The gapped duplex DNA approach to oligonucleotide-directed mutation construction.

Abstract:

:A simple and efficient method is described to introduce structurally pre-determined mutations into recombinant genomes of filamentous phage M13. The method rests on gapped duplex DNA (gdDNA) molecules of the phage M13 genome as the key intermediate. In this gdDNA, the (+) and the (shorter) (-) strand carry different genetic markers in such a way, that a rigorous selection can be applied for phage carrying the markers of the (-) strand. For introduction of the mutation, a synthetic oligonucleotide with partial homology to a target site within the single stranded DNA region is annealed to the gdDNA. The oligonucleotide subsequently becomes part of the (-) strand by enzymatic DNA gap filling and sealing. This physical linkage is preserved at the genetic level after transfection of a recipient E.coli strain deficient in DNA mismatch correction, so that the synthetic marker can be selected from the phage progeny independent from its potential phenotype. It is demonstrated that by this method mutants can be constructed with marker yields in excess of 70%.

journal_name

Nucleic Acids Res

journal_title

Nucleic acids research

authors

Kramer W,Drutsa V,Jansen HW,Kramer B,Pflugfelder M,Fritz HJ

doi

10.1093/nar/12.24.9441

subject

Has Abstract

pub_date

1984-12-21 00:00:00

pages

9441-56

issue

24

eissn

0305-1048

issn

1362-4962

journal_volume

12

pub_type

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