Reliability of RAPD fingerprinting of three basidiomycete fungi, Laccaria, Hydnangium and Rhizoctonia

Abstract

Randomly amplified polymorphic DNA (RAPD) profiles are currently being developed for Laccaria and Hydnangium species and Rhizoctonia solani. The technique is increasingly being used to differentiate fungal isolates. As for the polymerase chain reaction (PCR) from which it was derived, the conditions necessary for reproducible RAPD products have received attention. However, in contrast to the PCR reaction, the technique relies on non-specific primers and as a consequence the reaction conditions are not necessarily as specific as they are in PCR. Compared with PCR products, RAPD fingerprints were therefore more sensitive to reaction and thermocycle conditions. RAPD products produced using 10 and 17–23 mer primers were visualized on ethidium bromide stained polyacrylamide electrophoresis gels. Factorial experiments showed RAPD patterns were altered by changes in various reaction mixture components including the concentration of non-DNA impurities, the number and concentration of primers and the DNA polymerase enzyme type, source and concentration. These factors, particularly the enzyme source, reacted differently with the magnesium chloride concentration. Fluorescent dye labelled primers were used with internal lane standards in a DNA sequencing system to assess accurately the molecular weights and relative amounts of reaction products. Attachment of the fluorescent label appeared to favour the synthesis of some fragments compared with others on patterns visualized on ethidium bromide stained non-denaturing polyacrylamide gels. The temperature at which DNA was denatured in the first cycles altered the fingerprints. Reaction mixture temperatures of 94° or higher, compared with 91–93°, caused loss of visual yield of some products, particularly those greater than 500 base pairs, and increased the yield of others. Reproducibility of RAPD patterns, when the reaction mixture and temperature profile factors were varied, was facilitated by cross reference to fluorescently labelled bands separated with internal lane standards in a DNA sequencing system. Reproducibility of fingerprint patterns in a standard reaction mixture was achieved, with different thermocycle programmes and in different thermocyclers when the temperature profile was reproduced, suggesting that particular RAPD fingerprints may be reproduced in any laboratory provided the same set of reaction and thermocycle conditions are used.