Download Procedure - DNA Interactive

Analysis of the Bronze Mutation in Maize
Barbara McClintock's observation that regions of DNA could jump, or "transpose",
would challenge the simplistic view of how a genome was supposed to work. In the mid
1940's most of her colleagues assumed the genome was a static entity- stable genes,
replicating faithfully as cells divided and organisms developed. Observations made by
McClintock at Cold Spring Harbor Laboratory told a radically different story. Although
not immediately accepted by many of her fellow researchers, McClintock's transposable
DNA elements, popularly known as "jumping genes", would offer an explanation to
gene expression patterns in plants that Mendel's Laws failed to provide. 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 later, 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 purple/red 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.
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.
1
Lab Flow and Options
Amplification of the bz-2 locus can be broken into three parts:
I.
II.
III.
Isolating DNA from maize tissue using the Edward's extraction protocol.
Amplifying the bz-2 locus by PCR. During this step, specific reagents are used
to analyze both possible alleles- wild type and Ds knock out.
Analyzing the amplified DNA by gel electrophoresis.
The laboratory is organized by part, with instructor notes, preparation, and experiment
protocol. A separate Results and Discussion is provided for this lab.
The following table will help you plan and integrate the three parts of the experiment:
Part
I. DNA Isolation
Day
1
II. PCR Amplification
2
III. Gel Analysis
3
4
Time
60 min.
30-60 min.
30 min.
30 min.
70+ min.
70 min.
30 min.
30 min
30+ min.
20+ min.
20 min.
20 min.
Activity
Pre-lab: Prepare and aliquot reagents
Isolate maize DNA
Pre-lab: Prepare and Aliqout Reagents
Set-up PCR reactions
Post-lab: Amplify DNA in thermal cycler
Prepare agarose gel solution and cast gels
Load DNA samples into gels
Post-lab: Electrophoresis
Post-lab: Stain gels
Post-lab: De-stain gels
Post-lab: Photograph gels
Procedure I: Isolating DNA From Maize
Reagents
Equipment & Supplies
Shared Items
Edward's Extraction Buffer, 1.5
ml
test
tube, Microcentrifuge
polypropylene
400 l
100-100 µl micropipet and
Isoprpanol, 400 l
tips
Tris/EDTA (TE) Buffer
.1- 20 l micropipet and tips
Disposable pellet pestle
2
Pre-lab Preparation

Plant maize seeds and allow for 2 week growth period
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. Add 400 l of Edward's Extraction Buffer.
3. Grind briefly (to remove tissue from pestle).
4. Vortex 5 seconds; leave at room temperature for 5 minutes.
5. Microfuge for 2 minutes.
6. Transfer 350 l of supernatant to a fresh tube.
7. Add 350 l of isopropanol, mix, leave at room temperature for 3 minutes.
8. Microfuge for 5 minutes, decant, air dry pellet for 10-15 minutes.
9. Resuspend DNA pellet in 100 l of TE Buffer.
10. Template DNA can be used immediatly or stored at -20C.
PART II: AMPLIFYING DNA BY PCR
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, 200 M of each dNTP.
Primer/Loading Dye Mix
This mix incorporates the appropriate primer pair (0.67m/l), 13.9% sucrose, and
0.0082% cresol red in tris-low EDTA (TLE) buffer (10mM Tris-HCl, pH 8.0; 0.1 mM
EDTA).
3
Setting Up PCR Reactions
The lyophilized Taq polymerase in the Ready-To-Go PCR Bead becomes active
immediately upon addition of the primer/loading mix. In the absence of thermal cycling,
“nonspecific priming” allows the polymerase to begin generating erroneous products,
which can show up as extra bands in gel analysis. Therefore, work quickly, and initiate
thermal cycling as soon as possible after mixing PCR reagents. Be sure the thermal
cycler is set and have all experimenters set up PCR reactions coordinately. Add
primer/loading dye mix to all reaction tubes, then add each student template, and begin
thermal cycling immediately.
To insure maximum specificity, some experimenters employ a "hot start" technique
where one reagent is withheld from the reactions until the samples are cycled to the
initial denaturing temperature. You can perform a “hot start” by adding the DNA
template during the first denaturation step. To employ a “hot start”, one can program
an extended first denaturation of 10 minutes, or stop cycling and restart after adding
template. A simpler alternative is to set up reactions on ice, start the thermal cycler,
and then place the tubes in the machine as the temperature approaches the denaturing
set point.
