Download procedure - DNA Interactive

Student Protocol
Analysis of Genotype/Phenotype using the Arabidopsis clf-2 mutation
This laboratory uses a rapid method to isolate DNA from plant tissue for genotyping maize by
using the Polymerase Chain Reaction (PCR) method (Edwards, et al., 1991). You will amplify a small
region of the BRONZE (BZ) gene to identify the genotype of the plant with respect to the gene. Maize is
diploid, so a single plant contains two copies of the BZ gene. Thus, there are three possible genotypes
with respect to the CLF gene for any given plant: homozygous-mutated/mutated, heterozygousmutated/wild type, and homozygous-wild type/wild type. If the gene is mutated, amplification of the locus
will produce a 750 bp product. If the gene is in the wild type state, amplification of the locus will produce a
smaller 250 base pair product. Once PCR has been performed, the genotype of a given plant is
determined by using agarose gel electrophoresis to analyze the base pair lengths of the PCR products
amplified from the BZ locus. The genotype can then be compared to the phenotype of the plant from
which the DNA was isolated. The BZ gene encodes a glutathione S-transferase that performs the last
genetically defined step in the maize anthocyanin biosynthesis pathway. Mutations to this gene lead to the
accumulation of an anthocyanin precursor that confers a bronze pigmentation onto the ears and stalk of
corn.
INTRODUCTION
The Discovery of “Jumping Genes”
In the mid 1940's most geneticists assumed the genome was a static entity with stable genes,
replicating faithfully as cells divided and organisms developed. Observations made by Barbara McClintock
at Cold Spring Harbor Laboratory told a radically different story. McClintock observed that regions of DNA
could jump, or "transpose". This observation challenged the simplistic view of how a genome was
supposed to work. McClintock's transposable DNA elements, popularly known as "jumping genes", offered
an explanation to gene expression patterns in plants that Mendel's Laws failed to provide. However, her
work was not immediately accepted by many of her fellow researchers. It took the development of
recombinant DNA methods and the discovery of transposable elements in everything from bacteria to
humans, to give her ideas wide acceptance. In 1983, three decades after her work was first published,
she was awarded the Nobel Prize for her work. Currently, transposable elements are one of the most
powerful tools in the study of plant growth and development.
McClintock's first clue to the existence of mobile DNA elements came from studies of a maize
locus where chromosomal breakage occurred (McClintock, 1951). She called this region of chromosomal
breakage "Dissociator” (Ds). For the Ds element to break from the chromosome, it required the presence
of a second element that she called "Activator" (Ac). Using a classical genetics approach, McClintock was
able to demonstrate how Ac and Ds interacted to affect gene expression. In maize, the wild type corn
kernel is purple. McClintock observed that Ds was not at its normal chromosome location in a strain of
maize that possessed a kernel pigment mutation. Unable to produce the pigment anthocyanin, this mutant
strain produced ears of corn with white kernels instead of purple kernels. When the mutant strain was
crossed to strains containing Ac, Ds transposition caused the wild type kernel color to be restored,
producing purple speckles on a white kernel background. To McClintock, this suggested Ds transposition
could inactivate gene expression by jumping into genes or reactivate gene expression by jumping out of
genes. Furthermore, McClintock observed that Ac has the ability to transpose and cause insertion
mutations. Unlike Ds though, Ac has the ability to jump independently. She hypothesized that independent
mobility of Ac would explain the instability associated with Ac mutations. Due to their ability to control gene
expression, she referred to Ac and Ds as “Controlling Elements”. Years later, her description of the Ac/Ds
system would serve as a model to explain the speckled and variegated gene expression patterns so
common to plants.
The BRONZE (BZ) Gene
This lab will analyze the BRONZE (BZ) gene of maize to demonstrate the molecular relationship between
genotype and phenotype. The Bronze-2 gene encodes a glutathione S-transferase that performs the last
genetically defined step in the maize anthocyanin biosynthesis pathway. Mutations to this gene lead to the
accumulation of an anthocyanin precursor that confers a bronze pigmentation onto the ears and stalk of
corn. Wildtype plants have red/purple anthocyanins located in the cell vacuole, whereas bz-2 mutants
have a tan/bronze color dispersed throughout the cell cytosol (Dooner, et al., 1985).
