Nucleic Acids Research, Vol 27, Issue 5 1323-1330, Copyright © 1999 by Oxford University Press
A Cole-Strauss, H Gamper, WK Holloman, M Munoz, N Cheng and EB Kmiec
Chimeric oligonucleotides consisting of RNA and DNA residues have been
shown to catalyze site-directed genetic alteration in mammalian cells both
in vitro and in vivo. Since the frequency of these events appears to be
logs higher than the rates of gene targeting, a process involving
homologous recombination, we developed a system to study the mechanisms of
chimera-directed gene conversion. Using a mammalian cell-free extract and a
genetic readout in Escherichia coli, we find that point mutations and
single base deletions can be corrected at frequencies of approximately 0.1%
and 0.005%, respectively. The reaction depends on an accurately designed
chimera and the presence of functional hMSH2 protein. The results of
genetic and biochemical studies reported herein suggest that the process of
mismatch repair functions in site-directed gene correction.
ARTICLES
Targeted gene repair directed by the chimeric RNA/DNA oligonucleotide in a mammalian cell-free extract
Department of Microbiology and Immunology, Jefferson Center for Biomedical Research, Thomas Jefferson University, 700 Butler Avenue, Doylestown, PA 18901, USA.
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