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Nucleic Acids Research, Vol 24, Issue 17 3467-3468, Copyright © 1996 by Oxford University Press

ARTICLES

Directed mutagenesis of YAC-cloned DNA using a rapid, PCR-based screening protocol

RM Tucker and DT Burke
Department of Human Genetics, University of Michigan Medical School, Ann Arbor 48109, USA.

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.
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