Banding Profile of Rep-PCR Experiments Differs With Varying Extension Times and Annealing Temperatures

Reproducibility has been a disadvantage for the rep-PCR technique, used for analyzing microbial contamination. The rep-PCR DNA fingerprint technique used for bacterial strain fingerprinting, was analyzed to elucidate the molecular mechanism by which certain PCR products are amplified preferentially over others. The analysis of rep- PCR products was done by gel electrophoresis after the reactions were run under a variety of elongation times and annealing temperatures. Two rep-PCR primers, BOX A1R and (GTG), were tested under a variety of elongation 5 times to determine the cause of lack of reproducibility. Decreasing elongation time had the effect of decreasing the number and intensity of resulting bands when the resulting product was analyzed on gel electrophoresis. BOX A1R was further analyzed by performing several PCRs under a variety of temperatures. At higher temperatures, o particularly at a 60 C annealing temperature, additional bands were observed, suggesting that secondary structure of the DNA template may have an impact on what products are formed during PCR.