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Targeted gene repair directed by the chimeric RNA/DNA oligonucleotide in a mammalian cell-free extract.

TitleTargeted gene repair directed by the chimeric RNA/DNA oligonucleotide in a mammalian cell-free extract.
Publication TypeJournal Article
Year of Publication1999
AuthorsCole-Strauss A, Gamper H, Holloman WK, Muñoz M, Cheng N, Kmiec EB
JournalNucleic Acids Res
Volume27
Issue5
Pagination1323-30
Date Published1999 Mar 1
ISSN0305-1048
KeywordsAnimals, Base Sequence, Cell Line, Cell-Free System, DNA, DNA Primers, DNA Repair, Mammals, Nucleic Acid Heteroduplexes, Polymorphism, Restriction Fragment Length, Recombination, Genetic, RNA
Abstract

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.

Alternate JournalNucleic Acids Res.
PubMed ID9973621
PubMed Central IDPMC148319
Grant ListR01 HL58563-01A1 / HL / NHLBI NIH HHS / United States

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