2020.4.13

Eurofinsgenomics RAPD 10-mer Kits and Primers
Product Description
Eurofinsgenomics RAPD 10-mer Kits contain 10-base oligonucleotide primers for use in genetic
[1]

[2]

mapping (Williams et al. ) and DNA fingerprinting (Welsh and McClelland ). Eurofinsgenomics
presently has 1,200 different 10-base primers in stock. These primers are sold in kits of 20
sequences each and are designated "Kit A" through "Kit Z," "Kit AA" through "Kit AZ," and "Kit BA"
through "Kit BH." The oligos were selected randomly from a group of sequences with a 60% to 70%(G+C) content and no self-complementary ends.

Principle of Technique
A single 10-base oligonucleotide primer is used to amplify genomic DNA. A DNA amplification
product is generated for each genomic region that happens to be flanked by a pair of 10-basepriming sites (in the appropriate orientation), which are within 5,000 base pairs of each other.
Amplification products are analyzed by electrophoresis. Genomic DNA from two different individuals
often produce different amplification fragment patterns. A particular DNA fragment which isgenerated for one individual but not for another represents a DNA polymorphism and can be used as(1)

a genetic marker. These markers are inherited in a Mendellian fashion . In mapping studies, thesegregation of these markers among the progeny of a sexual cross can be used to construct a
genetic map. In fingerprinting studies, the banding patterns are compared directly to allow strain
determination, usually without the need to correlate band differences with particular properties.

Methods
Each Eurofinsgenomics 10-mer sample tube contains enough primer for approximately 1000
amplification reactions. Each 10-mer sample tube should be resuspended in sterile water or TE
buffer at pH=7. For long term stability, we recommend that you subdivide each 10-mer sample into
several aliquots, dry each aliquot, and store at -20°C. Use clean disposable plasticware for alltransfers.
For DNA amplification, the following conditions are those originally recommended by Williams et al.

(1)

Amplification reactions are performed in a volume of 25 μl containing 10 mM Tris-Cl, pH 8.3, 50 mM
KCl, 2 mM MgCl2, 0.001% gelatin, 100 μM each of dATP, dCTP, dGTP, and dTTP, 5 picomoles [addユーロフィンジェノミクス株式会社〒101-0065
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1 mL, take out 1 μL and add 9 μL of water. Of this solution take 1 μL for the reaction] of a single

10-base primer, 25 ng of genomic DNA, and 0.5 units of Taq DNA polymerase (Perkin Elmer Cetus).
Amplification should be performed on a top quality thermal cycler programmed for 45 cycles of 1
minute at 94°, 1 minute at 36°, and 2 minutes at 72°. Amplification products are analyzed byelectrophoresis in 1.4% agarose gels and detected by staining with ethidium bromide.
More recently published methods recommend an annealing temperature between 33° and 35°.
(9)

Nadeau et al. , have recommended slightly different conditions than those of the original procedure.
Nadeau recommends the use of 25 picomoles [add 200μL, take out 1 μL and add 9 μL of water. Of
this solution take 1 μL for the reaction] of primer and 100 ng template (a fivefold and fourfold
increase over Williams' recommendations, respectively.) These conditions may improve thereproducibility of the results of this method.

Number of Amplification Products
The number of different amplification products for each reaction depends upon the primer sequence,
the genomic sequence, and the genome size. Assuming that the priming sites are randomly
distributed throughout a genome, probability theory predicts that the number of amplification-9

products will be approximately 2.5 x 10 x G, where G is the size of the haploid genome in base pairs.
9For example, lettuce has a haploid genome size of approximately 2 x 10 base pairs, so the above
formula predicts that a typical reaction should yield between 5 and 10 bands, depending on theextent of genetic heterozygosity. This prediction is in close agreement with actual results in lettuce
obtained by Michelmore et al. (5). However, for much smaller genome sizes, such as that of E. coli
6(G = 4 x 10 base pairs), the above formula predicts that most primers should generate no bands atall. Nevertheless, several laboratories have reported multiple amplification products from prokaryotic
DNA. Such results can only be explained on the basis of mismatch between the primer and the DNA(1)

template .

Naming of Primers and Markers
In order to prevent ambiguity in the naming of primers from different sources, Eurofinsgenomics
attaches the prefix "OP" to the names of all of its primers. For example, the fourth sequence in Kit H
is labeled "OPH-04." To refer to a specific polymorphic amplification product, we recommend the(3)convention used by Paran et al. which is to add a subscript denoting its size to the primer name.
For example, an 800 bp amplification product produced by primer OPAC-01 would be called

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"OPAC-01800."

