Optimization of random-amplified polymorphic DNA producing amplicons up to 8500 bp and revealing intraspecies polymorphism in Leishmania infantum isolates.

In the case of investigation of polymorphism in closely related strains, the highest possible complexity of the patterns obtained by random-amplified polymorphic DNA PCR (RAPD-PCR) is required to assure revealing of limited polymorphism. In the present work, most parameters (reaction components concentration, additives, different polymerases, and thermal profiles) affecting RAPD-PCR were examined, in an effort to increase pattern complexity. A long PCR thermal profile, betaine as cosolvent, and Dynazyme EXT polymerase produced longer amplicons and higher pattern complexity, revealing polymorphism among Leishmania infantum isolates from infected dogs originating from Northern Greece (Macedonia).