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

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.

journal_name

Nucleic Acids Res

journal_title

Nucleic acids research

authors

Osinga KA,Van der Bliek AM,Van der Horst G,Groot Koerkamp MJ,Tabak HF,Veeneman GH,Van Boom JH

doi

10.1093/nar/11.24.8595

subject

Has Abstract

pub_date

1983-12-20 00:00:00

pages

8595-608

issue

24

eissn

0305-1048

issn

1362-4962

journal_volume

11

pub_type

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