Maize Karyotyping and FISH Manual
Foreword
This manual contains the methods used by the Birchler lab for maize
karyotyping and in situ hybridization to chromosomes at the University of
Missouri. These procedures have evolved from those developed by Kato et al.
(2004). Contributions to the methods and the manual were made by Akio Kato,
Jonathan Lamb, Matthew Bauer, Weichang Yu, Leah Roark, Fangpu Han,
Patrice Albert, Tatiana Danilova, Zhi Gao, Seth Findley, and Robert Gaeta. The
particular reagents noted in the text are not an endorsement of particular brand
names. In fact, in our experience, there is great latitude in the reagents that
work in these procedures. Citation to these methods should be to the primary
literature. The methods are described in the references listed at the end of the
narrative.
Revised June, 2015 by Patrice Albert
Contents
Page
Fluorescence in situ Hybridization . . . . . . . . . . . . . . . . . 3
Preparation of DNA sequences for labeling . . . . . . . . . . . . 4
Mini-gel electrophoresis. . . . . . . . . . . . . . . . . . . . . 7
Probes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
Enzyme stock solutions. . . . . . . . . . . . . . . . . . . . . . 9
Direct labeling -- Small chromosomal targets. . . . . . . . . . .10
Direct labeling -- Large chromosomal targets. . . . . . . . . . .12
Processing non-column-purified probes . . . . . . . . . . . . . .14
Preparation of metaphase spreads from root tissue . . . . . . . .15
Signal detection of directly labeled probes . . . . . . . . . . .18
Pachytene FISH . . . . . . . . . . . . . . . . . . . . . . . . . . .22
Single Gene/Small Target Detection . . . . . . . . . . . . . . . . .24
Retroelement Genome Painting . . . . . . . . . . . . . . . . . . . .26
Method for Pollen FISH in Maize. . . . . . . . . . . . . . . . . . .27
Endosperm Chromosome and Nuclei Spreads. . . . . . . . . . . . . . .28
A Cocktail of Many Colors. . . . . . . . . . . . . . . . . . . . . .30
5' End-labeled Oligos as FISH Probes . . . . . . . . . . . . . . . .32
Developing a New Karyotyping Cocktail for Another Plant Species. . .34
Rolling Circle Amplification of BAC DNA for Nick-translated Probes .36
Alternate Strategy for Labeling of Small (Repeat) Probes . . . . . .41
Preparation of Metaphase Spreads from HiII Embryonic Type-II Callus.42
Getting Good Spreads from an Adult Corn Plant. . . . . . . . . . . .44
Streamlined FISH Protocol for Large Targets. . . . . . . . . . . . .45
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . .46
Appendices:
A. Maize probe cocktail plasmids. . . . . . . . . . . . . . . . .47
B. Maize probe cocktail DNA . . . . . . . . . . . . . . . . . . .48
C. Minimization of background . . . . . . . . . . . . . . . . . .49
D. Kits and reagents. . . . . . . . . . . . . . . . . . . . . . .50
E. Source information . . . . . . . . . . . . . . . . . . . . . .51
F. Solutions. . . . . . . . . . . . . . . . . . . . . . . . . . .52
G. Nitrous oxide gas chamber. . . . . . . . . . . . . . . . . . .54
2
Fluorescence in situ Hybridization
The Basics
The FISH (fluorescence in situ hybridization) process is analogous to that of a Southern
hybridization. The general process of FISH is presented below. The third step is optional;
the fourth will improve the signal-to-background ratio for red- and far-red-labeled probes.
Step 1.
A m p lif ic a t io n o f p r o b e D N A
sequence by PCR
↓
Step 2.
L a b e l D N A b y n ic k
t r a n s la t io n
↓
Step 3.
E v a lu a t i o n o f p r o b e q u a lit y
b a s e d o n D N A f r a g m e n t s iz e
↓
Step 4.
P u r if ic a t io n o f p r o b e
↓
Step 5.
H y b r id iz a t io n
↓
Step 6.
D e t e c t io n
3
Preparation of DNA sequences for labeling
The sequence to be labeled is usually a plasmid or cosmid, but can be a non-cloned PCR
product. If using genomic DNA as a PCR template, gel excision and DNA purification is
strongly recommended to remove residual genomic DNA before making a probe.
Chromosomal targets containing high copy numbers of repeated sequences, such as knob,
CentC, Cent4, and the maize B-specific repeat are easily detected with the method below.
For labeling targets less than 2-3 kb, see the Single Gene Detection section.
Plasmid DNA for PCR can be purified from a bacterial culture using either standard
molecular biology protocols or a kit (see Appendix D for product information).
1. Amplification of plasmid DNA by PCR
For FISH, a relatively large amount of DNA is necessary for each slide (20-100 ng/slide).
Therefore, 10-100 µg of the DNA insert should be amplified prior to the experiment.
Amplification of plasmid DNA is preferred over re-amplification of PCR-product DNA
because the latter will, over time, introduce and amplify errors.
PCR primers
Most of the common plasmids (pUC18, pBluescript, etc.) contain common primer
sequences that can be used for PCR amplification (such as T3, T7, M13F, M13R,
SP6, etc.). These primers are available by ordering them as custom primers [e.g.,
25 nmol synthesis from Integrated DNA Technologies (http://www.idtdna.com) or
Sigma-Aldrich (http://www.sigmaaldrich.com)].
It is also possible to design primers from the DNA sequence. The Whitehead
Institute for Biomedical Research/MIT Center provides a software program that
can be used to determine optimal primer pairs. The address of the web page is
http://www.bioinfo.ut.ee/primer3/. Also see http://www.primer3plus.com.
See A p p e n d i x A for plasmids and primers used to identify maize chromosomes. Use a hot-start PCR protocol. Either of the following work well. The recipes are given for a
single 100-µL reaction, but if a large number of probes will be made, set up 4-8 reactions.
Q i a g e n : HotStarTaq DNA polymerase (Cat. No. 203205) gives consistent PCR results and
high yields. Assemble the following reagents in a PCR tube at room temperature or on ice.
10x PCR buffer, Qiagen
MgCl2 15 mM
dNTP mix 25 mM each
Primer 1
100 µM
Primer 2
100 µM
Plasmid DNA 10 ng/µL
Sterile ddH2O
Q solution
Qiagen Taq polymerase
10.0 µL
6.0 µL
0.8 µL
0.5 µL
0.5 µL
1.0 µL
60.8 µL
20.0 µL
0.4 µL
(2 µL if 10 mM each)
(nuclease free water is recommended)
100.0 µL
Mix the solution by pipetting or gentle shaking. Spin down.
4
Thermal cycler program:
94˚C 5 min
(denature plasmid DNA)
↓
94˚C 10 sec
5 5 ˚ C 2 0 s e c (anneal DNA; dependent on melting temperature of primers)
72˚C 1 min
(extension, 1 min per 1 kb of product)
(35 cycles)
↓
7 2 ˚ C 7 m i n (extend DNA fragments to complete DNA strands)
4˚C soak
Start the reaction using the hot start procedure (see Qiagen manual).
S i g m a : To save set up time, use Sigma JumpStart ReadyMix REDTaq DNA polymerase
(Cat. No. P-0982). Components may be mixed at room temperature.
2x JumpStart Taq Mix
Primer 1
100 µM
Primer 2
100 µM
Plasmid DNA 10-50 ng/µL
Sterile ddH2O
50.0 µL
0.5 µL
0.5 µL
1.0 µL
48.0 µL (nuclease free water is recommended)
100.0 µL Mix thoroughly and spin down.
Thermal cycler program:
9 4 ˚ C 2 -5 m i n (denature plasmid DNA)
↓
94˚C 30 sec
55˚C 30 sec
(anneal DNA; dependent on melting temperature of primers)
72˚C 1 min
(extension, 1 min per 1 kb of product)
(35 cycles)
↓
7 2 ˚ C 7 m i n (extend DNA fragments to complete DNA strands)
4˚C soak
Notes:
Promega's GoTaq Green Master Mix (M7122) has also been used successfully.
If Invitrogen's Taq DNA polymerase (Invitrogen Cat. No. 10342-020) is used, the
PCR product should be resin-purified because a component of the kit has a UV absorbency
similar to that of DNA and co-precipitates with the amplified DNA.
5
2. DNA precipitation of PCR product
1. Transfer the PCR solution to a 1.6-mL tube.
N o te : If unheated lid was used and mineral oil is present, first
remove most of the oil with a pipet. Then pipet the solution onto a
small strip of parafilm. Transfer the drop several times. More oil
will be removed with each successive transfer.
2. Add 0.1 volume of 3 M sodium acetate (pH 5.2) and 2.5 volumes of 100% ethanol.
3. Mix by vortexing.
4. Put at -20˚C for 2 hours to overnight (longer is okay) or at -80˚C for 15 minutes.
5. Centrifuge at 16,000 rcf for 25-30 minutes.
6. Pour off the supernatant.
7. Add about 1 mL of 70% ethanol to rinse the DNA pellet. Carefully pour off the
ethanol. Repeat, removing as much 70% ethanol as possible.
8. Centrifuge briefly and use a pipet to remove the remaining 70% ethanol.
N o te : Drying the pellet is optional. Overly dry pellets are
hard to dissolve.
9. Add 50-200 µL of 1x TE (or nuclease-free water) and thoroughly dissolve the DNA.
The DNA concentration of a 1-µL sample is measured using a Nanodrop spectrophotometer.
(If you want to further check the DNA concentration, run a dilution series with an
appropriate DNA mass ladder in a mini gel.) Recipes in the remainder of this manual are
based on a DNA concentration of 200 ng/µL.
For a rapid purification of PCR products, kits are commercially available, for example
Promega Gel and PCR cleanup kit (catalog # A9282). Up to 500 µL of PCR product can be
run through each column. Check DNA concentration.
N o t e : If you cannot obtain consistent spectrophotometer results, please consider the
following tips.
Thick DNA pellets are difficult to dissolve in water. Vortex thoroughly.
Use TE rather than distilled water to dilute the DNA solution.
If you used a PCR purification kit which contains resin, the resin residue may interfere with the measurement. Resin may be removed by centrifugation.
