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

Mycological Research (1999), 103:1361-1368 Cambridge University Press

Copyright © The British Mycological Society 1999Research Article

The generation of specific DNA primers using random amplified polymorphic DNA and
its application to Verticillium dahliae
K.-N. LI a1 , D. I. ROUSE a1 c1 , E. J. EYESTONE a1 and T. L. GERMAN a1
a1
Department of Plant Pathology, University of Wisconsin, Madison WI, U.S.A.

Abstract

A DNA fragment apparently unique to Verticillium dahliae was found by comparing RAPD profiles of V.
dahliae to those of other closely related fungi. RAPD analyses were performed on eight V. dahliae, six V.
albo-atrum and three V. tricorpus isolates to identify DNA sequences specific to V. dahliae. RAPD primer E20
(AACGGTGACC) yielded a 567 bp band shared only by V. dahliae isolates. This band from isolate V14 was
cloned and sequenced. No significant sequence similarity was found between this amplicon and any other
nucleic acid sequence in the databases. Based on the sequence information, a pair of PCR primers, VDS1
(5[prime prime or minute]-CACATTCAGTTCAGGAGACGGA-3[prime prime or minute]) and VDS2 (5[prime
prime or minute]-CCTTCTACTGGAGTATTTCGG-3[prime prime or minute]) was designed. PCR tests showed
that VDS1 and VDS2 amplified the expected fragment of DNA from 62 V. dahliae isolates from diverse hosts
and geographical origins, but not from any other sources of DNA tested, including DNA from the most
closely related species, V. albo-atrum. Southern blot analysis showed that the PCR product specifically
hybridized to V. dahliae genomic DNA. An internal control was constructed for competitive PCR and used to
develop a detection and quantification assay for V. dahliae, for which the detection limit was determined.

http://www.oligo.net/

OLIGO Primer Analysis Software is the essential tool for designing and analyzing sequencing and
PCR primers, synthetic genes, and various kinds of probes including siRNA and molecular beacons.
Based on the most up-to date nearest neighbor thermodynamic data, Oligo's search algorithms find
optimal primers for PCR, including TaqMan, highly multiplexed, consensus or degenerate primers.
Multiple file batch processing is possible. It is also an invaluable tool for site directed mutagenesis.
For each primer or primer pair, Oligo's various analysis windows show a multitude of useful data,
such as DNA and RNA secondary structure, dimer formation, false priming and homology, internal
stability, composition and physical properties. With Oligo you can analyze open reading frames
down to predicted molecular weight and pKa of proteins, and search for restriction enzyme sites, not
only in DNA but also in reverse-translated proteins.
The first version of the software appeared on the market in 1989. It went through several
modifications, and the last one, the change from version 6 to 7, was the most comprehensive. Oligo
7 can automatically select multiplex primers, process sequence files in batch modes, automatically
design PCR primers to cover multiple DNA regions in just one search and automatically find
primer/probe sets for real time PCR or finds nested primers sets. Oligo search protocols (scoring
system) may be customized in detail, so you may optimize the results according to your specific
needs.
Oligo runs on Macintosh and Windows and it may be downloaded from this site. Click on the
Tutorials link above to see Oligo 7 general overview and the major windows screen designs.

A new method for RAPD primers selection based on primer

bias in nucleotide sequence data

J.J. Lia, b, G.L. Peia, c, H.X. Panga, A. Bilderbeckd, S.S. Chene and S.H. Taoa, b, ,

a
School of Life Sciences, Northwest A&F University, Yangling, Shaanxi 712100, China
b
Institute of Bioinformatics, Northwest A&F University, Yangling, China
c
Shaanxi Key Laboratory of Agriculture Molecular Biology, Yangling, China
 d
Department of Experimental Psychology, University of Oxford, England
e
UF Genetics Institute, University of Florida, Gainesville, FL, USA
Received 17 November 2005;

revised 28 April 2006;

accepted 9 May 2006.

Available online 16 May 2006.

Abstract
Sequence analysis has proved that decamer nucleotides, used as primers of RAPD (random amplified polymorphic
DNA), differ with each other greatly in number of annealing sites in the Arabidopsis thaliana genome. It is called the
‘primer bias’ by the authors. The biased primers produce a highly variable number of amplicons by polymerase chain
reaction (PCR). The number of amplicons is proved to correlate with the number of annealing sites. Therefore, a
statistical method is proposed for selecting efficient primers based on the primer bias in the genomic sequence. The
method was tested by experiment in A. thaliana genome, and the results demonstrate that the method outperforms
routine methods and can substantially increase the efficiency of RAPD methodologies. We also proved that the
expressed sequence tags (ESTs) show a highly coincident bias pattern with that of the whole genomic sequence, and
can therefore be used to assess efficiencies of primers for species whose genomic sequence data are currently
unknown.

Keywords: RAPD; Primer bias; Primer selection; Annealing sites