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5'-3' exonucleases in phosphorothioate-based oligonucleotide-directed mutagenesis
J R Sayers1, W Schmidt, F Eckstein
1Max-Planck-Institut für Experimentelle Medizin, Abteilung Chemie, Göttingen, FRG.
Abstract:
The application of T7 and lambda exonuclease to phosphorothioate-based oligonucleotide-directed mutagenesis was investigated. Oligonucleotide primers designed to introduce single or double base mismatches, an insertion or a deletion (each of 16 bases) were annealed to M13 phage derivatives. Double stranded closed circular DNA (RF IV) containing phosphorothioate internucleotidic linkages in the (-)strand was prepared enzymatically from these templates. A nick was introduced into the (+)strand of the hetroduplex DNA. This nicked DNA (RF II) was subjected to treatment with T7 or lambda exonuclease. Both of these enzymes were able to degrade almost all of the viral (+)strand when presented with DNA containing one or two base mismatches. Repolymerisation of the DNA after the gapping reaction, followed by transfection into E. coli cells gave mutational efficiencies of up to 95%. In the case of RF II DNA prepared with insertion or deletion primers these exonucleases could only partially degrade the viral (+)strand but were nevertheless highly efficient in such mutagenesis experiments.
Insights
T7 and lambda exonucleases efficiently create mutations using phosphorothioate-based oligonucleotide-directed mutagenesis. This method achieves high mutational efficiencies, up to 95%, for base mismatches, insertions, and deletions.
Area of Science:
- Molecular Biology
- Biotechnology
Background:
- Oligonucleotide-directed mutagenesis is a powerful tool for genetic engineering.
- Phosphorothioate linkages offer unique properties for DNA manipulation.
Purpose of the Study:
- To investigate the efficacy of T7 and lambda exonucleases in phosphorothioate-based oligonucleotide-directed mutagenesis.
- To assess the efficiency of these enzymes in introducing various types of mutations.
Main Methods:
- Annealing mutagenic oligonucleotide primers to M13 phage derivatives.
- Enzymatic preparation of double-stranded closed circular DNA (RF IV) with phosphorothioate linkages.
- Nicking the (+)strand of heteroduplex DNA (RF II).
- Exonuclease treatment with T7 or lambda exonuclease.
- Repolymerization and transfection into E. coli cells.
Main Results:
- T7 and lambda exonucleases effectively degraded the viral (+)strand in DNA with single or double base mismatches.
- High mutational efficiencies (up to 95%) were achieved for base mismatch mutagenesis.
- Exonucleases partially degraded the viral (+)strand for insertion/deletion mutagenesis but remained highly efficient.
Conclusions:
- T7 and lambda exonucleases are highly effective tools for phosphorothioate-based oligonucleotide-directed mutagenesis.
- The method demonstrates broad applicability for introducing various genetic modifications with high efficiency.