If using cuvettes, use only one (or a transmission-matched set). 6
Mini-gel electrophoresis
After amplification, PCR product size and specificity are checked using gel electrophoresis.
1. Prepare a 1% agarose gel
Add 0.7 g of agarose to 70 mL of 1x TAE buffer (adjust volume of agarose solution based on
size of gel). Heat the solution in a microwave oven for a few minutes until it starts boiling
(If using a screw cap container, keep the lid loose!). Mix the solution well during heating.
When the agarose is completely dissolved, you will not see any intact particles. Place comb
in a plastic casting tray. When the agarose has cooled to about 50˚C, pour it into the tray.
After the gel is set, carefully remove the comb. If not needed for DNA extraction, gels can be
made in advance and stored in TAE at room temperature (or refrigerated) for several
months.
2. Run and stain the gel
After placing the gel in an electrophoresis box, 2-5 µL of the PCR product are mixed with
loading buffer and inserted into a well. An appropriate DNA size marker (e.g., 2-Log ladder)
is also loaded. (Note that some Taq mixes already contain loading buffer and dye.)
Run
the gel at ~100V (5 V/cm distance between electrodes) for about 30 min. The dyes in the
loading buffer indicate the approximate position of the DNA (bromophenol blue co-migrates
with 300-bp DNA fragments). See Taq Mix product information for sizes of dye markers
used.
To stain the DNA, gently shake the gel in a 1X TAE solution containing ethidium bromide
at a concentration of 0.5 µg/mL (alternatively, ethidium bromide is mixed with the agarose
before pouring the gel). Ethidium bromide is a mutagen and highly hazardous; wear gloves.
Any materials contaminated with ethidium bromide are disposed of as hazardous
materials.
If a PCR product of the correct size is observed, the DNA is precipitated as previously
described. If the results are unsatisfactory, optimize the PCR conditions.
Optimization of PCR conditions
Points for consideration:
-primer sequences
-annealing temperature
-magnesium ion concentration
7
Probes
Everything containing fluorochromes is light sensitive. Labeled dNTPs, nick translation
reactions, purification columns, probe DNA, and hybridization slides should to be stored in
the dark and exposed to bright light as little as possible.
The fluorochromes used most frequently to label probe DNA include coumarin (blue
emission spectrum), fluorescein or Alexa Fluor 488 (green), Texas Red (red), and cyanine 5
(far red). [Note: The Birchler lab is currently using a custom synthesis of coumarin- 5-dUTP
by Perkin Elmer because blue fluorochromes are not readily available.] Column purification
of green and blue probes is not required. Purification of red and far red probes is suggested,
but not absolutely required. (Not doing so may result in higher levels of non-specific
hybridization.) Protocols for making column- and non-column-purified probes are provided.
The direct labeling procedure utilizes a nick translation reaction in which dNTPs are
incorporated into the PCR product of the sequence of interest. Generally, only one of the
four dNTPs is tagged with a fluorochrome. Non-labeled dNTPs are added to the reaction as
a mix made by adding appropriate amounts of each concentrated dNTP in nuclease-free (or
autoclaved, ultra-pure) water. Nick translation involves the activities of two enzymes.
DNase I randomly nicks the template DNA. The second enzyme, DNA polymerase I, has
two activities — an exonuclease activity that removes DNA at the nick and a polymerase
activity that fills in the lesion.
The nick translation procedure can be modified to alter the degree of incorporation of
conjugated nucleotides into the probe. If sensitivity is not an issue, less DNA polymerase I
is sufficient for adequate labeling. However, if the chromosomal target sequence is small,
such as a transgene or single-copy endogenous gene, then a higher incorporation rate of the
labeled dNTP is desired. Increasing the amount of DNA polymerase up to about 20 times
the amount in a "standard" reaction will produce a brighter probe (Kato et al., 2006). These
findings have been incorporated into the labeling protocols in this manual.
For detecting small targets on the chromosomes, the signal-to-noise ratio must be
maintained as high as possible. In addition to the degree of labeling, the size of the probe
also affects signal brightness. Ideal probe length (after nick translation) is from
approximately 50-300 bases. In our experience, fragments much larger than 400 bases
cause background fluorescence. If probes from long templates are being produced, addition
of extra DNase to the labeling procedure will result in a probe that is of the desired length.
Because antibody-based (indirect labeling) detection methods also increase the background
noise due to non-specific adherence to chromosomes, the use of probes that are directly
labeled with a fluorescent molecule is preferred.
8
Enyzme stock solutions
DNA Polymerase I is purchased "ready-to-use" at 10 U/µL (Invitrogen #18010-025).
DNase I can be purchased ready-made or as a powder. DNase from different sources has
different levels of activity. This also seems true for different lot numbers of the same
product. See box for preparation of a stock solution from the powdered form of the enzyme.
Preparation of a working stock DNase solution (100 mU/µL)
Prepare 2x DNase buffer and 1x buffer (50% glycerol).
2x DNase buffer
100 mM Tris HCl pH 7.5
10 mM MgCl2
0.2 mM phenylmethylsulfonylfluoride (optional, see note below)
2 mM 2-mercaptoethanol
200 µg/mL bovine serum albumin
(Phenylmethylsulfonylfluoride is hazardous. If used, this substance is
dissolved in ethanol first and then added to the buffer.)
Primary stock: 10000 U of DNase I (Roche, Cat. No. 04536282001) are
dissolved in 0.5 mL 2x DNase buffer on ice. Add 0.5 mL glycerol and mix
gently. Concentration is 10 U/µL.
Intermediate stock: Add 20 µL primary stock to 80 µL of cold 1x DNase
buffer (50% glycerol). Concentration is 2 U/µL.
Working stock: Add 10 µL of the 2 U/µL DNase solution to 190 µL cold 1x
DNase buffer (50% glycerol). Concentration is 100 mU/µL.
Store all solutions at -20˚C. Freezing smaller volume aliquots of the
more dilute stocks is recommended.
The activity of the diluted DNase is relatively unstable. Check the
activity before use. 10 mU DNase in 10 µL DNase buffer digests 1 µg
DNA (1 kb) below 300 bp within 10 min at room temperature.
N o t e : During column chromatography, nick translation buffer (which
contains a high concentration of EDTA) is exchanged for TE, and the
unincorporated nucleotides are removed (stay in column). A spin column
method is also available, although it tends not to be quantitative.
9
Direct labeling for SMALL chromosomal targets (less than 30 kb)
1 . N i c k t r a n s l a t i o n . Assemble the following on ice (22.9 µL reaction volume). [To date,
Texas Red, fluorescein, Alexa Fluor 488, and Cy5 are the only labeled dNTPs that have
been tested for labeling targets less than 30 kb. Texas Red is the most sensitive and it
therefore recommended for detecting the smallest targets. See "Single Gene/Small Target
Detection."]
DNA 2 µg (200 ng/µL)
10x Nick translation buffer
Labeled-dNTP (1 mM)
Non-labeled dNTPs (2 mM each, mixed)
10.0 µL
2.0 µL
0.5 µL
2.0 µL
Add the following enzymes and thoroughly mix by pipetting. Do not vortex.
DNA polymerase I (10 U/µL)
8.0 µL
DNase (100 mU/µL)
0.4 µL
Incubate at 15˚C for 2 hours. The incubation may be performed in a PCR machine. Set
the machine to hold at 1-4˚C after the two-hour incubation for "overnight" reactions. If a
thermal cycler is not available, cold water in a covered styrofoam box works as well.
2 . O p t i o n a l s t o p p in g p o in t . Completed reactions may be stored at -20˚C in the dark.
To continue, add 2 µL of stop buffer (0.5 M EDTA, pH 8.0); this step is also optional.
3. Probe purification (Texas red- and cyanine 5-labeled sequences).
Column preparation. One column is generally used to purify 1- 5 µg of probe DNA
(maximum capacity ~8 µg). The tip of a Pasteur pipet is blocked with silane-treated
glass wool (insert using two pairs of forceps and push toward tip with narrow diameter
inoculating loop handle). With the pipet held at an angle, fill with 1x TE-saturated
Bio-Gel P-60 using a plastic transfer pipet. Set the Pasteur pipet in a 1.6-mL tube
(slant okay). TE will begin to flow into the tube. Empty as needed. Continue adding gel
until the top of the settled gel is just above the constriction in the pipet. (Excess gel can
be removed by pipetting while TE is present.) Add TE to wash the column and ensure
proper flow. Wait until all of the TE has run through the column before proceeding to
next step. (In the meantime, label collection tubes.)
Column purification.
Slowly add probe to the column. Cover with a cardboard box and wait for a minute.
Add:
50 µL of 1x TE. Elute. Discard the eluate.
350 µL of 1x TE. Elute. Discard the eluate. Move column to a new 1.6-mL tube.
350 µL of 1x TE. Elute. Save eluate. Move column to a second 1.6-mL tube.
350 µL of 1x TE. Elute. Save eluate.
If purifying >2 µg DNA, a third elution may be needed to ensure collection of all
the labeled probe.
10
4. Ethanol precipitation of probe.
Add 3 µL autoclaved salmon sperm DNA (10 mg/mL) to each of the two tubes to aid
precipitation.
Add 0.1 sample volume (35 µL) of 3M sodium acetate (pH 5.2) and 2.5 sample volumes
(875 µL) of 100% ethanol.
Mix by vortexing.
Store the tube at -20˚C for at least 2 hours (overnight or longer is okay).
Centrifuge at 16,000 rcf for 30 min. Pellets should be colored.
Pour off the supernatant. Use care. Pellets can dislodge.
Rinse the DNA pellets twice with 70% ethanol. After the last rinse, remove as much of
the ethanol as possible.
Centrifuge briefly, then remove the remaining ethanol.
Dissolve the damp pellets in a combined volume of 10 µL (not 10 µL each) 2x SSC,
1x TE buffer (pH 7.6) for a probe concentration of 200 ng/uL. Time, brief heat
treatment at 65˚C, and/or vortexing may be needed to dissolve the DNA.
N o te s:
Air-drying the pellet is optional. Overdrying the pellet will
make it much harder to dissolve the labeled DNA.
Any probe remaining on the wall of the 1.6-mL tube should be
washed down when 2x SSC, 1x TE is added and again when
DNA is dissolved.
