Detecting Epigenetic DNA Methylation in Arabidopsis

211403
211403P
211404
211404P
211405
211405P
Detecting Epigenetic
DNA Methylation
in Arabidopsis
Detecting Epigenetic DNA Methylation in Arabidopsis
IMPORTANT INFORMATION
Storage: Upon receipt of the kit, store primer mix, RNAse solution, McrBc enzyme, GTP, BSA, and DNA
marker pBR322/BstNI in a freezer (approximately –20°C). All other materials may be stored at room
temperature (approximately 25°C).
Use and Lab Safety: The materials supplied are for use with the method described in this kit only. Use of
this kit presumes and requires prior knowledge of basic methods of gel electrophoresis and staining of DNA.
Individuals should use this kit only in accordance with prudent laboratory safety precautions and under the
supervision of a person familiar with such precautions. Use of this kit by unsupervised or improperly
supervised individuals could result in injury.
Printed material: The student instructions, pages 5–20, as well as the CarolinaBLU™ and GelGreen™
staining protocols on pages 27 and 28 may be photocopied as needed for use by your students.
Note: The potting soil in this kit cannot be shipped to some states. As a substitute, use Fafard® Seed
Starter Potting Mix if available. Miracle-Gro® Seed Starting Potting Mix may also be used, but the plants
will not grow as vigorously.
DNA
KITS
Learning Center
Copyright © 2013, DNA Learning Center, Cold Spring Harbor Laboratory. All rights reserved.
REAGENTS, SUPPLIES, AND EQUIPMENT CHECKLIST (6 groups)
Included in the kit:
DNA extraction and amplification (all kits):
£ Arabidopsis seeds from Ler (Landsberg erecta) wild
type
£ Arabidopsis seeds from fwa-1 (flowering
wageningen) mutant
£ Planting flat and tray
£ Plastic dome lid
£ Potting soil
£ 12 mL nuclei lysis solution
£ 250 µL RNAse solution, 5 mg/mL
£ 3.5 mL protein precipitation solution
£ 20 mL isopropanol (100%)
£ 30 mL ethanol (70%)
£ 2 mL DNA rehydration buffer
£ 700 µL FWA primer/loading dye mix
£ 15 plastic pestles
£ 25 *Ready-to-Go™ PCR beads in 0.2-mL tubes
£ 130 µL pBR322/BstNI markers (0.075 µg/µL)
£ 200 µL 10x McrBc Buffer
£ 1 mL sterile distilled water
£ 20 μL McrBc enzyme 10 U/µL (store on ice)
£ 15 μL guanosine 5’ – triphosphate (GTP) 100 mM
£ 30 μL BSA 10 mg/mL
£ Mineral oil
£ Instructor’s Manual with reproducible
Student Lab Instructions
**Electrophoresis kits with GelGreen™ stain (Kits 211405
and 211405P) also include:
£ 8 g agarose
£ 150 mL 20x TBE buffer
£ 100 µL GelGreen™ Nucleic Acid Gel Stain
£ 12 gloves
£ 6 staining trays
£ 8 mL 5 M NaCl
Needed but not supplied:
£ Tweezers, scissors, or scalpel
£ Water baths/heatblocks (65°C and 37°C
needed)
£ Micropipets and tips (1 µL to 1000 µL)
£ Microcentrifuge for 1.5-mL tubes
£ 1.5-mL microcentrifuge tubes, polypropylene,
8 per group
£ Microcentrifuge tube racks
£ Thermal cycler, programmable
£ Electrophoresis chambers
£ Electrophoresis power supplies
£ UV or blue light transilluminator (GelGreen™
staining)
£ White light box (CarolinaBLU™ staining,
optional)
£ Camera or photo-documentary system
(optional)
£ Vortexer (optional)
£ Permanent markers
£ Containers with cracked or crushed ice
£ Computer with Internet access
£ Microwave, hot plate, or boiling water bath
**Electrophoresis kits with CarolinaBLU™ staining
(Kits 211404 and 211404P) also include:
£ 8 g agarose
£ 150 mL 20 x TBE buffer
£ 7 mL CarolinaBLU™ Gel & Buffer Stain
£ 250 mL CarolinaBLU™ Final Stain
£ 12 gloves
£ 6 staining trays
*Ready-to-Go™ PCR Beads incorporate Taq
polymerase, dNTPs, and MgCl2. Each bead is
supplied in an individual 0.2–mL tube.
**Electrophoresis reagents must be purchased
separately for Kits 211403 and 211403P.
Copyright © 2013, DNA Learning Center, Cold Spring Harbor Laboratory. All rights reserved.
