Preparation of a Nonradioactive DNA Probe for Detection of the APH (3')-II Gene

Authors: Armağan KOÇER SAĞIROĞLU, Semra KOCABIYIK

Abstract: As an alternative to the enzymatic methods a DNA probe was developed for testing microorganisms suspected of containing aminoglycoside 3'-phoshphotransferase [APH (3')] resistance determinants. In this method, a nonradioactive DNA probe specific for APH (3')-II was used to screen five enzymatically defined bacterial strains for the presence for the APH (3')-II structural gene. An approximately 0.92 kb Pst I fragment was isolated from a cloned APH (3')-II gene to be used as a probe. This fragment was labeled using dig-dUTP. As a result of this study, the DNA probe technique can identify the APH (3')-II subclass enzyme with remarkable specificity among other isoenzymes. Compared to conventional methods, including phosphocellulose paper binding assay, this nonradioactive method proved remarkably sensitive and specific, much faster and safer to handle.

Keywords: DNA probe, Aminoglycoside 3'-phosphotransferase, Aminoglycoside resistance, Southern Blot.