Place pictures of the bronze and WT maize corn here.
REFERENCES
1. Edwards, K., Johnstone, C. and Thompson, C. (1991). A Simple and Rapid Method for the Preparation
of Plant Genomic DNA for PCR Analysis. Nucleic Acids. Res. 19: 1349.
2. McClintock, B. (1951). Chromosome Organization and Genic Expression. Cold Spring Harbor Symp.
Quant. Biol. 16:13-47.
3. Dooner, H. K., E. Weck, S. Adams, E. Ralston, M. Favreau et al., (1985) A molecular genetic analysis
of insertion mutations in the bronze locus in maize. Mol. Gen. Genet. 200:240-246.
Part I: ISOLATING DNA
PROCEDURE
1. Grind tissue in a microfuge with plastic pestle for 1 minute. Note: It is important to use tissue from
young plants. The quantity is also important. Best results come from using the opposite end of a
1000 µl micropipette to “punch” a whole in the leaf. The resulting leaf disk is the ideal size.
2. Grind the plant tissue forcefully in the microcentrifuge tube using the plastic pellet pestle. Grind for
approximately 1 minute. The sample should look like green liquid when it is fully ground.
3. Add 400 L of Edward's Extraction Buffer to the ground plant tissue.
4. Grind briefly (to remove tissue from the pellet pestle and to liquify any remaining pieces of tissue).
5. Vortex the tube for 5 seconds; leave at room temperature for 5 minutes.
6. Microcentrifuge the tube containing the ground plant tissue and Edward’s buffer for 2 minutes. After 2
minutes any insoluble plant tissue should form a tight pellet at the bottom of the tube.
7. Transfer 350 L of the supernatant to a fresh tube. This supernatant contains the desired DNA. Make
sure not to disturb the pelleted plant tissue when transfering the supernatant.
8. Add 400 L of isopropanol to the DNA containing supernatant, mix, and leave at room temperature for
3 minutes. This step is to precipitate the DNA.
2
9. Microcentrifuge the tube with the isopropanol and supernatant for 5 minutes with the hinge of the tube
against the back wall of the rotor. Carefully remove the supernatant completely. The pellet should be
located on the side that was against the back wall of the centrifuge (the side with the hinge) during the
spin. It will be small and may be very difficult to see. Air dry the pellet for 10 minutes to remove any
remaining isopropanol.
10. After drying, resuspend the DNA pellet in 100 l of TE Buffer.
11. Centrifuge the DNA in TE for 1 minute to pellet any plant material that did not go into solution. You will
use 2.5 L of this supernatant as the template DNA for the PCR reactions.
12. The DNA can be used immediatly or stored at -20C. During use keep the DNA on ice.
PART II: AMPLIFYING DNA BY PCR
Important Note
This procedure uses two PCR reactions to analyze each plant. Since the Ds element insertion
creating the bz-2 mutation is so large, detecting the mutation by amplifying across the Ds element is
impractical. This lab uses an alternative approach involving 2 distinct primer sets. One primer set (bz1/Ds)
contains a primer that anneals to the BZ gene and a primer that anneals to the Ds element at the mutated
bz-2 locus. This primer set amplifies only the mutated allele. The second primer set (bz1/bz2) amplifies
the wild type BZ allele. If the plant demonstrates a normal phenotype, it should have a least one copy of
the wild type allele. Therefore, the primer set amplifying the wild type allele is useful as a positive control
for template DNA quality. It helps insure that any lack of amplification by the first primer set is a result of
the absence of the Ds element and the accompanying mutation and not of a problem with template
quantity or quality. The PCR products from the two primer sets are different in size- 750 bp for the bz1/Ds
primer set and 250 bp for the bz1/bz2 primer set. This allows for easy resolution of the two alleles by 2%
agarose gel electrophoresis.
