RELIABILITY OF RAPD FINGERPRINTING OF 3 BASIDIOMYCETE FUNGI, LACCARIA, HYDNANGIUM AND RHIZOCTONIA

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 isol ates. As for the polymerase chain reaction (PCR) from which it was der ived, the conditions necessary for reproducible RAPD products have rec eived attention. However, in contrast to the PCR reaction, the techniq ue relies on non-specific primers and as a consequence the reaction co nditions 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 a nd 17-23 mer primers were visualized on ethidium bromide stained polya crylamide electrophoresis gels. Factorial experiments showed RAPD patt erns were altered by changes in various reaction mixture components in cluding the concentration of non-DNA impurities, the number and concen tration of primers and the DNA polymerase enzyme type, source and conc entration. These factors, particularly the enzyme source, reacted diff erently with the magnesium chloride concentration. Fluorescent dye lab elled primers were used with internal lane standards in a DNA sequenci ng system to assess accurately the molecular weights and relative amou nts of reaction products. Attachment of the fluorescent label appeared to favour the synthesis of some fragments compared with others on pat terns visualized on ethidium bromide stained non-denaturing polyacryla mide gels. The temperature at which DNA was denatured in the first cyc les altered the fingerprints. Reaction mixture temperatures of 94 degr ees or higher, compared with 91-93 degrees, caused loss of visual yiel d of some products, particularly those greater than 500 base pairs, an d increased the yield of others. Reproducibility of RAPD patterns, whe n the reaction mixture and temperature profile factors were varied, wa s facilitated by cross reference to fluorescently labelled bands separ ated with internal lane standards in a DNA sequencing system. Reproduc ibility of fingerprint patterns in a standard reaction mixture was ach ieved, with different thermocycle programmes and in different thermocy clers when the temperature profile was reproduced suggesting that part icular RAPD fingerprints may be reproduced in any laboratory provided the same set of reaction and thermocycle conditions are used.