Vilgalys Lab, Duke
University
RAPD (Random Amplified Polymorphic DNA) markers were originally designed for use in the genetic mapping technique described by Williams et al. (1). The basic RAPD strategy employs a single, simple (10-15 base) oligonucleotide primer to amplify genomic DNA using the polymerase chain reaction. A particular short arbitrary sequence is likely to occur frequently in the genome; PCR products are produced in regions of the genome flanked by two internally oriented primer sites within 5,000 base pairs of each other. 5000 bp is the practical limit of PCR. Amplification products are analyzed by electrophoresis on conventional agarose gels. Because the PCR is dependent on proper positioning of primer sites along the genome of different individuals, RAPD markers may be used as genetic markers and for "fingerprinting" of genetically unique individuals. The most common use of RAPDs has been for construction of genetic maps using Mendelian progeny. Other applications in systematics and population genetics are also possible. Depending on the species, from 50% to 98% of most 10-base primers should yield PCR products which are useful as genetic markers.
Primers for screening RAPD markers may be synthesized privately, however, one reliable commercial source is Operon Technologies, Inc., which has 500 random primers available (1000 Atlantic Ave, Suite 108, Alameda CA 94501, tel 415-865-8644, fax 415-865-5255). Their primers are sold in kits of 20 sequences per kit. Almost any short oligonucleotide may be used for amplification of RAPD markers. To be generally useful for a variety of species, however, primers should conventionally have a (G + C) content between 60-70%, and have no self-complimentary ends.
RAPD reactions are typically performed in a total volume of 25 µl, containing 10 mM Tris-CI, pH 8.3, 5O mM KCI, 2 mM MgCI2, 0.001% gelatin, 100 µM each of dATP, dCTP, dGTP, and dTTP, 15 ng of a single RAPD primer, 25 ng of genomic DNA template, and 0.5 units of Taq DNA polymerase (Perkin Elmer Cetus). Amplification should be performed on a good quality thermal cycler programmed for 45 cycles (1 minute at 94 C,1 minute at 36 C, and 2 minutes at 72 C) using the fastest available transitions between each temperature. Amplification products are analyzed by electrophoresis in 1.4% agarose gels and detected by staining with ethidium bromide.
Recipes for RAPDs
The following protocol employs primers supplied by Operon along with the GenAmp core kit supplied by Perkin-Elmer/Cetus. The enzyme (Amplitaq) is from Perkin-Elmer/Cetus. Genomic DNA for PCR is obtained from minipreps, diluted to 0.1 ng/µl in distilled water.
The following recipes make a total of 200 µl worth of reactions, which is enough for 8-20 individual reactions depending on how you slice it:
1. Thaw all the reagents, primer stocks and templates (except the enzyme) on ice (if at room temp, then use immediately and replace in freezer a soon as possible).
2. For each primer mix in the following
order (be sure that the cocktail is mixed before adding the Amplitaq:
| Recipe | |
| GenAmp kit | |
| dH20 | 34 µl |
| 10X PCR buffer | 20 µl |
| 25 mM MgCl2 | 20 µl |
| dNTPs | 4 x 4 µl of each |
| primer (16.5 µg/ml) | 8 µl |
| Amplitaq (2.5 U/µl | 2 µl |
| total volume | 100 |
| Total
per individual reaction
(cocktail + DNA) |
1
part cocktail
+ 1 part DNA |
Mix briefly, and dispense the cocktail to the bottom of the appropriate number of labelled 500 µl PCR tubes.
3. Add the appropriate amount of diluted template DNA to each tube (see table for ratios). Then add 1 drop sterile light mineral oil. Cap and spin briefly to bring down the reagents.
4. Prepare slots in thermocycler by adding one drop of mineral oil. Place tubes into slots. Run the thermocycler. (The following instructions are for the Mycology Lab Perkin Elmer Thermocycler: Turn on machine. Select "file", then punch in number "11", followed by "enter", followed by "start". After 45 cycles (about 5 hrs) the machine will link to a 4 C soak cycle.)
5. After the PCR, add 2-3 µl of 10X loading buffer to the side wall of each tube. Cap tubes, and "quick-spin" down the loading dye to the bottom of the tubes. Vortex or flick each tube to mix in the loading dye, and briefly centrifuge again to bring everything down.
6. Electophoresis: prepare a 1.4% agarose gel (300-400 µl for large gels) in 1X TBE buffer with 0.5 µg/ml EthBr. Load the entire sample into a single well on the gel. (Hint: Unless you plan to do other things, at this stage its OK to use a single yellow pipet tip to load all of the samples). Using a clean pipet tip, load 20 µl of MW markers into one or more lanes (lambda EcoRI/Hind-III digested DNA). Run gels at 75-100 Volts for 2-3 hrs until bands are visible.
7. Photograph and label gels with date, names of primers, etc., when done.
RAPD marker nomenclature
Primers made by Operon are identified by the letters "OP" in front of the names of each primer series (A-Y). For example, the fourth sequence in Operon Kit H is labelled "OPH-04". One convention used for naming specific amplification products recommended by Paran et al (2) is to adda subscript denoting the marker size to the name of the primer. For example, an 800 bp amplification product produced by primer OPC-06 would be called OPC-06800.
Trouble-Shooting with RAPDs
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RAPD references
1 . Williams, J.G.K., Kubelik, A.R., Livak, K.J., Rafalski, J.A., and Tingey, S.V. (1990). DNA polyrnorphisms amplified by arbitrary primers are useful as genetic markers. Nucl. Acids Res. 18: 6531-6535.
2. Paran, I.,Kesseli, R.,and Michelmore, R. (1991). Identification of RFLP and RAPD markers linked to downy mildew resistance genes in lettuce using near-isogenic lines. Genome, in press.
3. 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.
4. Michelmore, R.W., Paran, I.,and Kesseli, R.Y. (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, in press.
5. Welsh, J., and M. McClelland (1990). Fingerprinting genomes using PCR with arbitrary primers. Nucl. Acids Res. 18:7213-7218.
6. Hedrick, P. (1992). Shooting
the RAPDs. Nature 355: 679-680.