In silico and RFLP analysis of HSP70

The HSP70 gene PCR products (10 ml each) were digested with the HaeIII (BsuR1) restriction enzyme using the conditions recommended by the manufacturer (MBI Fermentas, Vilnius, Lithuania). At first, this enzyme was tested against sequences of reference strains (Fasta format genes downloaded from GenBank) and searched for restriction sites using Restriction Mapper 3 (www.restrictionmapper.org). Theoretically, obtained fragments from digestion with HaeIII would be expected to create different patterns as follows: L. major (351, 307, 246, 152, 99, 47, 41, 40, 34, and 2 bp), L. tropica (354, 338, 246, 150, 99, 80, 41, 40, 21, 13, and 8 bp), and L. infantum (338, 307, 246, 152, 99, 80, 53, 47, 41, 40, and 13 bp). Digestion was performed in a total of 30 ml 1× optimal buffer, using 1U HaeIII restriction enzyme. Reactions were incubated at 37°C and completely analyzed by electrophoresis in a 3% small fragment agarose gel. The gels were subsequently ethidium bromide stained and subjected to electrophoresis along with the Gene Ruler TM 50 bp DNA Ladder (MBI Fermentas) as a reference DNA size marker.