Store probe at -20˚C in the dark.
11
Direct labeling for LARGE chromosomal targets (more than 100 kb)
1 . N i c k t r a n s l a t i o n . Assemble the following on ice (43.25-50.25 µL reaction volume).
DNA 5 µg (200 ng/µL)
10x Nick translation buffer
Labeled-dNTP (1 mM)
Non-labeled dNTPs (2 mM each, mixed)
Sterile ddH20 (optional)
25.0 µL
5.0 µL
1.0 µL
5.0 µL
7.0 µL
Add the following enzymes and thoroughly mix by pipetting. Do not vortex.
DNA polymerase I (10 U/µL)
6.25 µL
DNase (100 mU/µL)
1.0 µL
Incubate at 15˚C for 2 hours. The incubation may be performed in a PCR machine. Set
the machine to hold at 1-4˚C after the two-hour incubation for "overnight" reactions. If a
thermal cycler is not available, cold water in a covered styrofoam box works as well.
2 . O p t i o n a l s t o p p i n g p o i n t . Completed reactions may be stored at -20˚C in the dark.
To continue, add 5 µL of stop buffer (0.5 M EDTA, pH 8.0); this step is also optional.
3. Probe purification (Texas red- and cyanine 5-labeled sequences)
Column preparation. Make one column for each nick translation reaction. The tip of a
Pasteur pipet is blocked with silane-treated glass wool (insert using two pairs of
forceps and push toward tip with narrow diameter inoculating loop handle). With the
pipet held at an angle, fill with 1x TE-saturated Bio-Gel P-60 using a plastic transfer
pipet. Set the Pasteur pipet in a 1.6-mL tube (slant okay). TE will begin to flow into the
tube. Empty as needed. Continue adding gel until the top of the settled gel is just above
the constriction in the pipet. (Excess gel can be removed by pipetting while TE is
present). Add TE to wash the column and ensure proper flow. Wait until all of the TE
has run through the column before proceeding to the next step. (In the meantime, label
collection tubes.)
Column purification.
Slowly add probe to the column. Cover with a cardboard box and wait for a minute.
Add:
50 µL of 1x TE. Elute. Discard the eluate.
350 µL of 1x TE. Elute. Discard the eluate. Move column to a new 1.6-mL tube.
350 µL of 1x TE. Elute. Save eluate. Move column to a second 1.6-mL tube.
350 µL of 1x TE. Elute. Save eluate. Move column to a third 1.6-mL tube.
350 µL of 1x TE. Elute. Save eluate.
There might not be any probe in the last eluate. If so, the contents of that tube may
be discarded after the centrifugation step in Section 4.
12
4. Ethanol precipitation of probe.
Add 5 µL autoclaved salmon sperm DNA (10 mg/mL) to each of the three tubes to aid
precipitation.
Add 0.1 sample volume (35 µL) of 3M sodium acetate (pH 5.2) and 2.5 sample volumes
(875 µL) of 100% ethanol.
Mix by vortexing.
Store the tube at -20˚C for at least 2 hours (overnight or longer is okay).
Centrifuge at 16,000 rcf for 30 min. Pellets should be colored.
Pour off the supernatant. Use care. Pellets can dislodge.
Rinse the DNA pellets twice with 70% ethanol. After the last rinse, remove as much of
the ethanol as possible.
Centrifuge briefly, then remove the remaining ethanol.
Dissolve the damp pellets in a combined volume of 25 µL (not 25 µL each) 2x SSC,
1x TE buffer (pH 7.6) for a probe concentration of 200 ng/uL. Time, brief heat
treatment at 65˚C, and/or vortexing may be needed to dissolve the DNA.
N o te s:
Air-drying the pellet is optional. Overdrying the pellet will
make it much harder to dissolve the labeled DNA.
Any probe remaining on the wall of the 1.6-mL tube should be
washed down when 2x SSC, 1x TE is added and again when
DNA is dissolved.
Store probe at -20˚C in the dark.
13
Processing non-column-purified probes
P r o b e s l a b e l e d w i t h c o u m a r i n , f l u o r e s c e i n , o r A l e x a F l u o r 4 8 8 d o n o t b e n e f it
s ig n if ic a n t l y f r o m c o l u m n p u r i f i c a t i o n . T h e y a r e e t h a n o l p r e c i p i t a t e d .
1 . N i c k t r a n s la t io n r e a c t i o n . Complete as previously described.
2 . O p t i o n a l s t o p p i n g p o i n t . Store the reaction at -20˚C in the dark.
3. Continuation:
a) Thaw reaction. Select volumes for continued processing based on the scale of the
reaction. I m p o r t a n t : If you make a 5-µg probe of a small target, i.e., use 20 µL
of DNA polymerase, calculate the amounts needed based on the actual volume.
2 µg
b) Add 1x TE (pH 7.6) to a total volume of 100 µL
77.1 µL
5 µg
56.75 µL
c) Vortex, centrifuge briefly, and transfer entire reaction
volume to a 1.6-mL tube.
d) Add autoclaved salmon sperm DNA (10 mg/mL)
3 µL
5 µL
10 µL
10 µL
250 µL
250 µL
e) Add 0.1 volume 3M sodium acetate (pH 5.2)
f) Add 2.5 volumes of 100% ethanol
g) Mix by vortexing. Put tube at -20˚C for at least 2 hrs
(overnight or longer) or -80˚C for 15 min to precipitate DNA.
h) Centrifuge at 16,000 rcf for 30 min.
i) Pour off the supernatant.
j) Rinse the pellets twice with 70% ethanol. After the last rinse,
remove as much of the ethanol as possible. Centrifuge briefly,
then remove the remaining ethanol.
k) Dissolve the damp pellets in 2x SSC, 1x TE buffer (pH 7.6)
10 µL
for a probe concentration of 200 ng/µL. Time, brief heat
treatment at 65˚C, and/or vortexing may be needed to
dissolve the DNA.
N o te s:
Air-drying the pellet is optional. Overdrying the pellet will
make it much harder to dissolve the labeled DNA.
Any probe remaining on the wall of the 1.6-mL tube should be
washed down when 2x SSC, 1x TE is added and again when
DNA is dissolved.
l) Store at -20˚C in the dark.
14
25 µL
Preparation of metaphase spreads from root tissue
1. Supplemental information
Use the nitrous oxide - enzymatic maceration method (Kato, 1999) to make slides for FISH
from plant tissue. Root tips can be obtained from newly germinated seeds, from young
plants, or from new growth on root-pruned plants. Root tips to be nitrous oxide treated are
placed in 0.5-mL or 1.5-mL tubes, each with a hole punched in the lid to facilitate gas
exposure and lightly misted with water to prevent desiccation. Longer treatment times
generally result in chromosomes that are more condensed.
The length of time the root meristematic tissue is digested depends on the number and size
of the tissue pieces, and the degree of chromosome spread desired. The tissue can be left
intact, cut transversely into several pieces or cut both longitudinally and transversely.
Whatever the size, quickly transfer the pieces into the enzyme solution. Ideal conditions for
different lines will vary. For smaller diameter roots, two or three roots per enzyme tube
might be needed to produce adequate cell density.
To make ~10 mL of the enzyme solution, mix together (on ice):
0.1 g of pectolyase Y-23 (1% w/w),
0.2 g cellulase Onozuka R-10 (2% w/w)
9.7 g of 1x citric buffer
Quickly dispense as 20-µL aliquots into thin-walled 0.5-mL PCR tubes,
quick freeze on dry ice, and store at -20˚C.
Note: Concentrations might need to be adjusted when changing an enzyme
source or lot number. The Birchler lab is currently using 3% cellulase, 1.5%
pectolyase. If background cytoplasm is a problem, as is with some maize lines,
increase the cellulase concentration to 4%.
Our version of a humid chamber consists of a shallow, open-top cardboard box, subdivided
into sections that will easily accommodate the width and number of microscope slides to be
processed. All surfaces are lined with multiple layers of paper towels (stapled into position),
which are moistened with 'distilled' water prior to use. Slides placed in the chamber are
elevated on a dry plant stake. Covering the chamber with a large Kimwipe (either damp or
dry) is optional. The chamber is reused.
Prepare slides using the suspension dropping method, which is better for FISH than the
smear-scraping technique. Signal intensity is stronger, background level is lower, and the
incidence of non-overlapping chromosomes is higher with the dropping method.
The pattern of dropped cells is dependent upon the individual and can include a single 6-9
µl drop in the center of the slide or a grid of smaller drops to cover the same area (e.g., 3 x 3).
Also, two hybridizations can be set up on the same slide by positioning the dropped cells
such that they can be covered individually by a 22 x 22 mm plastic cover slip for
hybridization, yet fit under a 24 x 60 mm glass cover slip for viewing. Single sample
preparations are viewed through 24 x 50 mm cover glass.
15
2. Preparation of root tips
Germinate kernels in moist vermiculite or on nearly wet Kimwipes for 2-3 days at 30˚C, or
harvest young roots from plants. Choose roots 1-5 cm in length (2 - 4 cm ideal; longer end of
range if plant to be kept). Keep roots moist.
Cut 1-1.5 cm of root tip and treat with N20 at 10 atm for 1-3 hr.
Fix root tips in ice cold 90% acetic acid for 10 min (not longer than 1 hr).
[Optional stopping point: Store in 70% ethanol at -20˚C. Roots keep well for years.]
Soak root tips in 2-3 changes of reverse-osmosis-purified water for 10-20 min on ice to
remove ethanol (longer if stored for an extended period). [Citric buffer soak not required.]
Remove the sticky substance at the root tip by rolling on dry filter paper (moistening filter
paper with citric buffer, optional).
Cut off the tip of the root cap. Discard. (This step and the next may be performed on a dark
surface to facilitate identification of the root cap and actively dividing region.)
Cut off about 0.5-1.0 mm of the section containing the actively dividing region (more opaque,
about 2 mm) and transfer to a tube containing 20 µl ice cold enzyme solution.
Incubate at 37˚C for 30-60 min (species-, line-, size-, and age-dependent). Plunge tube into
ice and fill with cold 70% ethanol. Invert to mix well. Return to ice.