Troubleshooting
Not all amplifications products arise from perfect pairing between primer and DNA template.

Amplification products arising from mis-pairing may still be reproducible and may be useful genetic
markers. However, we suspect that these mismatched markers are more sensitive to slight changes
in the temperature cycle, so we strongly suggest using identical amplification conditions whencomparing results.

The DNA amplification method described above is unusual in that it uses very short (10-base)primers, which have less specificity than longer primers. As a result, this method is quite sensitive tosmall variations in the temperature cycle, particularly the annealing temperature. Since the actual
temperatures delivered to the tubes by different thermal cyclers may differ significantly, it is often
necessary to refine the recommended temperature program in order to optimize this method to yourparticular thermal cycler. If no amplification products are seen, it may be necessary to adjust
temperatures downward. If too many products are seen, it may be necessary to adjust temperatures
upward.
Occasionally, a "smear" of amplification products is observed, and this may be converted todiscretely sized bands by adjusting the concentration of the polymerase, the primer, or the genomicDNA.
It is important that your genomic DNA is relatively free of single strand breaks since such breaks will
prevent amplification. Treat your genomic DNA very gently to prevent shearing. In particular, avoid
repeated boiling or freeze-thawing of your genomic DNA samples.
Although we have chosen oligonucleotide sequences which do not have self-complementary ends,
some of our 10-mers are still capable of forming complicated secondary structures which can lead tothe production of amplification artifacts. These artifacts tend to appear only if one intentionally omits
the genomic DNA to test a "reagent blank." These artifacts have now been directly observed
Fortunately, such artifacts are not produced when genomic DNA is included.

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(10)

.

Uses in Genetic Mapping
Of those kits already tested, our customers report getting useful genetic markers from about 50% to98% of our 10-mer sequences, depending on the species.

A variety of mapping and fingerprinting strategies which employ this technique have appeared in thescientific literature and are listed below (1-10). This technology has been reviewed by Rafalski et(6)al. .

References
1. Williams, J.G.K., Kubelik, A.R., Livak, K.J., Rafalski, J.A., and Tingey, S.V. (1990) DNA polymorphisms
amplified by arbitrary primers are useful as genetic markers, Nucl. Acids Res. 18, 6531-6535.
2. Welsh, J. and McClelland, M. (1990) Fingerprinting genomes using PCR with arbitrary primers, Nucl.
Acids Res. 19, 303-306.
3. Paran, I., Kesseli, R., and Michelmore, R. (1991) Identification of RFLP and RAPD markers linked todowny mildew resistance genes in lettuce using near-isogenic lines, Genome 34, 1021-1027.
4. Martin, G.B., Williams, J.G.K., and Tanksley, S.D. (1991) Rapid identification of markers linked to a
Pseudomonas resistance gene in tomato by using random primers and near-isogenic lines, Proc. Natl.
Acad. Sci. USA 88, 2336-2340.
5. Michelmore, R.W., Paran, I., and Kesseli, R.V. (1991) Identification of markers linked to disease
resistance genes by bulked segregant analysis: a rapid method to detect markers in specific genomic
regions using segregating populations, Proc. Natl. Acad. Sci. USA 88, 9828-9832.
6. Rafalski, J.A., Tingey, S.V., and Williams, J.G.K. (1991) RAPD markers-a new technology for genetic
mapping and plant breeding, AgBiotech News and Information 3, 645-648.
7. Reiter, R.S., Williams, J.G.K., Feldman, K.A., Rafalski, J.A., Tingey, S.V., and Scolnik, P.A. (1992)
Global and local genome mapping in Arabidopsis thaliana by using recombinant inbred lines andrandom amplified polymorphic DNAs, Proc. Natl. Acad. Sci. USA 89, 1477-1481.
8. Woodward, S.R., Sudweeks, J., and Teuscher, C. (1992) Random sequence oligonucleotide primers
detect polymorphic DNA products which segregate in inbred strains of mice, Mammalian Genome 3,73-78.
9. Nadeau, J.H., et al. (1992) Multilocus markers for mouse genome analysis: PCR amplification basedon single primers of arbitrary nucleotide sequence, Mammalian Genome 3, 55-64.
10. Higuchi, R., Fockler, C., Dollinger, G. and Watson, R. (1993) Kinetic PCR analysis: real-timemonitoring of DNA amplification reactions, Bio/Technology 11, 1026-1030.

ユーロフィンジェノミクス株式会社〒101-0065
東京都千代田区西神田3丁目8-1 千代田ファーストビル東館12F
TEL 03-6631-0105 Web : www.eurofinsgenomics.jp