Ic. Tommerup et al., RELIABILITY OF RAPD FINGERPRINTING OF 3 BASIDIOMYCETE FUNGI, LACCARIA, HYDNANGIUM AND RHIZOCTONIA, Mycological research, 99, 1995, pp. 179-186
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.