PCR INHIBITION ASSAY FOR DNA-TARGETED ANTIBIOTICS

DNA amplification by polymerase chain reaction (PCR) should be inhibit ed if the target for amplification region in the template DNA is nicke d or cut. Based on this premise, we established a sensitive and differ ential assay using PCR to detect antibiotics that act on DNA. After te mplate lambda DNA (10 pg) was incubated with antibiotics (10 similar t o 20 ng) at 37 degrees C for 30 minutes in a 5 mu l reaction volume, a PCR assay (10 mu l reaction volume; 25 similar to 30 cycles) was perf ormed under the conditions we modified, resulting in amplification of a 500 bp fragment of lambda DNA which was monitored by agarose gel ele ctrophoresis. Among the several antibiotics examined, the anthracyclin es, bleomycin, D-cycloserine and mitomycin C clearly inhibited the PCR amplification reaction. whereas actinomycin D and ofloxacin did not. Preincubation of template DNA in the presence of Fe++ was necessary fo r bleomycin and cycloserine to exhibit marked inhibition of PCR. Mitom ycin C exhibited the inhibition in the presence of DTT and Cu+. By con trast, non-DNA-acting antibiotics (200 ng) such as aminoglycosides, be ta-lactams, and macrolides showed no inhibition. The PCR-amplified fra gment from lambda DNA was not degraded by incubation with the antibiot ics (20 ng) that inhibited PCR. Furthermore, ethylacetate extracts of the cultured broths of actinomycetes proved to be suitable as samples for this PCR inhibition assay.