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Nucleic Acids Research, 1983, Vol. 11, No. 24 8595-8608
© 1983

MOLECULAR BIOLOGY

In vitro site-directed mutagenesis with synthetic DNA oligonucleotides yields unexpected deletions and insertions at high frequency

K.A. Osinga, A.M. van der Bliek, G. Van der Horst, M.J.A.Groot Koerkamp, H.F. Tabak, G.H. Veeneman and J.H. van Boom

Section for Molecular Biology, Laboratory of Biochemistry, University of Amsterdam Kruislaan 318, 1098 SM Amsterdam, The Netherlands Organic Chemistry Laboratory, Gorlaeus Laboratories, State University Leiden P.O. Box 9502, 2300 RA Leiden, The Netherlands

Received October 4, 1983. Revised November 18, 1983. Accepted November 18, 1983.

We have used in vitro site-directed mutagenesis with synthetic DNA oligonucleotides to introduce single nucleotide mutations in yeast mtDNA. In addition to the expected DNA alterations we also recovered with high frequency mutants with large deletions and insertions which arose through interaction with the synthetic DNA fragment. Characterization of a number of these by DNA sequence analysis has permitted reconstruction of the mutagenic events. In all cases, the DNA fragment had base paired with non-adjacent DNA sequences sometimes more than 1000 nucleotides apart from each other on the target strand. The products of such interactions cannot be avoided due to the non-stringent annealing conditions during complementary DNA strand synthesis. However, deliberate mispairing can be directed precisely, as shown by our ability to specifically delete the 1143-bp intron from the yeast mitochondrial gene coding for large ribosomal RNA with a synthetic DNA fragment consisting of the sequence of the exon borders flanking the intron.


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