Optimization of arbitrarily primed PCR for the identification of bacterial isolates

Arbitrarily primed polymerase chain reaction (AP-PCR) has been used extensively for genetic mapping, and the identification of bacterial isolates. To ensure that the results will be reproducible and due to true genetic variations, the AP-PCR reaction conditions must be optimized. In this study, three cultured bacterial isolates were screened with 100 arbitrary primers. Of these, five were chosen for the optimization study. The parameters optimized included: the operating conditions of the thermal cycler, the agarose gel concentration, the annealing temperature, and the concentrations of Tag polymerase enzyme, magnesium chloride, primer, and template. The final optimized PCR reaction conditions were 1 x buffer (3.5 mM MgCl2, 10 mM Tris-HCl, 50 mM KCl and 0.1 mg ml(-1) gelatin), 200 mu M dNTP, 0.4 mu M primer, 2.5 U AmpliTaq(R) (Perkin-Elmer Cetus) polymerase enzyme, and 5 mu l of template (at least 10(6) lysed bacterial cells). The Perkin-Elmer Gene-Amp(TM) 9600 PCR System was used with the following cycling conditions; a 94 degrees C 15 s denaturation step, a 45 degrees C 15 s annealing step, and 72 degrees C 30 s extension step for a total of 35 cycles. Reproducible, unique fingerprints were generated for the three isolates using each of the five arbitrary primers.