Include the figure showing the primer location here.
Ready-To-Go PCR BeadsTM
Each PCR bead contains reagents so that when brought to a final volume of 25 L the reaction
contains 1.5 units of Taq polymerase, 10 mM Tris-HCl (pH 9.0), 50 mM KCl, 1.5 mM MgCl2, and 200 M
of each dNTP.
Primer/Loading Dye Mix
This mix incorporates the appropriate primer pair (0.25 picomoles/L of each primer), 13.9% sucrose,
and 0.0082% cresol red in Tris-low EDTA (TLE) buffer (10mM Tris-HCl, pH 8.0; 0.1 mM EDTA).
PROCEDURE
1.
Use a micropipet with a fresh tip to add 22.5 µL of the bz1/bz2 primer/loading buffer mix to a PCR
tube containing a Ready-To-Go PCR Bead. Tap the tube very gently with your finger to dissolve
the bead. Label the tube appropriately.
3
2.
Use a micropipet with a fresh tip to add 22.5 µL of the bz1/Ds primer/loading buffer mix to a
second PCR tube containing a Ready-To-Go PCR Bead. Tap the tube very gently with your finger
to dissolve the bead. Label the tube appropriately.
3.
Use a fresh tip to add 2.5 µL of Maize DNA (from Part I) to each reaction tube. Mix by gently
pipetting up and down. If necessary, pool the reagents by pulsing in a microcentrifuge or by
sharply tapping the tube bottom on the lab bench.
4.
Add one drop of mineral oil to the top of the reactants in the PCR tube. Be careful not to touch the
dropper tip to the tube or reactants, or subsequent reactions will be contaminated with DNA from
your preparation. Note: Thermal cyclers with heated lids do not require the use of mineral oil.
5.
Store all samples on ice or in the freezer until you are ready to amplify according to the following
program. Program the thermal cycler for 30 cycles according to the following cycle profile. The
program may be linked to a 4°C hold program to hold the samples at 4°C after the 30 cycles are
completed.
Denaturation step: Time Temp
Annealing step: Time – Temp
Extension step: Time – Temp
30 sec - 94C
30 sec - 58C
30 sec - 72C
PART III: ANALYZING AMPLIFIED DNA BY GEL ELECTROPHORESIS
PROCEDURE
1. Prepare a 1X concentration of TBE by adding the contents of the bottle of 20X concentrated stock (150
mLs) to 2850 mL of deionized or distilled water. Mix thoroughly.
2. Seal the ends of the gel tray with masking tape and insert the comb. Prepare a 2% agarose gel in 1X
TBE as follows. Add 8 g of agarose to 400 mL of 1X TBE and heat in a boiling water bath
(approximately 15 minutes), on a hotplate, or in a microwave (approximately 5-10 minutes) until the
agarose is completely dissolved. You should no longer see agarose particles floating in solution. Allow
the agarose to cool so that you can touch the container without burning yourself before pouring it into
the gel tray (55-65C). (If boiling hot agarose is poured into the gel trays without cooling to the touch it
shortens the lifetime of the gel trays.) When the agarose has cooled, pour it into the tray to form a gel
approximately one quarter inch thick. Allow the gel to solidify completely. The gel should be cloudy
when it is completely solidified. This takes at least 20 minutes.
3. Place the gel into the gel rig and cover it with 1X TBE buffer.
4. Combine both PCR products (bz1/bz2 and bz1/Ds) into a fresh 1.5 mL test tube. If you used mineral
oil, either remove the mineral oil prior to doing this, or place your pipet tip below the mineral oil to pipet
up the PCR products and leave the mineral oil behind in the original tube. Pipet up and down to mix.
Use a micropipet with a fresh tip to add 30 µL of the PCR sample/loading dye mixture of the two
4
reactions into your assigned well of a 2% agarose gel. (IMPORTANT: Expel any air from the tip before
loading, and be careful not to push the tip of the pipet through the bottom of the sample well).
