Effects of preservation methods, parasites, and gut contents of black flies (Diptera: Simuliidae) on polymerase chain reaction products.
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 > or = 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 amplications. 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.
Koch
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DA
Department of Biology, Nebraska Wesleyan University, Lincoln 68504, USA.
Duncan
G A
GA
Parsons
T J
TJ
Pruess
K P
KP
Powers
T O
TO
D000818:Animals; D004272:DNA, Mitochondrial / Q000302:isolation & purification; D005766:Gastrointestinal Contents; D007814:Larva / Q000235:genetics; D009245:NADH Dehydrogenase / Q000235:genetics; D016133:Polymerase Chain Reaction; D019105:Random Amplified Polymorphic DNA Technique; D012843:Simuliidae / Q000235:genetics* / Q000254:growth & development; D013048:Specimen Handling; D014021:Tissue Preservation / Q000379:methods*
D004272:DNA, Mitochondrial; C098483:NADH dehydrogenase subunit 4; D009245:NADH Dehydrogenase