Remove ethanol/enzyme mixture. Fill tube with 70% ethanol. [Optional stopping point –
store digested, intact root meristem tissue at -20˚C in the 70% ethanol.]
Remove as much ethanol as possible (invert tube, blot edge, and/or use pipet tip). Tissue
will most likely stick to interior of tube.
Add 20-30 µL of freshly prepared 90% acetic acid - 10% methanol or 100% acetic acid.
Carefully break the root section with a rounded off dissecting needle.
Stir with needle and tap the tube with your finger several times to suspend the cells.
Upon addition of acetic acid or methanol, keep cells on ice only as long as is necessary to
drop the cell suspension onto a slide and to observe the spreads.
To store cell preparation at -20˚C for a short period of time, remove 70% ethanol
and replace with 100% ethanol. Repeat the 100% ethanol wash two more times to
remove all water.
Replace ethanol with 20-30 µL of freshly prepared 90% acetic acid - 10%
methanol (doesn’t work if cells are in 100% acetic acid). Break the root sections
with a rounded off dissecting needle. Stir with needle and tap tube to suspend cells.
Store at -20˚C. This method is not recommended when single-gene probes will be
used.
16
3. Drop and cross-link cells
Label slides onto which cells will be dropped (Tough Tag positioned near end works well.).
Drop 6-8 µL of the cell suspension on a microscope slide in a humid chamber.
Requirement of the humid chamber for drying the cell suspensions is
dependent on ambient humidity. In Missouri, use of the chamber is not
necessary during the humid summer months. However, if not used during the
drier times of the year, the solvent drop will not evaporate, and the cells will
not spread. Therefore, year-round use of the humid chamber is recommended.
When dry, view through the 10x objective lens of a compound microscope to select slides
with the best spreads (quality and number) for hybridization.
Cross-link chromatin to slides by exposure to UV light. Set cross-linker to "optimum."
A total energy of 120-125 mJ per square cm is delivered.
Cross-linked slides can be used immediately for hybridization or stored for later use.
See options in text box.
The following are rough guidelines. Hybridization signal quality will decrease
with time, with strong signals being less affected.
Room temperature – about a week.
4˚C – longer but limit not determined.
-20˚C – four to six months when hybridizing with the standard multi-copy
probes [slides stored in frost-free freezer; ice forms on slides in
non-frost-free freezer].
For very small targets, e.g. single genes, it may be necessary to use slides that
are prepared from freshly digested cells that have not been stored. To
maximize the signal quality, store root tips in 70% ethanol until ready to
proceed with the FISH procedure.
17
Signal detection of directly labeled probes
D e n a t u r e , 1 0 0 ˚ C , 5 m i n ↓
H y b r i d i z a t i o n , 5 5 ˚ C , 4 h r t o o v e r n ig h t
↓
W a s h in 2 x S S C R T , b r ie f ly t o 5 m i n
↓
S t r i n g e n t w a s h , 2 x S S C , 5 5 ˚ C , 1 0 -3 0 m i n
↓
Apply Vectashield (may contain DAPI or PI)
↓
O b s e r v a t i o n N o t e : In other protocols, ethanol dehydration (series of 70%, 90%, and 100%,
-20˚C) is usually conducted on denatured slides. The method presented here is
simpler and gives equal or superior results.
1. Preparation
Make probe cocktail. Determine which probes will be needed and at what concentration.
The total volume of probe cocktail per slide should be 6-10 µL, the balance of the
volume (if any) being 2x SSC, 1x TE. Start with 8 µL.
Smaller volumes will not adequately cover the area under the 22-mm square
cover slip. Factors to consider when choosing the volume of probe to use are
given below, but trial and error will provide the answer.
1. Probe type – can use smaller volumes of oligo probes than of nicktranslated probes because the former spread better (less DNA).
2. Number of probes in cocktail – if many, may need to look at more
spreads to find one that’s publication quality.
3. Number of metaphase spreads on the slide – if few or if there are many
clumps of un- or under-digested cells, it would be better to use a larger
volume. (Cell clumps prevent the cover slip from lying flat, which causes the
probe to accumulate around the clumps, thereby resulting in some areas
receiving an inadequate amount of probe.)
4. Target – Localization of a single gene on a chromosome will require
more probe than determining presence/absence of a multiple-copy sequence
on a nucleus.
It is seldom necessary to use more than 1 µL of a 200 ng/µL probe per slide, the
exception being 1.5-2 µL of a single-gene probe.
For information pertaining to concentrations of the maize probes described in Kato
et al. (2004), see Appendix B.
Drop, dry, and cross-link cells. Select the best slides for hybridization as described above
if not already done.
18
Preheat boiling water bath (can use electric skillet), but not insert tray assembly.
Water bath insert: a small aluminum tray, the bottom of which is covered
with a wet layer of Kimwipes (use RO purified water). Slides and tubes are
pressed firmly on the Kimwipes (good contact needed) and are covered with
a plastic pipet tip box lid. The entire assembly floats in a larger tray
containing boiling water. Cover everything with aluminum foil at the
beginning of the denaturation step. Fill insulated bucket with ice. Gather all necessary items and equipment.
Needed after hybridization: a Coplin jar containing 2x SSC preheated to 55˚C.
Alternatively, heat in a microwave just before needed. Heat the solution
slowly to allow the glass to warm without breaking.
2. Denaturation
The method shown below can be used for detection of any target but is primarily used for
single-gene detection. The probe and chromosomal DNAs are denatured separately.
If the hybridization is not to very small targets, the probe cocktail can be applied directly to
the slide and denatured in situ (see text box at the end of this section). This occasionally
increases background fluorescence but saves time when many slides are to be screened.
a) Pipet ~8 µL of denatured salmon sperm DNA (85 ng/µL 2x SSC, 1x TE made from
autoclaved 10 mg/mL DNA in TE stock) onto the center of each cell spread. Using
forceps, apply 22 x 22 mm plastic cover slip; one of the corners or an edge hangs over the
edge of the slide (facilitates removal).
b) Denature root tip DNA (on slide) and probe DNA cocktail (in 200-µL thin-wall PCR
tube) by floating in the boiling water bath (see above) for 5 min (100˚C).
While waiting, prepare to chill probes and slides – turn metal slide tray
upside down and plunge into ice such that good contact is made. Also,
prepare a small beaker of slushy ice in which to quick chill the probe.
c) Remove insert tray. Take care not to get burned when removing foil. Transfer tray to
benchtop using tongs or a large pair of forceps. Keep the plastic lid parallel to the
benchtop as it is being removed to avoid getting condensation on the slides.
d) Quick cool the probe(s) in the ice slush and the slides on the pre-chilled metal for one to
two minutes. Keep probes in the dark.
e) Spin probes briefly to return volume to bottom of tube. Return to slushy ice.
f)
With fine-tipped forceps, lift plastic cover slip (or lift off, flip over, and set down). Pipet
6-10 µL of probe onto the cells and replace cover slip in same orientation. (Cells should
not be allowed to dry.)
g) Place slides in a humid storage container (reusable airtight 'food' container lined with
Kimwipes moistened with 2x SSC) for 4 hr to overnight at 55˚C.
19
h) Place slide(s) in a Coplin jar containing room-temperature 2x SSC briefly (up to 5 min)
to remove the cover slip and excess probe. Slides can be placed back to back to increase
the processing capacity of the jar.
i)
Using forceps, transfer slides to a Coplin jar containing 55˚C 2x SSC and wash for 20
min at 55˚C. The temperature and SSC concentration can be varied for different
stringency requirements (See text box below).
S tri n ge n t w ash
The second wash of this protocol is a stringent wash. By changing the
temperature and salt concentration, one can change the stringency. The
addition of 50% formamide further increases the stringency, but is not
necessary in most cases. Wash for 10-30 min.
42˚C,
55˚C,
60˚C,
65˚C,
50˚C,
55˚C,
60˚C,
j)
2x SSC
2x SSC
2x SSC
2x SSC
0.1x SSC
0.1x SSC
0.1x SSC
low stringency
medium stringency
high stringency
very high stringency
medium stringency
high stringency
very high stringency (most signals will disappear)
Remove slides. Flick to remove excess SSC from the top of the slide and blot to remove it
from the bottom and edges of the slide (SSC remaining on slide surface will dilute
Vectashield).
k) Apply one drop of pre-mixed Vectashield mounting medium with DAPI and carefully
apply a 24 x 50 mm glass cover slip. (Vectashield without DAPI is used with the full
probe cocktail or when detecting blue fluorescent probes. Alternatively, DAPI
containing Vectashield is diluted 1/20 or 1/40 with DAPI-free Vectashield to lightly
stain chromosomes but still allow detection of strong blue signals.)
l ) Acquire images. Slides may be stored in a slide box at 4˚C (before or after image
acquisition). Line box with foil and dry Kimwipe to absorb excess mounting medium and
immersion oil.
I n si tu d e n atu r ati o n m e th o d:
Pipet 6-10 µL of probe onto each slide.
Apply 22-mm square plastic cover slips (no need for overhang).
Transfer slides to insert tray as described above and float the assembly in the boiling
water bath for 5 min.
Remove slides and transfer directly to the humid storage container (step “g” above).
Follow steps “h” through “l”.
20
3. Microscopic observation
Before using the microscope, make sure you have been instructed on its use by someone
who knows the instrument well.
Frequent turning on and off of a mercury bulb will shorten its life. Keep the bulb lit for at
least 30 min once you have turned it on, and wait 30 min after you turn off the bulb before
turning it on again.
Extreme care is necessary when cleaning an objective lens. If you have not received
training, please refrain from doing it.
If you cannot focus the microscope in dark field, go to low magnification in bright field and
adjust focus at the edge of the label.
Locate spreads using a low magnification oil objective lens (20-40x). A lens designed for
fluorescence is best. The larger the numerical aperture, the more light it will transmit.
Acquire images with a 100x oil lens. In addition to the above comments, this lens should be
flat field and color corrected to get the best images.
The filter set in use can be determined by observing the color of the excitation light as
viewed between the objective lens and slide. Peaks listed numerically.
