full issue - Association of Biotechnology and Pharmacy

Current Trends in Biotechnology and PharmacyVol. 5 (2) 1183-1192 April 2011. ISSN 0973-8916 (Print), 2230-7303 (Online)1185checked by agarose gel electrophoresis.Strain typing: Identity of the strains used wereconfirmed using the hsp70 gene PCR-RFLP aspreviously described (21, 22). Briefly, hsp70amplicons (approximately 500 ng) were digestedeither with HaeIII or BccI in final volume of 20µl, then loaded in 1.5% agarose gel and visualizedwith ethidium bromide staining using standardprocedures (23). Restriction fragments patternswere compared to previously published results(21, 22).AFLP reactions and data analysis: FluorescentAFLP kits (Applied Biosystems, USA) were usedfor the whole procedure, which include EcoRI+3 and MseI +3 selective primers (Regular PlantGenomes kit, P/N 4303050), EcoRI+2 andMseI+3 selective primers (Small Plant Genomeskit, P/N 4303051), and EcoRI+0+1+2 andMseI+0+1+2 selective primers (AFLP MicrobialFingerprinting Kit, P/N 402948). Restriction –digestion reactions were performed asrecommended by the manufacturer. Briefly, 50ng of DNA were incubated in 11 µl reactioncontaining 1X T4 DNA ligase buffer containingATP, 50 mM NaCl, 45 µg/ml acetylated BSA, 1µl MseI adaptor, 1 µl EcoRI adaptor, 5 u EcoRI(BioLabs, USA), 5 u Tru9I (Promega, USA), and64 u T4 DNA Ligase (BioLabs, USA), for 2 h at37ºC. After incubation, these reactions werediluted with 189 µl of 20 mM Tris-HCl, 0.1 mMEDTA, pH 8.0. Preselective amplifications wereperformed combining 4 µl of diluted restriction –ligation reaction with 1 µl preselective primersand 15 µl amplification core mix (P/N 402005,Applied Biosystems, USA). Thermal cycling was72ºC x 2 min, 30 cycles of (94ºC x 20 sec, 56ºC x30 sec, 72ºC x 2 min), 60ºC x 30 min, and hold at4ºC. Ten microliters of this reaction were dilutedwith 190 µl of TE as above. Selectiveamplifications reactions were prepared with 3 µlof diluted preselective reaction, 1 µl selective MseIprimer (5 µM), 1 µl selective EcoRI primer(1µl) and 15 µl amplification core mix. Atouchdown thermal cycling protocol was followedaccording to the manufacturer, with the exceptionthat the number of extension cycles at 72ºC wasincreased to 40. All reactions were loaded in aGenetic Analyzer 3130xl using GeneScan 500(ROX) as internal size standard. Detection andsizing of peaks were conducted with GeneMarkerv. 1.91 software (Softgenetics, USA) using thestandard AFLP procedure recommended by themanufacturer (24).Results and DiscussionTo assess the identity of the biologicalmaterial, all the Leishmania strains used for theexperiments were genotyped using PCR – RFLPof heat-shock protein 70 gene. All the strainsanalyzed show HaeIII and BccI patternsconsistent with previous typing results (see Fig. 1for a representative agarose electrophoresisresult).The strategy followed to assess theusefulness of AFLP on Leishmania panamensisFig. 1. Representative image of agarose gel electrophoresisof HaeIII and BccI digestions of hsp70 amplicon for theLeishmania species used at this study. M: size standards;bp: base pairs; P9 and P4: L. panamensis isolates; G: L.guyanensis reference strain; N: undigested hsp70 amplicon;H: HaeIII digestion products; B: BccI digestion products.AFLP analysis of Leishmania panamensis.