett
S H
SH
Leem
L
L
Stubbs
N
N
Kouprina
V
V
Larionov
D000818:Animals; D003001:Cloning, Molecular / Q000379:methods*; D004247:DNA / Q000235:genetics*; D011905:Genes, ras / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics; D051379:Mice; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification*; D018741:Replication Origin / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics; D019076:Transgenes / Q000235:genetics
D011993:Recombinant Fusion Proteins; D004247:DNA