Directed mutagenesis of YAC-cloned DNA using a rapid, PCR-based screening protocol.
We have developed a system which facilitates the rapid modification of yeast artificial chromosome (YAC) insert DNA. Specific modifications, such as deletions, insertions and point mutations, can be generated by a two-step allele replacement method using the yeast translational suppressor, SUP4-o, as both a positive and negative selection. The introduction of the SUP4-o gene was successful in 4 out of 24 selected transformant colonies, while the subsequent homologous elimination occurred in 2 out of 30 colonies. The use of a simple, short-range PCR assay rapidly identified the correct events among the genetically selected isolates and should be generally applicable to YAC modifications.
Tucker
R M
RM
Department of Human Genetics, University of Michigan Medical School, Ann Arbor 48109, USA.
Burke
D T
DT
D000818:Animals; D018244:Chromosomes, Artificial, Yeast / Q000235:genetics*; D016153:Genes, Suppressor; D051379:Mice; D008810:Mice, Inbred C57BL; D016297:Mutagenesis, Site-Directed; D016133:Polymerase Chain Reaction / Q000379:methods*; D012641:Selection, Genetic; D017384:Sequence Deletion; D014170:Transformation, Genetic