Enzymatic mutation detection: enrichment of heteroduplexes from hybrid DNA mixtures by cleavage-deficient GST-tagged endonuclease VII

S Golz; B Kemper

doi:10.1093/nar/27.15.e7

Enzymatic mutation detection: enrichment of heteroduplexes from hybrid DNA mixtures by cleavage-deficient GST-tagged endonuclease VII

Nucleic Acids Res. 1999 Aug 1;27(15):e7. doi: 10.1093/nar/27.15.e7.

Authors

S Golz¹, B Kemper

Affiliation

¹ Institut für Genetik, Universität zu Köln, Zülpicher Strasse 47, 50674 Köln, Germany.

Abstract

A method for the enrichment of heteroduplex DNAs from hybrid DNA mixtures by endonuclease VII is reported. The procedure is based on the ability of a GST-fused cleavage-deficient mutant endonuclease VII (EVII-N62D(GST)) to bind to mismatching nucleotides in heteroduplex DNAs identical to the wild-type enzyme. The GST tag was used for stable immobilisation of the protein to Glutathione Sepharose 4B. This enables the material to withstand the repeated rounds of binding steps required for enrichment of heteroduplex molecules from appropriate samples.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Base Pair Mismatch
DNA / genetics*
DNA / isolation & purification
Electrophoresis, Polyacrylamide Gel
Endodeoxyribonucleases / genetics
Endodeoxyribonucleases / metabolism*
Glutathione Transferase / genetics
Mutation*
Nucleic Acid Heteroduplexes

Substances

Nucleic Acid Heteroduplexes
DNA
Glutathione Transferase
Endodeoxyribonucleases
endodeoxyribonuclease VII