Excitation nm (color)
Emission nm (color)
DAPI
345 (UV/violet)
455 (Blue)
Coumarin
400 (UV/violet)
445 (Blue
Alexa Fluor 488
490 (Blue green)
525 (Green)
Texas Red
593 (Green)
612 (Red)
Cyanine 5
650 (Red)
668 (Far red)
Acquire images using the software provided with your microscope.
Process images using Adobe Photoshop or similar software.
21
Pachytene FISH
Immature tassels are fixed in 3:1 ethanol:acetic acid for 24 hours at room
temperature. The tassels are rinsed and stored in 70% ethanol at 4°C or -20°C.
Each floret on the tassel contains two groups of three anthers. Each of the three
anthers is at the same stage of meiosis. By examining one of the three, the stage
of the remaining two can be determined. The anthers can be dissected out of the
glumes and kept in 70% ethanol. The anther to be staged is placed on a dry slide
and a drop of aceto-carmine is added. Iron dissecting probes are used to smash
apart the anther. Large pieces of debris are removed from the slide and a glass
cover slip is applied. The slide is heated with an alcohol lamp until just before
the stain begins to boil. This helps flatten cells and darkens the chromosomal
staining.
After correctly staged anthers are located, they are placed in 1X citric buffer on
ice for at least 5 minutes. The anthers are added to a tube of enzyme cocktail (the
same as used for root tip digestion) and incubated at 37°C for 20-50 minutes
(typically 40 minutes is appropriate).
The enzyme solution is rinsed away by filling the tube with cold 1X TE. The TE
is removed and the anthers are gently rinsed in 100% ETOH several times (to
ensure that all the water is eliminated). Add 35 µL of 3:1 acetic acid:methanol
and break apart the anthers with a dull dissecting probe. Flick the tube to
further separate cells. About 8 µL of cell suspension is dropped onto a glass slide
in a humid chamber and allowed to dry. Examination of the cell spreads will
confirm the correct staging and spreading of the meiotic cells.
Pachytene chromosomes do not usually separate from their cytoplasm during the
spreading procedure. As a result, there tends to be higher background after FISH
than for mitotic chromosome spreads. If the cytoplasm appears dark (without
staining) under a light microscope, then the background after FISH is usually
strong. However, there is not always strong background and for intense signals
(knob, CentC, NOR etc.) the background is not a problem. For smaller targets,
only slides containing pachytene spreads with little or no visible cytoplasm
should be used for FISH.
Other labs have described various treatments to reduce or eliminate the
background of pachytene spreads in order to detect small signals. For example,
22
one method involves treating the slides with pepsin. We have not tested these
treatments but have been able to detect small signals on preparations that have
little cytoplasm. Also, increasing the length of incubation of anthers in the
enzyme cocktail seems to reduce the cytoplasmic background.
The slides are UV-cross-linked and FISH is performed as described for mitotic
spreads.
23
Single Gene/Small Target Detection
The above-described procedures consistently allow detection of chromosomal
targets greater than approximately 3 kb. Even smaller sequences can be
detected, although on a lesser percentage of cells in a given preparation. For
these smaller targets, we recommend labeling probe DNA with Texas Red, which
usually results in a brighter signal compared to probes labeled with the other
fluorochromes. Probes should be hybridized to fresh cell preparations (i.e., they
are digested, suspended and dropped on the same day) with selection of
preparations of the best quality (with abundant chromosome spreads, free of cell
walls and cytoplasmic remains). Using high probe concentration or fewer cells
per slide and a long hybridization time (overnight) are also helpful.
The average size of a maize gene is 4 kb, so many maize genes can be visualized
with FISH. However, detection of maize genes smaller than 3 kb or having
repetitive elements in their introns requires a modified strategy. To localize such
targets, repeat-free “pooled” probes that hybridize to adjacent unique
chromosomal regions (clustered genes or regions within one gene) are produced.
Repetitive elements can be identified in a sequence with various software
programs available online, for example, www.repeatmasker.org or
www.girinst.org. Because these programs can be limited by the completeness of
repeat element databases, presumptive "unique" sequences as determined by
BLAST analysis are hybridized to confirm that they are not repetitive. The
regions within "unique" sequences with homology to mRNAs or cDNAs of rice,
sorghum or other plant species have the most promise to be single-copy,
conserved genic regions and can be expected to be present in different maize
lines. These sequences can be used to design PCR primers and can be amplified
using genomic DNA or BAC DNA as a template. By FISH testing each PCR
product to be included in the final pooled collection separately, sequences that
hybridize throughout the genome because they contain repetitive element
homology can be identified and eliminated. Finally, to produce a pooled probe,
selected PCR products can be labeled with a fluorochrome by nick translation
separately or together as an equal molar ratio mix.
Because processed mRNA has had the introns removed, cDNAs can be
successfully used as probes for genes containing repetitive elements in their
introns. Searching the public databases for large maize cDNAs (greater than 4
kb) revealed many candidate genes. Several of these have been successfully used
as FISH probes.
24
Many genes in the maize genome are present as tandem arrays. These gene
families make good FISH targets because a single member of the family can be
used as a probe, which hybridizes to many locations in the cluster. This approach
has been used to label the rp1 and rp3 rust resistance gene clusters as well as
two alpha-zein clusters and an expansin gene cluster (Lamb et al., 2007).
Further, the selected DNA regions can be cloned and amplified using standard
primers (M13 or T7, SP6). In this step it is recommended to prepare the PCR mix
in a laminar hood and purify the PCR product in columns with caps to exclude
contamination from clones of highly repetitive elements such as knobs or NOR.
Because of the long exposure time used to visualize single gene probes, even low
concentrations of labeled contaminants can give bright signals during FISH.
25
Retroelement Genome Painting
Because the maize genome contains retrotransposon families that are present in
thousands of copies, probes prepared from them can be used to paint maize
chromosomes. The retroelement families have differentially expanded in the
various Zea lineages as well as in the sister genus Tripsacum (Lamb and
Birchler, 2006). Retrotransposons used as FISH probes label chromosomes from
different species with different intensities, allowing the chromatin from different
species to be distinguished in interspecific hybrids.
This approach can be used as an alternative to, and has some advantages over,
the commonly used Genomic In Situ Hybridization (GISH) technique. Because
the retrotransposons are present in different copy number, using them directly
as probes allows the chromosomes to be distinguished based on fluorescence
intensity. Additionally, because knob heterochromatin sequences are so
abundant, it is not possible to block hybridization to the knob regions.
To distinguish maize and Z. diploperennis, it is possible to use a number of
different retrotransposon probes. The long terminal repeats (LTRs) from Grande
or Huck work particularly well for this purpose. To distinguish maize from
Tripsacum, most common maize retrotransposons will work. Grande, Huck,
Prem1, Prem2, Opie and others will work well. Cinful1 is present at high copy
number in both genera.
To identify new retrotransposons that paint Tripsacum chromatin but not maize,
a library of random DNA sequences from Tripsacum was produced and screened.
The library was prepared by nebulizing genomic DNA from Tripsacum, repairing
the ends and ligating into a standard cloning vector. Twenty-five clones were
chosen for FISH screening based on strong hybridization to Tripsacum genomic
DNA. Four of these clones contained elements that strongly hybridized to
Tripsacum chromosomes but only weakly to maize chromosomes. Other clones
contained a 5S ribosomal gene, knob sequences, or retrotransposons, which
hybridized equally well to both maize and Tripsacum chromosomes. Because
FISH screening is rapid and simple, this approach could be easily adapted to
other species with genomes that are enriched with repetitive elements.
26
Method for Pollen FISH in Maize
1. Mature pollen is collected in greenhouse or field.
2. The pollen is fixed in ethanol:acetic acid (3:1) overnight at -20ºC.
3. Wash the fixed pollen with 70% ethanol and store at -20ºC.
4. Take a suitable amount of pollen in a 1.5-mL tube, wash the pollen in 2X SSC
three times, each for 5 minutes.
5. Wash the pollen in 10 mM HCl for 10 mins.
6. Treat the pollen with 1X (50 µg/mL) or 4X Pepsin (in 10 mM HCl) for 10-30
mins at 37ºC.
7. Wash the pollen with 2X SSC three times, each for 5 mins.
8. Place 10 µL hybridization mixture in the tube containing pollen (the mixture
contains 200 ng of nick translated probe, 2X SSC and 50% formamide,
denatured for 10 mins at 100ºC and then placed on ice for 5 mins).
9. Place the tube in an 80ºC heat block for 6 mins.
10. Place the tube in the dark at 37ºC for 22-24 hrs.
11. Stain the pollen with DAPI (Vectashield H-1200, 200 ng/mL diluted 4X) and
leave for 1-2 hrs at 4ºC.
12. Observe with microscope and photograph.
27
Endosperm Chromosomes and Nuclei Spreads
Maize endosperm tissue can be harvested at various time points after
pollinations. For chromosome spreads, endosperm tissue should be harvested
9-11 days after pollination (DAP). Once the ear is removed1, wet it with distilled
H2O, and very loosely cover with plastic wrap around it. This is done to keep the
ear moist, but must be loose enough to allow access for air and nitrous oxide
(N2O) gas. Place the ear in a N2O chamber, and treat for three hours at 10 Kpa.
If the endosperm is harvested more than 12 DAP, few mitotic cells exist, so the
N2O treatment can be skipped. After three hours, remove ear from chamber and
cut out roughly a 1-mm3 piece of tissue from the central region of the endosperm.
Fix in ice cold 90% acetic acid for a minimum of ten minutes, and then transfer
the tissue to ice cold 1X TE for 5 minutes. Transfer to 20 µL of digestion buffer
(2% cellulase, 1% pectolyase, 10 mM EDTA) and incubate at 37°C for 25-35
minutes. The digestion time can vary depending on the size of the block of tissue
and the time point at which it was harvested. Usually 25 minutes of incubation
is sufficient.
The digestion buffer is replaced with 1X TE (Tris-HCl, EDTA) and incubated on
ice for 5 minutes. After removal of TE, 100% ethanol is added. The tissue is
broken up by gently flicking the tube. Do not vortex if chromosome spreads are
desired. The sample is pelleted by centrifugation in a tabletop centrifuge (~6000
x g) for 30 seconds. Ethanol is removed, and two to four drops of spreading
solution is added (27:3 acetic acid:methanol) using a glass Pasteur pipette. The
sample is gently mixed, and then using a glass Pasteur pipette one to two drops
are placed on a glass slide. Incubate the slides at room temperature in a shallow
cardboard box lined with wet paper towels for two hours or until slides are dry.
