A tool for functional plant genomics: Chimeric RNA/DNA oligonucleotides cause in vivo gene-specific mutations

Peter R. Beetham, Peter B. Kipp, Xenia L. Sawycky, Charles J. Arntzen, Gregory D. May

Research output: Contribution to journalArticlepeer-review

167 Scopus citations

Abstract

Self-complementary chimeric oligonucleotides (COs) composed of DNA and modified RNA residues were evaluated as a means to (i) create stable, site- specific base substitutions in a nuclear gene and (ii) introduce a frameshift in a nuclear transgene in plant cells. To demonstrate the creation of allele- specific mutations in a member of a gene family, COs were designed to target the codon for Pro-196 of SuRA, a tobacco acetolactate synthase (ALS) gene. An amino acid substitution at Pro-196 of ALS confers a herbicide-resistance phenotype that can be used as a selectable marker in plant cells. COs were designed to contain a 25-nt homology domain comprised of a five- deoxyribonucleotide region (harboring a single base mismatch to the native ALS sequence) flanked by regions each composed of 10 ribonucleotides. After recovery of herbicide-resistant tobacco cells on selective medium, DNA sequence analyses identified base conversions in the ALS gene at the codon for Pro-196. To demonstrate a site-specific insertion of a single base into a targeted gene, COs were used to restore expression of an inactive green fluorescent protein transgene that had been designed to contain a single base deletion. Recovery of fluorescent cells confirmed the deletion correction. Our results demonstrate the application of a technology to modify individual genetic loci by catalyzing either a base substitution or a base addition to specific nuclear genes; this approach should have great utility in the area of plant functional genomics.

Original languageEnglish (US)
Pages (from-to)8774-8778
Number of pages5
JournalProceedings of the National Academy of Sciences of the United States of America
Volume96
Issue number15
DOIs
StatePublished - Jul 20 1999

ASJC Scopus subject areas

  • General

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