RANDOM AMPLIFIED POLYMORPHIC DNA-POLYMERASE CHAIN REACTION BASED DIFFERENTIATION OF SOME SPECIES OF THE GENUS ANOPHELES (CULICIDAE: DIPTERA)

摘要

The present paper deals with the results of RAPD-PCR studies on 6 species of the genus Anopheles viz., AH. stephensi type form, An. fluviatilis sibling species T, An. culcifacies sibling species A, B and C and An. subpictus sibling species A. The genomic DNA was extracted from individual specimens of the species by following the standard ammonium acetate precipitaiton technique and purified by using Sephadex G-25 spin column. Three different random primers with the following base pair composition viz., 10JB- 5'-ACCGCGAAGG-3', 11JB- 5'-GTCCCGACGA-3' and 12JB- 5'-TGATCCCTGG-3' were used for the amplification reactions. The thermocycler was programmed for one cycle of initial denaturation (hot start) at 94°C for 5 min, 45 cycles of denaturation, annealing and extension at 94°C for 1min, 38°C for 1 min and 72°C for 2 min respectively and 1 cycle of final extension at 72°C. The amplified products were then analyzed by agarose gel electrophoresis on 1.2% agarose gel. The base pair lengths of the amplified DNA fragments were calculated by using standard 100 bp DNA ladder (gene ruler) and Quantity One software. The data thus obtained was analyzed in relation to those DNA fragments which were "unique" and "conserved" among all the individuals of a given species. As a result of this, out of 3 primers, only 12 JB was able to amplify conserved fragments from the DNA of all the species covered in the present study. Accordingly, as many as 6 bands with base pair length of 200,411,985. 1230,1292 and 1392 were found to be confined to An. stephensi type form, An. subpictus A, An. fluviatilis T, An. culicifacies C, B and A respectively.