Using Random Amplified Polymorphic DNA (RAPD) Analysis to Examine Phylogenetic Distance Among Different Plant Species



Background
 

Molecular markers are commonly used by plant biologists to perform a number of tasks, including the genetic fingerprinting of plant varieties, determining similarities among inbred varieties, mapping of plant genomes, and establishing phylogeny among plant species. New techniques for the extraction, purification, and amplification of plant DNA are being developed on a regular basis, enabling researchers to decrease preparation time and obtain readily reproducible results. Plants can now be compared at the molecular level in several ways, via examination of restriction fragments, identification of isoenzymes (protein/gel electrophoresis), or products of the polymerase chain reaction (PCR).
 

One technique which can provide useful data for the comparison of plant types is random amplified polymorphic DNA (RAPD). This is a modified PCR technique involving the amplification of whole-plant DNA extracted from leaves or other plant organs. In the RAPD technique, multiple 10 base pair (bp) oligonucleotide primers are added each to an individual sample of DNA which is then subjected to PCR. The resulting amplified DNA markers are random polymorphic segments with band sizes from 100 to 3000 bp depending upon the genomic DNA and the primer. This technique is sensitive, fast, requires the use of no radioactive probes, and is easily performed. RAPD markers are limited in their usefulness, however, in that they are dominant, so it is necessary to prepare many closely linked markers to insure reliable comparisons among plant populations.
 
 
 

Materials
 

DNA Extraction:

21 sterile microcentrifuge tubes                                             spinach
microcentrifuge                                                                     leaf lettuce
3 clean plastic pestles                                                           iceberg lettuce
1000 ml pipetman                                                                 romaine lettuce
200 ml pipetman                                                                   isopropanol
20 ml pipetman
1000 ml pipetman tips
0.5-10 ml ultra micro tips
extraction buffer (100 mM Tris, 500 mM KCl, pH 8.8)

PCR:

5' TCACCACGGT 3' primer                                                Taq polymerase
dNTPs                                                                                  45 - 1 ml tubes
amplification buffer                                                                15 - 0.5 ml  tubes
sterile deionized water                                                           1.5 mM MgCl2
thermal cycler

Electrophoresis:

50X TAE buffer
1X TAE buffer (5 ml 50X TAE / 250 ml DiH2O)
DNA grade agarose (1% gel = 1g / 99 ml 1X TAE)
ethidium bromide (5 ml / 50 ml gel)
transluminator
 

Procedure

A. DNA Extraction
 

1. Add 400 ml extraction buffer into a 1.5 ml sterile Eppendorf tube.

2. Use the lid of the Eppendorf to pinch out a disk of leaf material into the tube.

3. Homogenize the leaf tissue with a pestle that fits the tube tightly. Keep pestle clean!

4. Centrifuge extracts in a micro centrifuge for 1 minute, and transfer 300 ml of the supernatant
    to a fresh tube.

5. Add one volume (300 ml) of isopropanol, to precipitate the DNA for 5 minutes, then
    centrifuge for 5 minutes.

6. Discard the supernatant and air dry the sample completely (about 30 minutes on the bench).

7. Add 100 ml of H2O and allow the DNA to dissolve for 10 minutes or longer without agitation
    (recovery of DNA is improved by dissolving at 4o overnight).

8. Centrifuge sample for 1 minute and collect the supernatant. There should be enough DNA for
    direct RAPD analysis. This can vary and can be measured with a DNA dipstick (Invitrogen),
    or by running agarose gels with standard DNA samples. (note: samples containing extracted
    DNA in amounts greater than ~5 ng / ml or more than the recommended amount of Taq may
    produce smears rather than useful bands).

9. DNA can be stored at 4o C for at least 6 months, or longer at -20o C.

B. PCR Protocol

Unlike normal PCR which uses two, RAPD only uses one primer with an arbitrary sequence. Therefore, amplification in the RAPD process occurs anywhere along a genome that contains two complementary sequences to the primer which are within the length limits of PCR (~3 kb). These protocols work well for random 10-mers.

Each group will prepare three PCR primer reactions as follows.

1. Mix the following in a 0.5 ml microfuge tube:

Materials                                                (ml)                        Final Concentration

10X amplification buffer                                           2.5
0.5 mM dATP, 0.5 mM dTTP pH 7.0                      5.0                             0.1 mM each dNTP
0.5 mM dCTP, 0.5 mM dGTP solution
3 mM primer solution                                                3.0                             0.36 mM
Genomic DNA                                                         2.0                             25 ng/reaction
Taq DNA polymerase (0.75 units/ml)                        0.2                             1.5 units/reaction
sterile H2O                                                             10.5

2. Centrifuge for approximately 20 seconds to mix.

3. Layer 50 ml mineral oil on the top of each tube to prevent evaporation.

4. Place samples in the thermocycler. Cycle at 94oC for 1 minute, 36oC for 1 minute,
    72oC for 2 minutes. Run the reaction for 45 cycles (approximately 5 hours to complete).

C. Agarose Gel Electrophoresis Procedure

1. Prepare electrophoresis buffer and fill the electrophoresis tank.

2. Prepare casting mold by wrapping masking tape around the sides, making sure to fold the tape
    to cover the bottom of the plastic mold to prevent leaks.

3. Prepare agarose gel at desired concentration. Melt in microwave until boiling. Once agarose
    is dissolved, allow to cool to ~45oC before pouring (1 to 1.5 min.). Add 10 ml ethidium
    bromide to hot agar and swirl to mix just prior to pouring.

4. Insert comb and make sure no bubble are caught under the teeth. Remove comb GENTLY
    after gel hardens by pulling straight up. Remove tape, but leave the gel slab in the mold.

5. Place the gel in the electrophoresis tank and add sufficient buffer to cover it to a depth of
    about 1 mm.

6. Prepare DNA samples by mixing 25 ml of the PCR reaction mixture with 4 ml of the 6X
    loading buffer in fresh Eppendorf tubes.

7. Load samples into the wells in the gel with a micropipet (use a new tip with each sample),
    taking care not to cross-contaminate the wells.

8. Include a molecular marker (1 Kb ladder).

9. Set the voltage on the power supply to ~55 V and attach top cover, making sure that the
    positive and negative poles are placed correctly (wells are closest to the negative pole,
    DNA will migrate to the positive pole).

10. When the bromothymol blue marker dye has migrated to the mid-point of the gel, turn off
      the power and remove the gel.

11. Visualize DNA patterns by removing the gel slab from the mold and placing it on a
      UV transluminator. Record the pattern by photography or computer image capture.

Determining Relatedness/Identity of Plant Genotypes and Varieties
 

You can use many different computer programs, such as RAPD, PHYLIP, or PAUP to run relatedness analyses and produce trees. I am currently developing a method using the RAPD analysis programs.