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) analysis. 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 alleles, so it is necessary to prepare many closely linked markers to insure reliable comparisons among plant populations.
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
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
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)
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 C 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:
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 94o C for 1 minute, 36o C for 1 minute, 72o C for 2 minutes. Run the reaction for 45 cycles (approximately 5 hours to complete).
C. Agarose Gel Electrophoresis Procedure (1% 50 ml gel)
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 ~45o C before pouring (1 to 1.5 min.). Add 5 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
A. Data Entry
RAPD produces a large number of DNA bands of various sizes from each of the different samples which are prepared. These bands migrate according to size during electrophoresis. To analyze RAPD data, one must first count the total number of unique bands (thus if several lanes share a band, that band is only counted once toward the total) for each primer used. In this lab, we only use one primer, so this task is quite simple. Then, the presence or absence of each individual band is recorded for each lane on the gel representing a different plant sample. The easiest way to do this is to construct a matrix on paper with each sample representing one column and each band in one row. The presence of a band is recorded as a one (1) and the absence as a zero (0; see example below).
Example Data Matrix
The data can now be entered into the RAPDistance program as follows:
1. Start the program from DOS by entering C:\RAPD>RAPDIN, or by clicking on the RAPDIN icon on the Windows desktop.
2. You will be prompted to enter a name for your new datafile, then the number of samples (4 in the example below), a name for each sample (such as A, B, C, etc.), the number of "populations" (in our experiment we will have 1 population represented by four different plant species), the number of primers, the name of each primer, the length of each primer (the number of nucleotides), and the total number of bands produced by each primer (if thirteen bands are the maximum produced, this number would be 13.
After entering the data, the program will then prompt you to record whether each band is present or absent for each sample. You will enter a "1" for present, and a "0" for absent. At the completion of the data entry process, the data file will be stored as "filename.dat" where the filename is the name you gave previously .
3. Exit the program (in Windows, the DOS box will automatically terminate).
B. Producing the Genetic Distance Profile
1. From DOS, type in C:\RAPD\ RAPDALG, or click on the RAPDALG icon on the Windows desktop. When prompted, enter the name of the data file (lettuce.dat). The program will produce a new file, lettuce.njt which can be used to produce a dendogram (branching tree showing relatedness of each sample) based on genetic distance.
2. Exit the program.
3. From a text editor (Windows Write, MS Word, WordPerfect, etc.) find the file Tdraw.asc in the RAPD folder. When you open the file, you will see that it is a genetic distance dendrogram based on the data you entered.
Questions for Discussion
1. In this lab, one of the four samples examined exhibited the greatest genetic distance from the other three. Which sample was this? Why is the distance so great?
2. Which of the four samples are the most closely related?
NSF Award #9850325