Cross-link and proceed with FISH as described previously for root tissue.
The presence of starch interferes with viewing the fluorescent signals. Adding
more spreading solution will cause starch granules to be separated from the
nuclei, but also dilutes the sample, thereby decreasing the density of
chromosome spreads and nuclei on the slide.
If a large number of endosperms are to be harvested from one ear, it is easier to
fix the entire kernel. After N2O treatment, pick off the kernels, and cut off the
base to expose the endosperm tissue. Fix in 90% acetic acid for a minimum of
ten minutes, and then transfer to 70% ethanol for long-term storage at -20°C.
28
When the sample is taken out of storage, cut out 1 mm3 from the central region,
place in ice cold 1X TE, and continue as described above.
________________
1Do not harvest individual kernels and place them in the N O chamber, as this
2
will result in both decondensed chromosomes and a decrease in mitotic
chromosomes overall.
29
A Cocktail of Many Colors
The Birchler lab’s standard cocktail includes one example of combining colors.
Specifically, the maize ribosomal 5S gene is hybridized with two probes that
label the same sequence. One of these probes is tagged with Texas Red and the
other with Alexa Fluor 488 or fluorescein, which are green. The resultant signal
is yellow. This is true, however, only when the colors are mixed in the correct
ratios. By varying the concentrations of red and green, one can create a palette
that includes a deep red orange, orange, yellow, and lime green. Similarly, one
can create cyan (blue plus green), magenta (blue plus red), or pink (red plus
white, if a Cy5-labeled probe is pseudo-colored white). To get a feel for how
individual component colors affect the end result, one can experiment with
Photoshop’s section on color. Note that an entry of zero for each red, green, and
blue will result in black (absence of color), whereas 255 (full saturation) entered
for each will produce white. The color red is produced when 255 is entered for
red and zeros for green and blue. One can also select a color and note the
corresponding values for equal intensities of red, green, and blue. The basics of
the additive properties of light can be found in a variety of online sources.
The advantages of multiple colors for separate probes are obvious, but do
require some extra adjustments to obtain the desired result. In addition to
concentration, three factors affect color outcome.
1. The nick translation reaction used in our direct labeling procedure is
initiated by random events – the action of the DNase. Consequently, some
probes will produce stronger signals than others. Combining several probes
prior to determining the concentration needed to produce a specific color is
helpful. This problem may be eliminated by using 5ʹ′ end-labeled oligonucleotides as probes (see similarly titled section in manual).
2. The innate intensities of the fluorochromes must be taken into
consideration. Of those in common usage, Texas Red produces the strongest
signal, followed by Alexa Fluor 488 and fluorescein, Cy5, then coumarin. When
mixing colors, remember that equal concentrations will not contribute equally to
the end product, and that coumarin will only label very large targets.
3. Keep in mind that the background fluorescence from all the other probes
in the cocktail mix will affect your target color combination, as will DAPI if it is
used as a counter stain.
The last option for color correction is to modify the signal strengths of the
individual colors with Photoshop or a similar image processing software.
30
Below are colors frequently used in the Birchler lab and the final concentrations
of component probes. Note that the concentrations are provided as a starting
point and may not result in the exact color indicated.
Yellow (5S)
Orange (Cent4)
Teal (NOR 173)
16.0 ng/µL Alexa Fluor 488 + 8.0 ng/µL Texas Red
13.3 ng/µL Alexa Fluor 488 + 12.0 ng/µL Texas Red
0.8 ng/µL Alexa Fluor 488 + 18.7 ng/µL Cascade Blue
If using fluorescein-labeled green probes or coumarin-labeled blue probes, the
ratios are most likely similar.
31
5' End-labeled Oligos as FISH Probes
For routine screening of tissue samples, it is time consuming to continually
make probes for frequently targeted sequences. Alternatively, one might need to
check a large number of sequences for probe potential. In either case, it may be
more convenient to order 5' end-labeled oligonucleotides from companies such as
Integrated DNA Technologies (IDT), Life Technologies, or Sigma-Aldrich. Kits
are also available.
One will have to provide the company with the target sequence, usually 20-50
bases in length, and specify the fluorochrome. Each company has different fee
structures based on synthesis scale, the number of bases, the fluorochrome and
whether any special purification steps are required. Any oligo sequence will
have to be: 1) specific to the target and 2) screened so as to minimize
self-hybridization (i.e. dimerization and hairpins). Therefore, it will be
necessary to BLAST the candidate sequences against the organism’s database
as well as against those which are related if the genome has not yet been
sequenced or if there are many repeat sequences throughout the genome. Useful
web sites and probe design strategies can be found in the section “Single
Gene/Small Target Detection”.
Generally, because oligo probes can produce very strong signals, they may have
to be substantially diluted to working concentrations. As with nick-translated
probes, the final concentration used is dependent on probe signal strength and
target sequence copy number. Whereas the final concentrations of individual
nick-translated probes vary from 0.2-40 ng/µL, the 5' end-labeled oligos are used
at concentrations more in the range of 0.003-1 ng/µL. Because these oligos are
not very stable at the more dilute concentrations, it is advisable to immediately
make and freeze small-volume aliquots (10-20 µL) of the primary stock (500
ng/µL) and a few vials of a less concentrated intermediate working stock. Make
only enough final working stock for short-term use.
Oligo probes are single-stranded, so they do not require denaturation prior to
hybridization. (In fact, some are rendered less effective if exposed to high heat
conditions.) If your hybridization will include both nick-translated and 5'
end-labeled probes, add the oligo probes after the denaturation step, when the
slides and probe tube are on ice. Because fluor-labeled oligos are likely to be
initially used at relatively high concentrations during repeat screening
procedures, it is critical to minimize potential cross-contamination of samples
during both slide set-up and wash procedures.
32
5´ oligos for maize repeats:
Target
Fluorochrome
Sequence
CentC
Alexa 488
CCT AAA GTA GTG GAT TGG GCA TGT TCG
Knob
Alexa 488
TCG AAA ATA GCC ATG AAC GAC CAT T
1-26-2
Texas Red
G[TAG]18
A wide range of fluorochromes are available for conjugation to DNA
oligonucleotides, both in terms of emitted wavelength, as well fluorochrome
moiety within a given color range. Blue-emitting fluorochromes are generally
not an option due to lack of availability, insufficient signal strength and
excessive cost. This fact leaves the following three options:
Green fluors:
At present, the least expensive fluors are fluorescein-based (e.g.
6-carboxyfluorescein, 6-FAM, which is available from IDT). Because
fluorescein-tagged oligos do not require post-synthesis purification, they can be
synthesized at a small scale. While fluorescein is not as bright as red fluors, or
as resistant to photobleaching as Alexa Fluor 488 (another green option), it is
the most economical option.
Red fluors:
Texas red fluorochromes are generally the brightest option, and should be
reserved for lower-copy number repeats that are harder to detect. Texas red
tagged oligos do require a minimum synthesis scale (100 nM) and HPLC
purification, making them much more expensive. Within a given company,
multiple Texas red options might be available. (Texas Red 615, from IDT, works
quite well.)
Far-red fluors:
Cyanine 5 (Cy5) is a far-red fluorochrome that also works quite well, but should
be reserved for the highest copy number localized repeats. Cy5-tagged oligos
also require a minimum synthesis scale (100 nM) and HPLC purification.
33
Developing a New Karyotyping Cocktail
for other Plant Species
Developing a FISH-based karyotyping cocktail requires a collection of probes
that can be used in combination with morphological features to identify each
chromosome in a karyotype. Not only must individual probes work consistently
(they must be bright, low in background and relatively insensitive to minor
variations in hybridization conditions), but the collection itself must work
together. Karyotyping cocktails have been developed for numerous plant
species; they generally utilize some combination of probes developed from BACs,
repeat-subtracted gene collections, clustered repetitive elements and clustered
genes. Obviously, the utility of an individual probe type depends on the
organization and composition of the genome of interest. In maize, for example,
due to the high repeat content/low gene density, BAC probes are not a viable
option, because of the massive cross-hybridization with elements dispersed
throughout the genome. In contrast, in soybean, in which about half of the total
repeat content is concentrated in pericentromeric regions, low-repeat content
BACs can be used.
Each type of FISH probe has strengths and weaknesses. Because of their large
inserts (>100 kb), BAC FISH signals can generate excellent single-locus signals
in FISH; yet, also because of their large inserts, BACs with repetitive DNA can
generate genome-wide background in FISH. Even with a high level of
background, BAC FISH signals are often still apparent, and possibly useful.
Using the BAC protocols below, collections of BACs can be easily screened for
their utility in FISH/karyotyping.
Although single-copy gene sequences as small 2 kb can be detected in mitotic
chromosome spreads using nick-translated probes, the FISH signal is not
generally bright enough to be used in the context of a karyotyping cocktail
because most or all of the other cocktail components using the same fluorescent
label will be much brighter. One option is to develop collections of multiple
single-copy genes localized to a particular chromosomal segment. Another
option is to develop a single gene probe that targets clusters of highly
homologous genes. For example, both 18S and 5S ribosomal genes are
frequently present in gene clusters containing hundreds of copies of nearly
identical genes. Thus, even small (2 kb) nick-translated probes or
fluorochrome-labeled oligonucleotides can generate bright FISH signals. Other
gene clusters are also possibilities, but will have to be identified by candidate or
bioinformatic screening. For example, 1-kb probes developed from disease
34
resistance loci in soybean can be used because such genes are present in clusters
containing 10-30 copies within relatively confined (1 megabase) regions.
Repetitive DNA elements represent an extremely valuable resource for
karyotyping probes not only because repeats can be present in high-copy
clusters, but also because elements can have broad and variable distribution
across multiple or all chromosomes. Candidate repeat probes will have to be
identified by screening collections of genomic sequences obtained by
whole-genome shotgun sequencing, BAC-end sequencing etc. From an efficiency
perspective, the larger the sequence collection, the better. This is the case
because the most successful repeat probes will likely be developed from the more
abundant repeat classes, which can be best assessed using a large collection of
sequences. Two starting points for any repeat screens are (candidate)
centromeric repeats and trinucleotide repeats. The latter are a type of simple
sequence repeats common to many organisms; all 64 variants can be quickly and
inexpensively screened using a set of twelve fluorescein-labeled oligonucleotides.
