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 Buffer
Materials Amount Final Concentration
1 M Tris-HCL, pH 7.5
10.0 ml
200 mM
5 M NaCl
2.5 ml
250 mM
0.5 M EDTA
2.5 ml
25 mM
10% SDS (sodium dodecyl sulfate)
2.5 ml
25 mM
Water
32.5 ml
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
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).
Example Data Matrix:
| Band | Sample
1 |
Sample
2 |
Sample
3 |
Sample
4 |
| 1 | 1 | 0 | 1 | 1 |
| 2 | 1 | 0 | 0 | 0 |
| 3 | 1 | 1 | 0 | 0 |
| 4 | 1 | 0 | 1 | 0 |
| 5 | 1 | 1 | 0 | 0 |
| 6 | 1 | 1 | 0 | 0 |
| 7 | 1 | 1 | 0 | 0 |
| 8 | 1 | 1 | 1 | 1 |
| 9 | 1 | 1 | 1 | 0 |
| 10 | 1 | 0 | 0 | 1 |
| 11 | 1 | 0 | 0 | 0 |
| 12 | 1 | 1 | 1 | 0 |
| 13 | 1 | 1 | 0 | 1 |
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 above),
a name
for each sample (such
as A, B, C, etc.), the number of "populations" (in our experiment
we will have one 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 above, 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 (example, lettuce.dat).
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
phenogram (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
1. Which of the species are most
closely related? Which are the most distantly related?
2. Could this technique be adapted to determine relatedness of other plant species? How?