5. Load 20L of the molecular weight marker (pBR322/BstN1) into one well.
6. Run the gels at 130 V for approximately 30 minutes. Adequate separation will have occurred when the
cresol red dye front has moved at least 50 mm from the wells.
7. Once the loading dye has run the appropriate distance into the gel, stain the gels by soaking them in
stain. If you are using ethidium bromide, stain for 15 minutes. If you are using CarolinaBlu, see the
instructor for instructions. Use gloves when handling ethidium bromide or anything that has
ethidium bromide on it. Ethidium bromide is a known mutagen and care should be taken when
using and disposing of it.
8. Photograph gels
RESULTS AND DISCUSSION
1.
Observe the photograph of the stained gel containing your sample and those from other students.
Orient the photograph with the sample wells at the top. Interpret the band(s) in each lane of the gel.
Use the sample gel pictured below to help you.
750 bp,bz1/Ds
250 bp, bz1/bz2
a. Scan across the photograph of your gel and others as well to get an impression of what you see
in each lane. You should notice that virtually all student lanes contain one or two prominent
bands.
b. Now locate the lane containing the pBR322/BstN I markers. Working from the well, locate the
bands corresponding to each restriction fragment:1,857bp, 1,058 bp, 929 bp, 383 bp, and 121 bp
(may be faint or not visible at all).
c.
The amplification product of the mutant bz-2 allele (750 bp), which has been amplified using the
bz1/Ds primers, should align between the 929 bp marker and the 383 bp marker. The
amplification of the wild-type allele (250 bp), which has been amplified using the bz1/bz2 primers,
should align between the 383 bp marker and the 121 bp marker.
5
d. Establish the exact size of your PCR products as follows.
- Measure the distance that each marker DNA band migrated from the sample well by
measuring from the front of each well to the front edge of each band. The size of the DNA
fragments in your marker are as follows:1857 bp, 1058 bp, 929 bp, 383 bp, and 121 bp.
- Set up semi-log graph paper with the x-axis as the distance migrated by the DNA
fragments and the y-axis (the logarithmic axis) as base pair length of the fragments. Plot
the distance migrated versus base-pair length for each marker DNA fragment and
connect the data points with a line (This line will be referred to as the “standard curve”).
There is a linear relationship between the distance that a DNA band travels and the log of
its molecular weight. Since weight of a DNA fragment is proportional to the number of
base pairs in the fragment, base pairs are frequently used in place of molecular weight
when these types of calculations are done with DNA. Also note that since you are plotting
the molecular weight (in this case base pairs) on the log scale it is not necessary to take
the log of the molecular weight prior to graphing.
- Measure and record the distances migrated by the DNA bands in your PCR reaction. To
determine the size of your DNA bands, first locate the position on the x-axis that indicates
the distance migrated by that band. Then, use a ruler to draw a vertical line from this point
to its intersection with the “standard curve.” Next, extend a horizontal line from this
intersection point to the y axis. The number on the y-axis is the calculated base-pair size
of your PCR product.
d. It is common to see a second band lower on the gel. This diffuse (fuzzy) band is "primer dimer,"
an artifact of the PCR reaction that results from the primers overlapping one another and
amplifying themselves. Primer dimer is approximately 50 bp, and should be in a position ahead
of the 121 bp marker.
f. Additional faint bands, at other positions on the gel, occur when the primers bind to chromosomal
loci other than the bz-2 allele and give rise to "nonspecific" amplification products.
2. How would you interpret a lane in which you observe primer dimer, but not the 750 bp or 250 bp
bands?
3. Based on analysis of the agarose gel, what is the genotype of your plant?
4. Would you classify the bz-2 mutation as recessive or dominant? Explain your reasoning.
6
5.
Based on the genotypic distribution in the class, what genotype was the parental plant? Why may the
genotype distribution observed by your class deviate from what is expected under Mendel’s Laws of
inheritance?
7