Detecting Epigenetic DNA Methylation in Arabidopsis
CONTENTS
STUDENT LAB INSTRUCTIONS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5
INTRODUCTION
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5
LAB FLOW
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .7
METHODS
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .8
RESULTS AND DISCUSSION
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .14
BIOINFORMATICS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .15
INFORMATION FOR INSTRUCTOR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .21
CONCEPTS AND METHODS
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .21
LAB SAFETY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .21
INSTRUCTOR PLANNING, PREPARATION, AND LAB FINE POINTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .22
CarolinaBLU™ STAINING . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .27
GelGreen™ STAINING . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .28
ANSWERS TO DISCUSSION QUESTIONS
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .29
ANSWERS TO BIOINFORMATICS QUESTIONS
DNA
KITS
Learning Center
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .30
Copyright © 2013, DNA Learning Center, Cold Spring Harbor Laboratory. All rights reserved.
5
STUDENT LAB INSTRUCTIONS
INTRODUCTION
Although each cell of a multicellular organism carries the same genome,
different sets of genes are expressed in different cell types and at different
times. The spatial and temporal expression of genes must be tightly
controlled for proper growth and development of the organism.
Furthermore, expression patterns must also be stably inherited from
generation to generation. Many aspects of gene regulation are controlled by
specific DNA sequences within or outside genes. These regulatory DNA
sequences include promoters, which are typically located immediately
“upstream” of the genes they control, and enhancers, which may be distant.
In the 1940s, Conrad Waddington, a geneticist and developmental
biologist at the University of Edinburgh, coined the term “epigenetics” to
describe changes in gene expression that are not dependent on the DNA
sequence itself. The prefix “epi” means “on” or “above,” and thus
epigenetics describes genetic modifications that occur “on top of” the
DNA molecule, rather than within it.
Today, epigenetics is a growing field encompassing several different
mechanisms that regulate gene expression, including DNA methylation,
histone modification, and regulatory RNAs. DNA methylation, in which a
methylase enzyme adds a methyl group to a cytosine nucleotide, is an
important epigenetic mechanism in bacteria, animals, and plants.
Typically, methylation of a promoter prevents the binding of transcription
factors that are needed to initiate transcription, and thus “silences” a gene.
Patterns of methylation can be inherited, maintaining correct gene
silencing from generation to generation. The inheritance of an epigenetic
effect through one parental line is termed “imprinting.”
An epigenetic locus (epiallele) is usually maintained in a fully methylated
state, meaning that the corresponding nucleotide is methylated on both
strands of the DNA molecule. During semi-conservative replication, the
methyl group is retained on each of the original strands. Then a methylase
adds a new methyl group to the corresponding nucleotide on the newly
synthesized strands. This explains why methyl modifications (“marks”) are
heritable, even though they do not alter the DNA sequence.
Time to flowering is an
important phenotype in plants.
This experiment relates an
epigenetic genotype of the
FWA gene to a late-flowering
phenotype in the model plant
Arabidopsis thaliana. The FWA
gene encodes a homeodomaincontaining transcription factor
that controls flowering. Normal
flowering requires that both
copies of the gene be silenced
in the tissues of wild-type plants. FWA is silenced by DNA methylation of
repeated sequences in the promoter and 5’ region of the gene. Although
fwa-1 mutants have an identical gene sequence, demethylation of this
region allows transcription of the FWA gene and causes late flowering.
Copyright © 2013, DNA Learning Center, Cold Spring Harbor Laboratory. All rights reserved.
6
Detecting Epigenetic DNA Methylation in Arabidopsis
In this laboratory, wild-type Ler and mutant fwa-1 plants are grown, and
genomic DNA is extracted from normal and late-flowering plants. The
purified DNA is then incubated with the restriction enzyme McrBC. Unlike
classical restriction enzymes, McrBC is modification-dependent,
recognizing and cutting regions with methylated GC and/or AC
dinucleotides. Thus, the methylated, wild-type allele of the FWA gene is
cut by McrBC, but the demethylated mutant allele is not.
The McrBC-digested DNA and uncut controls are then amplified by
polymerase chain reaction (PCR), and the products are analyzed by
agarose gel electrophoresis. Primers spanning the FWA promoter and 5’
repeat region amplify a PCR product from the intact DNA from fwa-1
mutant plants but fail to amplify DNA from the digested wild-type plant.
Kinoshita Y., Saze H., Kinoshita T., Miura A., Soppe W.J., Koornneef M., Kakutani T. (2007).
Control of FWA gene silencing in Arabidopsis thaliana by SINE-related direct repeats.
The Plant Journal 49(1):38–45.
Martienssen R.A., Colot V. (2001). DNA methylation and epigenetic inheritance in plants
and filamentous fungi. Science 293(5532):1070–1074.
Soppe W.J.J., Jacobsen S.E., Alonso-Blanco C., Jackson J.P., Kakutani T., Koornneef M.
