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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.
Nucleic Acids Research
|February 11, 1988
Summary
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.