The reproducibility of random amplified polymorphic DNA (RAPD) profiles of Streptococcus thermophilus strains with XD9, M13 and OPI-02 MOD primers

Abstract

We optimized the conditions for generating random amplified polymorphic DNA (RAPD) profiles of Streptococcus thermophilus strains by using the polymerase chain reaction (PCR). Several factors can cause the amplification of false and non reproducible bands in the RAPD profiles. We tested three primers, OPI-02 MOD, M13 and XD9 throughout this study. In addition, we tested different concentrations of primer, DNA template and Taq DNA polymerase. We adjusted the ratio of the primer to DNA template. All the three primers yielded reproducible profiles on several days, under optimized concentrations of components and cycling parameters used. The bands of such profiles probably corresponded to perfect annealing sites amplified with good efficacy or present in multiple copies in the genome. Five months later, repeated experiment generated identical bands. However, extra faint bands were detected with M13 and XD9 primers, possibly, corresponding to nonspecific binding resulting from slight variation in temperature or calibration of the thermocycler. Therefore, OPI-02 MOD was determined as the most reliable primer for reproducible profiles of S. thermophilus strains.

Key words: Streptococcus thermophilus, random amplified polymorphic DNA (RAPD), DNA template, Taq DNA polymerase, OPI-02MOD, XD9, M13, optimization, reproducibility.