(2000). The late flowering phenotype of fwa mutants is caused by gain-of-function
epigenetic alleles of a homeodomain gene. Molecular Cell 6:791–802.
Waddington CH. (2012). The epigenotype. 1942. Int J Epidemiol. Feb;41(1):10-3.
doi: 10.1093/ije/dyr184.
DNA
KITS
Learning Center
Copyright © 2013, DNA Learning Center, Cold Spring Harbor Laboratory. All rights reserved.
Detecting Epigenetic DNA Methylation in Arabidopsis
7
I. PLANT ARABIDOPSIS SEEDS
WATER
seeds
PLANT
seeds
Wild-type
Mutant
2–3 WEEKS
Mutant
Wild-type
Mutant
Wild-type
II. ISOLATE DNA FROM ARABIDOPSIS
ADD
specimen
tissue
sample
ADD
nuclei
lysis
solution
ADD
more
nuclei lysis
solution
GRIND
sample
in
solution
INCUBATE
65°C
15 min
65°C
ADD
RNAse
CENTRIFUGE
4 min
CENTRIFUGE
1 min
TRANSFER
to fresh
tube with
isopropanol
REMOVE
ethanol
CHILL
on ice
5 min
VORTEX
ADD
protein
precipitation
solution
INCUBATE
37°C
15 min
MIX
CENTRIFUGE
1 min
DRY
pellet
10-15 min
ADD
rehydration
solution
ADD
ethanol
REMOVE
supernatant
REHYDRATE
at 65°C for
60 min
or 4°C
overnight
III. DIGEST PCR PRODUCTS WITH McrBC
TRANSFER
DNA
ADD
Restriction
Buffer
Master
Mix
ADD
McrBC
MIX
INCUBATE
in water
bath or
heat
block
STORE
at -20 °C
INACTIVATE
37°C
65°C
IV. AMPLIFY DNA BY PCR
ADD
primer
mix
ADD
mineral oil
(if necessary)
ADD
DNA
AMPLIFY
in thermal
cycler
STORE
at -20 °C
V. ANALYZE PCR PRODUCTS BY GEL ELECTROPHORESIS
POUR
gel
SET
20 min
LOAD
gel
ELECTROPHORESE
130 volts
30 min
Copyright © 2013, DNA Learning Center, Cold Spring Harbor Laboratory. All rights reserved.
Detecting Epigenetic DNA Methylation in Arabidopsis
8
METHODS
I.
To synchronize
germination, prior to
planting, store seeds for 2
days at 4°C.
PLANT AND GROW ARABIDOPSIS
To extract DNA from plant tissue, you must plant the Arabidopsis seeds 2–3
weeks prior to DNA isolation and PCR. You must provide continuous light
from a source no more than 1 foot above the plants.
Reagent
Supplies and Equipment
Arabidopsis seeds from wild type
Ler (Landsberg erecta)
and mutant fwa-1 (flowering wageningen)
Planting flats and tray
Plastic dome lid
Potting soil
water
1. Moisten the potting soil. Divide the flats into their three-celled
sections. Label half of the sections “ler-o” and the rest, “fwa-1.”
Fill planting cells evenly with soil, but do not pack tightly.
Arabidopsis seeds are very
tiny and difficult to handle,
so planting is not as simple
as it may seem.
2. Fit the planting cells into the tray, but leave empty space to facilitate
watering.
3. Carefully scatter seeds evenly on top of the soil. Plant each seed type
in the appropriately labeled sections.
a. Fold a 4- x 4-inch sheet of paper in half.
b. Place the seeds into the fold of the paper, and gently tap them
onto the soil.
c. Provide space between seeds (3⁄4-inch radius around the seeds), so
they will grow better and the plant phenotypes can be readily
observed. Plant approximately 2–3 seeds per planting cell.
Germination requires a
humid environment.
d. Record the date of planting.
4. At an empty space, add ½ inch of water to the tray. Water regularly to
keep soil damp, but do not allow soil to remain soggy.
5. Cover planting cells with the plastic dome lid to assist germination.
(Remove the cover 1–2 days after germination.)
6. Provide a constant (24 hours/day) fluorescent light source about 1
foot directly above the plants.
With 24-hour fluorescent
lighting, the phenotype can
be detected in 2–3 weeks.
7. Measure flowering time for wild-type Ler and mutant fwa-1 plants.
Record flowering time as the number of days from planting to the
emergence of anthers, the male structures producing yellow pollen.
(Examine plants regularly, looking for signs that the plant is
developing a stem. Once the stem begins to emerge, or bolt, check
daily for flowers.)
8. Continue on to Part II as soon as the late-flowering phenotype is
detected in mutant fwa-1 plants.
DNA
KITS
Learning Center
Copyright © 2013, DNA Learning Center, Cold Spring Harbor Laboratory. All rights reserved.

Download Report