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Effects Of Preservation Methods, Parasites, And Gut Contents Of Black Flies (Diptera: Simuliidae) On Polymerase Chain Reaction Products, D. A. Koch, G. A. Duncan, T. J. Parsons, K. P. Pruess, T. O. Powers

Department of Entomology: Faculty Publications

Molecular analysis of biological specimens usually requires extraction of high-molecular-weight DNA free of foreign DNA contaminants. DNA was extracted from black flies at different life stages that had been preserved by 4 methods: larvae and adults in ethanol, larvae in Carnoy’s solution, adults on card-points, and adults hand-swatted and sun-dried. Using specific primers for the mitochondrial ND4 gene, a 257-bp amplicon was obtained from specimens preserved by ethanol, card-point mounting, and sun-drying. Successful amplification often required DNA dilutions ≥ 1:20 (<1–10 ng). DNA from specimens preserved in Carnoy’s solution (ethanol: acetic acid, 3:1) yielded degraded DNA, resulting in fewer successful amplifications. Parasitic nematodes and, to a lesser extent, gut contents resulted in extra products when amplified with randomly amplified polymorphic DNA (RAPD) primers. Sufficient DNA was extracted from the head of a larva for a successful polymerase chain reaction (PCR), eliminating the need to remove the contaminating gut and parasites.