Although most of the DOS containing aminoglycosides are produced by Streptomyces, very little information is available about their biosynthesis. In the present paper, we report a method to isolate DOI synthase, a key enzyme in the biosynthesis of DOS, from aminoglycoside producer Streptomyces. PCR primers based on conserved region of DOI synthases were specific and reliable for the isolation of the biosynthetic genes of DOS containing aminoglycosides or the screening of the aminoglycoside producers. The use of DOI synthase as a probe could save both time and cost of cloning aminoglycoside biosynthetic genes.