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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.
Current Trends in Biotechnology and PharmacyVol. 5 (2) 1183-1192 April 2011. ISSN 0973-8916 (Print), 2230-7303 (Online)1188Table 2. Number of peaks detected for each combination of selective Mse (+0, =1, +2) primers onLeishmania panamensis.Msel selective primer0-MseI-A, and EcoRI-0-MseI-C, with a rangefrom 7 to 164 peaks (Table 3). The analysis ofseveral isolates allowed us to look at variationsamong L. panamensis isolates and between L.panamensis and reference L. guyanensis. Thesecomparisons revealed that an important proportionof the peaks were reproducibly polymorphic. Thecombinations showing the highest percentages ofpolymorphic peaks for these L. panamensisisolates were EcoRI-AA-MseI-T, EcoRI-T-MseI-0, and EcoRI-0-MseI-T, ranging from 7% to 57%(Table 3). The primer combinations tested showedmany peaks with the potential to clearlydifferentiate between both species. This possibilitywould require, however, the analysis of a highernumber of isolates from both species.These results are not in agreement withthose reported by Kumar et al. (25) forLeishmania donovani, where they found muchhigher proportions of polymorphic peaks and highernumber of alleles when selective +3 primers wereused. Those results, however, are not directlycomparable, as these authors explored differentspecies and did not give details about the allelescoring process. Here we have applied a ratherconservative approach by considering only peaksexceeding 100 fluorescence intensity units toassure reproducibility of electropherograms, evenat the expense of losing potentially useful peaks.In our hands, this procedure gave us good allelecalling reproducibility.Although a limited number of isolates wasanalyzed, our results support the idea that thereis substantial genetic diversity among L.panamensis isolates. As far as we know, this isthe first report on the evaluation of AFLP tocharacterize genetic diversity of Leishmaniapanamensis. Another species of the Vianniasubgenus, Leishmania peruviana, has shownvery low genetic variability when analyzed usingother markers, (26).Polymorphisms observed at AFLP peaksmay be the result of mutations at any of theCarlos M. Restrepo et al
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