The Long-PCR technique has recently proven to be a useful tool for the amplification of insect mitochondrial genomes based on a single PCR reaction (One-Step) or the amplification of large overlapping fragments (2-Steps or more). Successful whole mitochondrial genome amplification trough Long-PCR has important implications for both molecular and organismal evolutionary studies based on mitochondrial DNA analysis. This work provides an overview on Long-PCR primers construction and the usefulness of this approach for the analysis of Haematobia irritans mitochondrial genome. Long-PCR primers were designed based on 16S rDNA conserved sequences of dipteran species. H. irritans specific primers were combined with “universal” primers previously described for the insect mtDNAs, yielding two overlapping fragments of 8.5 and 9.1 Kb, which recovered the complete mitochondrial DNA molecule based on a 2-steps Long-PCR strategy. This 2-steps amplification reaction efficiently recovered the 9.1 Kb mtDNA fragment in other medical and veterinary important species such as Musca domestica (House fly), Cochliomya hominivorax (Screwworm fly), Chrysomya megacephala and Chrysomya putoria (both blowflies). Optimization of the One-Step amplification reaction of Haematobia irritans mtDNA is being conducted in order to provide an additional strategy for the amplification of the entire genome. This approach may be useful for further population genetics studies based on mtDNA markers and species-specific molecular identification of important pest species.
Financial support: CNPq/PROFIX ; FAPESP.
Species 1: Diptera Muscidae Haematobia irritans (Horn-fly, Mosca-dos-chifres)
Keywords: Long-PCR, Mitochondrial DNA
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