Centromeric repeats are also potentially present in widely variable copy number
and as sequence variants. In designing oligonucleotide probes (see section on 5´
End-labeled Oligos) based on centromeric (or other) repeat types, it is certainly
worth exploring sequence variants for distribution variation in the genome by
making probes in a second fluorochrome color (red).
35
Rolling Circle Amplification (RCA) of BAC
Miniprep DNA for Nick-translated Probes
FISH probes derived from BACs containing genomic DNA inserts represent a
powerful addition to the FISH tool kit. Collections of BACs, however, need to be
carefully screened for repeat content. By using the RCA-based BAC probe
protocol developed by Berr and Schubert (2006), microgram quantities of BAC
DNA can be generated for making nick-translated probes, facilitating screening
of large numbers of BACs.
Making BAC FISH probes consists of the following steps:
1) Small-scale overnight BAC cultures.
2) Kit-based miniprep DNA purification.
3) RCA using a small quantity (20 ng) of purified BAC DNA.
4) Nick-translation (NT) using an aliquot of the RCA reaction.
5) Purification of NT reactions using a commercial PCR/Gel clean-up kit.
BAC DNA Preps
To make BAC DNA, grow a 2-mL overnight culture (in LB + antibiotic
appropriate for the BAC vector) and work up 1.6 mL using a commercial
miniprep kit (e.g. the Promega Wizard Plus SV Miniprep DNA Purification Kit).
Elute the DNA in a small volume (30 µL). BAC DNA prepared in this way is
highly pure (run a minigel to visualize) and sufficiently high in yield (15-30
ng/µL) for multiple RCA reactions.
Rolling Circle Amplification (RCA)
To generate a sufficient quantity (micrograms) of BAC DNA to make NT FISH
probes, RCA is used. The following protocol is virtually unmodified from that
described in Berr and Schubert (2006) (see below).
RCA consists of three steps, which are carried out in a thermal cycler:
1) BAC DNA is first heat-denatured in the presence of thiophosphate-modified
random hexamer primers (called "TRP", below).
2) The denatured samples are cooled on ice.
3) RCA is carried out under isothermal conditions.
4) The reaction is terminated by heat-denaturation.
36
STEP 1: Denaturation
For each BAC, the following mix is assembled in a PCR tube (a master mix
may be used):
PRIMER MIX (5 µL)
DNA (20ng)
2X Annealing Buffer
1 mM TRP*
H2 O
Total primer mix volume:
1X
1 µL
2.5 µL
0.5 µL
1 µL
5 µL
NOTES
A
B
C
Incubate at 95°C for 3 minutes in a thermal cycler (with a heated lid, if
possible); transfer to wet ice for 5 minutes and then briefly spin down any
condensation in a microfuge.
STEPS 2+3: RCA and enzyme inactivation
Next, 15 µL of the following ENZYME MIX is added and mixed with the 5 µL
reaction above (total volume will become 20 µL). Mix after each addition, and
make a master mix, if desired:
ENZYME MIX (15 µL)
H2 O
dNTP Solution (4 mM each dNTP)
10X Phi29 DNA Pol. Buffer
Phi29 DNA Polymerase
Total enzyme mix volume:
1X
10.3 µL
2 µL
2 µL
0.7 µL
15 µL
Total volume primer mix + enzyme mix:
20 µL
NOTES
D
E
F
G
Transfer to a thermal cycler set up with the following profile:
8 hours at 30°C (RCA reaction); 10 minutes at 65°C (Enzyme
Denaturation); 4°C (storage).
The completed reactions can be stored at -20°C or 4°C.
See NOTE H.
37
On an agarose gel (below, 0.8%, largest marker is 10 kb), most of the RCA
product is too large/concatenated to enter the gel (it remains in the well).
Nick Translation and Purification
of BAC Probes
An aliquot of the heat-inactivated RCA reaction is then used directly in the
following Nick-Translation Reaction, which uses 2 µg of input RCA-amplified
BAC DNA. A typical RCA yield of 800 ng/µL thus requires 2.5 µL for 2 µg (See
note H).
Nick-Translation (20 µL)
RCA Product (for 2 µg)
H2 O
10X Nick Translation Buffer
2 mM [-C] dNTP Mix
1 mM Texas Red-5-dCTP*
DNA Polymerase I (10 U/µL)
DNAse I (0.1 U/µL)
Total volume
1X
2.5 µL
7.7 µL
2 µL
2 µL
0.4 µL
5 µL
0.4 µL
20 µL
NOTES
H
I
J
K
L
Incubate at 15°C for 2 hours and proceed to probe purification (below).
Probe Purification
Nick-translation reactions are then processed using Promega's Wizard SV Gel
and PCR Clean-Up Kit (or equivalent), using a 30-µL elution volume. Eluted
probes are then dried in a Speedvac (or equivalent) and resuspended in 20 µL of
2X SSC/1X TE. Probes purified in this way can retain a variable, but low level of
unincorporated Texas Red-5-dCTP that generally does not contribute
significantly to FISH background. Typically, using 2 µL of probe per slide is
38
sufficient to generate a strong signal in FISH. Because the probes are not
ethanol precipitated, numerous (>10) BAC probes can be combined into a single
cocktail. (The salmon sperm DNA used during ethanol precipitation can
compromise FISH signal if numerous probes are combined in a single
hybridization mix).
ADDITIONAL COMMENTS (See also NOTES, below)
The RCA reactions (stored at 4°C or -20°C) can develop a small white precipitate
over a prolonged storage period; avoid the pellet during pipetting. Because the
RCA product is used directly in the nick-translation, it is probably a good idea
not to deviate from the recommended amount of RCA product input, because
any residual dNTPs from the RCA could out-compete the labeled nucleotide in
the labeling reaction. Probes purified with Promega Wizard columns can retain
a variable amount of unincorporated label, which itself (in the absence of HMW
DNA) binds quite well to the column matrix. Thus, the purity of the probe seems
to depend on the efficiency of the previous reactions (high RCA yield, good
nick-translation efficiency). It is therefore recommended that a fraction (~5 µL)
of newly synthesized BAC probe be assessed on a 1.5% agarose, 1X TAE gel. The
gel is run without ethidium bromide, photographed, and then stained to
visualize DNA size markers. There should be two types of signal: 1) the
BAC-derived DNA probe, which runs as a smear from between ~100 bp and
~500 bp, and 2) a variable, but low amount of unincorporated Texas Red-5-dCTP,
which actually runs at ~ 500 bp marker.
NOTES:
A) The published protocol suggests that 5-20 ng, or even a small volume of
liquid culture may be used. Direct Texas Red-5-dCTP labeling of BAC DNA
during the RCA is also possible; see Berr and Schubert (2006) for details.
B) 2X Annealing Buffer = 80 mM Tris-HCl pH 8.0, 20 mM MgCl2.
C) Thiophosphate-modified random hexamer primers are denoted
5´-NpNpNpNpSNpSN-3´. When ordering online at IDT (Integrated DNA
Technologies), enter "NNNN*N*N". In the original protocol, HPLC purification
of the hexamers was recommended; however, with HPLC, the yield is severely
reduced, and the step does not seem to be necessary.
39
D) dNTP Solution = 4 mM dATP, 4 mM dCTP, 4 mM dGTP, 4 mM dTTP; freshly
diluted from 100 mM stock solution into H2O.
E) Phi29 DNA Pol. Buffer is supplied with enzyme when ordered from New
England Biolabs (NEB). The 1X (final=working) buffer, diluted from the 10X
stock is: 50 mM Tris-HCl, 10 mM (NH4)2SO4, 10 mM MgCl2, 4 mM dithiothreitol (DTT), pH 7.5 at 25°C. As noted in the technical information from NEB,
full enzyme activity requires (fresh) DTT. If the buffer is not fresh, then DTT
must be added.
F) Phi29 DNA Polymerase can be ordered from New England Biolabs: Cat. #
M0269L, supplied at a concentration of 10 U/µL. The polymerase may be less
stable than a typical molecular biology enzyme, so fresh enzyme (within its
expiration range) is recommended.
G) A master mix can be prepared, if desired.
H) The post-reaction A260 reading (via Nanodrop) is typically 700-800 ng/µL, a
value that might reflect some residual free nucleotides. However, this is the
number used to calculate the volume required for 2 µg (for the nick translation
reaction).
Thus, 2 µg of RCA-amplified BAC DNA requires 2.5 µL of an 800 ng/µL solution.
The total volume of BAC + H2O is 10.2 µL; modify the volume of H2O to
accommodate varying DNA concentrations.
I: The 2 mM [-C] dNTP Mix is a solution of: 2 mM dATP, 2 mM dGTP, 2 mM
dTTP (There is no unlabelled dCTP; freshly diluted from 100 mM stock solution
into H2O).
J: Fluorescein dUTP also works well for BACs; this nucleotide will require a
2 mM [-T] dNTP mix.
K: DNA Polymerase I is available as a 10 U/µL stock from Life Technologies,
NEB etc.
L: DNAse I is available from Roche.
Reference:
Berr A and Schubert I (2006) Direct labeling of BAC-DNA by rolling-circle
amplification. The Plant Journal 45:857-862.
40
Alternate Strategy for Labeling
of Small (Repeat) Probes
Life Technologies ULYSIS Labeling Technology
One powerful and rapid technique for screening repeat sequences is to use the
ULYSIS labeling technique (Molecular Probes/Invitrogen) to label DNA
amplicons of repeats generated by PCR. ULYSIS is a non-enzymatic method
that directly labels G residues of DNA using a platinum-fluorescent dye complex.
In this approach one µg (scalable) of DNA is denatured, labeled in a 15-minute
reaction and then purified over a microfuge-scale gel filtration column (e.g.
