COMPARISON OF THREE PROBE LABELLING METHODS TO DETECT PCR AMPLIFICATION PRODUCTS

Polymerase chain reaction amplifications are finding increased applications in environmental microbiology. The development of sensitive and specific methods to detect amplified products is necessary especially when these amplifications are conducted in the presence of the environmental matrix. Gene probes specific to the npt11 locus were prepared by nick translation, 5’end labelling and by a PCR driven amplification. These probes were tested against a 300 bp PCR amplified segment of the npt11 region of the transposable element Tn5. The nick translated probe was the most sensitive, though not as specific as the other two types of probes. Sensitivity and specificity were found to be dependent on the hybridization format (Southern blots versus dot blots), the number of amplification cycles and on the purity of the target sequence.