Cloning of the type Ib trimethoprim-resistant dihydrofolate reductase gene and preparation of a specific biotinylated DNA probe

Abstract

A 3.0-kb PstI/HindIII fragment encoding the type Ib trimethoprim-resistant dihydrofolate reductase (DHFR) was cloned into pUC18. Following successive subcloning, the type Ib DHFR gene was identified within a 500 bp HincII fragment. The 500 bp fragment was purified, biotinylated and tested for its suitability as a specific DNA probe in non-isotopic hybridisation experiments with standard plasmids carrying genes for the available known Gram-negative DHFR types. Under conditions of high stringency the probe was specific for the type Ib gene and hence will be useful for monitoring the spread of this particular trimethoprim resistance gene.