In Vitro Site-directed Mutagenesis with Synthetic DNA Oligonucleotides Yields Unexpected Deletions and Insertions at High Frequency

Overview

Journal Nucleic Acids Res

Publisher Oxford University Press

Specialty Biochemistry

Date 1983 Dec 20

PMID 6324090

Citations 14

Authors

K A Osinga

A M van der Bliek

G Van Der Horst

M J Groot Koerkamp

H F Tabak

G H Veeneman

J H VAN Boom

Affiliations

Soon will be listed here.

Abstract

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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Processing of yeast mitochondrial messenger RNAs at a conserved dodecamer sequence.

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