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)1187Digestions of Leishmania genomic DNAwith EcoRI and Tru9I (data not shown) showedthat most of the digested DNA was at sizes higherthan the useful range described for AFLP (50-500 base pairs). These higher than expected sizeswere anticipated due to the high GC content ofthe Leishmania genome and the fact that therecognition sites for these enzymes are AT richsequences. We proceeded to AFLP reactionsoptimizations by doing the selective primersscreening with one of the L. panamensis isolatesto cover all the possible combinations of availableEcoRI and MseI primers. The +3 and +2combinations (corresponding to the Regular PlantGenomes and Small Plant Genomes AFLP kits)generally yielded low numbers of peaks (Table1), consistent with the results of the restrictionanalysis of genomic DNA and the characteristicsof the Leishmania genome. Thus, further analysisincluded several L. panamensis isolates (P5, P6,P7, P8 and P9) and one reference L. guyanensisstrain, and were only done with the +2, +1 and +0corresponding to the Microbial FingerprintingAFLP kit. Although this kit contained 81 possibleselective primer combinations, we used only the67 producing more than ten (10) clear peaks(Table 2).The primer combinations producing thehighest numbers of bands on L. panamensis wereEcoRI-0-MseI-A, EcoRI-0-MseI-T, EcoRI-0-MseI-G, and EcoRI-C-MseI-0 (Table 3).Unexpectedly, some primer combinations renderedtoo few or no bands at all. The primercombinations yielding the highest cumulativenumber of bands were EcoRI-G-MseI-0, EcoRI-Table 1. Number of peaks detected for each combination of selective (- 3) primers on Leishmaniapanamensis.Msel selective primerAFLP analysis of Leishmania panamensis.