Thermal Cycling
PCR amplification from crude cell extracts is biochemically demanding, and requires the
precision of automated thermal cycling. However, amplification of bz-2 locus is not
complicated by the presence of repeated units. Therefore, the recommended
amplification times and temperatures will work adequately for all types of thermal
cyclers.
To hand amplify, simply set up three constant temperature water baths (or heat blocks)
at 94C, 58C, and 72C. Secure student reactions in a test tube rack, and rotate the
rack successively through the three baths for 30 seconds each. Stop after 30 cycles.
Procedure II: Amplifying Maize DNA by PCR
Reagents
Equipment & Supplies
Primer/loading buffer bz, 22.5 1-20 µl micropipet and tips
l
Primer/loading buffer bz-Ds,
22.5 l
Maize DNA 2.5 l
Ready to Bead (in reaction
tube)
Mineral oil (depending on
thermal cycler used)
4
Shared Items
Thermal cycler
Procedure
1.
Use a micropipet with a fresh tip to add 22.5 µl of the bz primer/loading buffer
mix to a PCR tube containing a Ready-To-Go PCR Bead. Tap tube with finger to
dissolve bead.
2.
Use a micropipet with a fresh tip to add 22.5 µl of the bz-Ds primer/loading buffer
mix to a PCR tube containing a Ready-To-Go PCR Bead. Tap tube with finger to
dissolve bead.
3.
Use fresh tip to add 2.5 µl of maize DNA (from Part I) to each reaction tube, and
tap to mix. Pool reagents by pulsing in a microcentrifuge or by sharply tapping
tube bottom on lab bench.
4.
Add one drop of mineral oil on top of 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 use of mineral oil.
5.
Store all samples on ice or in the freezer until ready to amplify according to the
following profile. Program thermal cycler for 30 cycles according to the following
cycle profile. The program may be linked to a 4°C to hold samples after
completing the cycle profile, but amplified DNAs also hold well at room
temperature.
Denaturing Time --Temp
Annealing Time – Temp
Extending Time – Temp
5
30 sec - 94C
30 sec - 55C
30 sec - 72C
PART III: ANALYZING AMPLIFIED DNA BY GEL ELECTROPHORESIS
Loading and Electrophoresing Samples
The object in these experiments is to let students determine the genotype of the
individual plants. The students can use the ratio of genotypes to determine the
genotype of the parental plant. So it pays to load as many samples possible on each
gel in adjacent wells. This allows students to quickly gauge the genotypic ratio.
However, analysis and sorting out anomalies will be greatly aided by adding at least
one lane of markers per row.
The bz-2 locus is easily resolved in a short run of only 2-3 centimeters. This means you
can double comb most mini-gel boxes with one set of wells at the top of gel and one set
in the middle.
Cresol Red Loading Dye
The cresol red and sucrose in the primer mix functions as loading dye, so that amplified
samples can be loaded directly into gels. This is a nice time saver. However, since it
has relatively little sugar and cresol red, this loading dye is more difficult to use than
typical loading dyes. So encourage students to load very carefully.
DNA Size Markers
Plasmid pBR322 digested with the restriction endonuclease BstN I produces fragments
that are useful as size markers in this experiment: 1,857bp, 1,058 bp, 929 bp, 383 bp,
and 121 bp. Use 20 µl of the DNA ladder per gel.
Viewing and Photographing Gels
View and photograph gels as soon as possible after appropriate destaining. Over time,
PCR products disappear from stained bands as they slowly diffuse through the gel.
Procedure III: Analyzing Amplified DNA by Gel Electrophoresis
Reagents
Equipment & Supplies
2% agarose gel
1.5 ml test tube, polypropylene
1X electrophoresis buffer
pBR322-BstNI markers
1 mg/ml ethidium bromide or
0.05% methylene blue
staining solution
Staining tray or weigh boat
.1- 20 l micropipet and tips
Disposable pellet pestle
6
Shared Items
Electrophoresis chamber
Electrophoresis power
Procedure
1.
Load each PCR reaction in a separate lane on the gel
2.
Use a micropipet with a fresh tip to add 20 µl PCR sample/loading dye mixture
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).
3.
Load 20 µl of the pBR322-BstNI size markers into one lane of gel.
4.
Electrophorese at 130 volts for 20 minutes. Adequate separation will have
occurred when the cresol red dye front has moved at least 50 mm from the wells.
5.
Gels may be stained either with 1 mg/ml ethidium bromide for 10 minutes or
0.05% methylene blue (or proprietary) stain for 30 minutes, followed by 20-30
minutes destaining with water.
7