RAPD-PCR is a wide-spread method for genetic fingerprinting of eukaryotes. Its major advantage is that absolutely no sequence information of the target genome is required.
Essentially all RAPD-Primers will give you a characteristic fingerprint of any target genome. Band numbers normally range between 2 and 10.
If fingerprinting is all you need, say for unequivocal recognizing natural isolates of a microorganism, you need not play around too much. Application of 3-6 random primers will normally do.
If you want to see correlations between RAPD band patterns and defined traits of your target organism some more efforts are required. A good advice is to start with 20 different primers and something like 6 target organisms of every phenotypic group that you need to characterize.
You may use any oligonucleotide as RAPD primer above a chain length of 9. It is essentially the annealing temperature of the PCR that defines the pattern, not so much the length of the primer.
For generating random sequences of a given chain length, you may use the 'RAPD-generator' in the Java applet on this page, written by Gebhard Wöstemeyer
. Do not rely on imagination - sequences by the 'RAPD-generator' are much more random than your own.
Good results were obtained with the following random primers for fungal DNA:
primer 6: 5´-GAAACAGCGG-3´
primer 8: 5´-GGAGCCCAC-3´
primer 14: 5´-GCCGTCTACG-3´
primer 17: 5´-GGCATCGGCC-3´
primer 21: 5´-GTGAGCGTC-3´
RAPD amplification mixtures contain in 50 µl
- approximately 25 to 50 ng of genomic DNA
- 40 ng nona- or decamer primer
- 16 mM (NH4)2SO4
- 50 mM Tris-HCl pH 8.8 (at 25 °C)
- 0.1% Tween 20
- 0.2 mM of each dNTP
- 3 mM magnesium chloride
- 1 U Taq Polymerase
We use the following temperature profile:
- initial denaturation step: 5 min -95 °C
- 30 cycles of:
primer annealing: 60 s - 32 °C
primer extension: 20 s - 72 °C
DNA denaturation: 20 s - 95 °C
- cool down to 20 °C
Amplicons are separated electrophoretically in 1.2 % agarose gels in 1 x TAE or for higher resolution in 5 % native polyacrylamide gels (5% w/v acrylamide, 0.17% w/v bisacrylamide, 0.08% w/v ammonium peroxodisulfate, 0.05% v/v TEMED) in 1 x TBE buffer for 1.5 h at 8 V/cm.
We have used RAPDs since 1992 for molecular diagnosis of plant pathogenic fungi. For background information and experimental details you may refer to the following publications.
Copies are available on request to Kerstin Voigt
or Johannes Wöstemeyer
- Schäfer C, Wöstemeyer J (1992) Random primer dependent PCR differentiates aggressive from non-aggressive isolates of the oilseed rape pathogen Phoma lingam (Leptosphaeria maculans). J Phytopathol 136:124-136
- Schäfer C, Wöstemeyer J (1994) Molecular diagnosis of the rapeseed pathogen Leptosphaeria maculans based on RAPD-PCR. In: Schots A, Dewey FM, Oliver R (eds) Modern assays for plant pathogenic fungi. CAB International Oxford, pp 1-8
- Voigt K., Schleier S., Brückner B. (1994) Genetic variability in Gibberella fujikuroi and some related species of the genus Fusarium based on random amplification of polymorphic DNA (RAPD). Curr Genet. 27: 528-535
- Voigt K., Wöstemeyer J. (1995) The combination of Gilbert/Maxam chemical sequencing and the dideoxynucleotide chain termination approach facilitates the construction of species-specific PCR primers based on diagnostic RAPD bands. Microbiol. Res. 150: 373-377.
- Schleier S., Voigt K., Wöstemeyer J. (1997) RAPD-based molecular diagnosis of mixed fungal infections on oilseed rape (Brassica napus): evidence for genus-and species-specific sequences in the fungal genomes. J. Phytopathology 145: 81-87
- Voigt K, Schleier S., Wöstemeyer J. (1997) Molecular diagnosis of rape seed pathogens. An experimental strategy for the development of taxon-specific genetic markers. In: Developments in Plant Pathology 11: Diagnosis ans Identification of Plant Pathogens (H.-W. Dehne et al., eds) Kluwer Academic Publishers, pp 195-198
- Voigt K., Schleier S., Wöstemeyer J. (1998) RAPD-based molecular probes for the blackleg fungus Leptosphaeria maculans (Phoma lingam): Evidence for pathogenicity group-specific sequences in the fungal genomes. J. Phytopathology 146: 567-576
- Voigt, K., Jedryczka, M., Wöstemeyer, J. (2001) Strain typing of Leptosphaeria maculans supports at the genetic level the multi-species concept of aggressive and non-agressive strains. Microbiol. Res., in press