BioRad Micro Bio-Spin P-30 or Princeton Separations Centri-Sep). Probes can
then be used directly in FISH. A drawback to this approach is that it requires
amplification and purification of microgram quantity of DNA (e.g. PCR
amplicon) prior to probe production. However, the strategy has several benefits.
First, because theoretically every G residue in the DNA is labeled (as opposed to
the single dye in oligos), individual probes can be much brighter. Second, kits
can be ordered with virtually any color fluor of interest (i.e. Alexa Fluor dyes
ranging from 488 to 660). Third, starting with purified amplicons, a large
number (25-50) of probes can be synthesized in a couple of hours.
41
Preparation of Metaphase Spreads from HiII
Embryogenic Type-II Callus
The nitrous oxide – enzymatic maceration method (Kato 1999; see page 15) that
is used to make slides for FISH from root tip preparations, can also be applied to
somatic embryos collected from maize HiII embryogenic Type-II callus tissue.
Type-II embryogenic callus can be initiated from immature (12-14 days after
pollination) maize embryos following aseptic collection and plating on N6E
medium (Frame et al., 2000). After incubation at 28°C in the dark for 1-4 weeks,
rapidly growing callus tissue is highly embryogenic (somatic embryos will
appear as numerous papules all over the surface of the callus tissue).
When the callus is vigorously growing, it is quite friable and small clusters of
embryos can easily be scraped off of the growing surface. In order to prepare
somatic embryos for FISH, numerous clusters were collected in moistened 0.5mL centrifuge tubes and gassed at 160 psi (1,100 kPa) in nitrous oxide in the
same manner as root-tip samples (see page 15). The tissue was gassed for a total
of 2.5 hrs, although shorter or longer times could be applied depending upon the
desired level of chromosome condensation. Following nitrous oxide treatment,
the samples were fixed in 90% glacial acetic acid on ice for 10 minutes. The
samples were then washed twice with 70% ethanol and stored in 70% ethanol at
4°C.
Clusters of somatic embryos were incubated in RO-purified H2O in 2-mL
centrifuge tubes for 10-20 min (on ice), and then transferred to tubes filled with
citric buffer for 10-20 min (on ice). Samples were gently vortexed several times
during both of these steps. Pieces of embryogenic callus were then briefly set on
dry Kimwipes to absorb extra moisture, and quickly transferred to a surface
where somatic embryos (small 1-2 mm papules) could be excised with a razor
blade. Somatic embryos were treated in the same 20-µL enzyme aliquots (1%
w/w pectolyase, 2% cellulase R-10 w/w in citric buffer) that are used for root tip
analysis. Between four and six somatic embryos were transferred to each of
several enzyme tubes on ice. The samples were incubated in a 37°C water bath
for ~35 minutes (shorter or longer times may be necessary depending upon the
age of the callus and the amount tissue in each tube). Samples were placed on
ice and immediately washed 3-4 times with 70% ethanol. The ethanol was
removed and ~30 µL of acetic acid:methanol (9:1) was added to each tube.
Samples were macerated with a probe, spotted on slides, and visualized
according to root time procedures (see page 15). Although the frequency of
metaphase spreads detected from callus tissue is relatively low (compared to
42
root tip preparations), several sample digestions can be processed in parallel in
order to obtain replicated FISH results for a given sample.
Frame, B. et al. 2000. Production of transgenic maize from bombarded Type II
callus: effect of gold particle size and callus morphology on transformation
efficiency. In Vitro Cell. Dev. Biol-Plant. 36:21-29.
43
Good Spreads from An Adult Corn Plant
1. Pull the plant out of the pot with soil attached and cut off the roots emerging
from the soil with scissors.
2. Put the plant back into the pot and water well.
3. Wait about 3-6 days and pull the plant out of the pot again.
4. Collect tips from "fast growing roots", that is, those roots that are
long and straight without secondary roots. Basically any root that looks
like the root from germinating seeds are worth collecting.
Old roots are almost NEVER worth collecting. Collect root tips from multiple
days. Once a good one is obtained, the rest that look like it that are collected at
the same time are usually good too.
Finally, some of the big root tips are very good with lots of spreads. One
has to digest them a bit longer or cut them in half but if good spreads are
obtained, many slides can be made from a single root tip.
44
Alternative FISH Protocol for Large Targets
This procedure combines a clean-up process together with the time-saving in
situ denaturation procedure discussed on page 20. Although this protocol can
also be used with smaller targets, the results are not as reproducible. However,
incorporation of clean-up steps 2-5 below into the separate denaturation
hybridization protocol (page 19) may increase the signal-to-noise ratio.
Upon completion of the digestion step:
1. Instead of using TE, fill the tube with 70% ethanol (use squeeze bottle if
many samples). Close cap and invert to mix. Set tube on ice. When tissue
settles, remove ethanol. Repeat.
2. Add 70-100 µL 70% ethanol. Use a blunt dissecting needle to "flatten" the
tissue. Flick tube or vortex briefly to separate cells. Return to ice.
3. Prepare a solution of 90% acetic acid - 10% methanol (~100 µL for three
tubes). Use glacial acetic acid and 100% methanol. Make fresh each time
(both components highly volatile).
4. Spin cells approximately 5 sec in tabletop mini microcentrifuge (2K x g).
5. Decant the ethanol, taking care to retain cells (some EtOH remains). Blot
opening of tube on a paper towel, then based on pellet size, add 15-30 µL of
the acetic acid-methanol solution (or use 100% acetic acid). Gently resuspend
cells (tap/flick tube, stir with pipet tip).
6. As in original protocol, drop 5-6 µL of each sample on a labeled microscope
slide in a humid chamber. Allow to dry, then cross-link using "optimal"
setting. Select best slides for hybridization.
7. Make probe cocktail mix (total volume 8-10 µL per slide). The larger
volume gives better coverage.
8. Pipet 8-10 µL of probe mix onto each slide then cover with a 22-mm plastic
cover slip (no need for over-hanging edge).
9. As in original protocol, place slides in humidified aluminum tray, cover, and
put in boiling water bath for 5 min. Transfer directly to humidified plastic
container for hybridization (4 hr to overnight, 55˚C).
10. Dip slides in 2x SSC at room temperature only long enough to pop off the
plastic cover slip, then wipe off the backs of the slides, and apply Vectashield
(with or w/o DAPI). Apply large glass cover slip. Note: The 20-minute 55˚C
rinse in 2x SSC specified in the original protocol is not needed unless target
is low copy number or too much background is observed.
Citation for the above protocol is:
Birchler JA, Albert PS and Gao Z (2008) Stability of repeated sequence clusters
in hybrids of maize as revealed by FISH. Tropical Plant Biology 1: 34-39.
45
References (Chronological Order)
Kato A (1999) Air drying method using nitrous oxide for chromosome counting in
maize. Biotech. Histochem. 3:160-166.
Kato A, Lamb JC and Birchler JA (2004) Chromosome painting using repetitive
DNA sequence as probes for somatic chromosome identification in maize. Proc. Natl. Acad.
Sci. USA 101: 13554-13559.
Kato A, Vega JM, Han F, Lamb JC and Birchler JA (2005) Advances in plant
chromosome identification and cytogenetic techniques. Curr. Opin. Plant Biol. 8:148-154.
Kato A, Albert PS, Vega JM and Birchler JA (2006) Sensitive FISH signal
detection using directly labeled probes produced by high concentration DNA polymerase
nick translation in maize. Biotech. Histochem. 81: 71-78.
Lamb JC, Kato A, Yu W, Han F, Albert PS and Birchler JA (2006) Cytogenetics
and chromosome analytical techniques. IN: Floriculture, Ornamental and Plant
Biotechnology: Advanced and Topical Issues. Jaime A. Teixeira da Silva. Global Science
Books, London.
Lamb JC and Birchler JA (2006) Retroelement Genome Painting: Cytological
visualization of retroelement expansions in the genera Zea and Tripsacum. Genetics 173:
1007-1021.
Bauer MJ and Birchler JA (2006) Organization of endoreduplicated chromosomes
in the endosperm of Zea mays L. Chromosoma 115: 383-394.
Yu W, Lamb JC, Han F and Birchler JA (2007) Cytological visualization of DNA
transposons and their transposition pattern in somatic cells of maize. Genetics 175: 31-39.
Lamb JC, Danilova T, Bauer MJ, Meyer J, Holland JJ, Jensen MD and Birchler JA
(2007) Single gene detection and karyotyping using small target FISH on maize somatic
chromosomes. Genetics 175: 1047-1058.
Lamb JC, Meyer JM, Corcoran B, Kato A, Han F and Birchler JA (2007) Distinct
chromosomal distributions of highly repetitive sequences in maize. Chromosome Research
15: 33-49.
Lough AN, Roark LM, Kato A, Ream TS, Lamb JC, Birchler JA and Newton KJ
(2008) Mitochondrial DNA transfer to the nucleus generates extensive insertion site
variation in maize. Genetics 178: 47-55.
Danilova TV and Birchler JA (2008) Integrated cytogenetic map of mitotic
metaphase chromosome 9 of maize: resolution, sensitivity and banding paint development.
Chromosoma 117: 345-356.
Birchler JA, Albert PS and Gao Z (2008) Stability of repeated sequence clusters in
hybrids of maize as revealed by FISH. Tropical Plant Biology1: 34-39.
46
Appendix A. M aize probe cocktail plasm ids
Sequence
Vector
Primer sequence*
PCR product
Bacteria†
Cent4
pBluescript
M13 F + R
800 bp
DH5 alpha
CentC
pBluescript
M13 F + R
540 bp
DH5 alpha
MR77 (TR-1)
pGem-T
M13 F + R
MR 77 F + R
400 bp
DH5 alpha
2-3-3
pGem-T
M13 F + R
BEH 2F
700 bp
DH5 alpha
2 kb
DH10B
NOR 173
pBluescript
M13 F + R
ladder§
pGem-T
M13 F + R
EBH 1F
700 bp
DH5 alpha
Knob 3-copy
pGem-T
M13 F + R
knob F + R
540 bp
Stbl4
1-26-2
pGem-T
M13 F + R
EBH 1F
500 bp smear
Stbl4
4-12-1
pGem-T
M13 F + R
BEH 2F
1 kb
DH5 alpha
pBluescript
M13 F + R
1.1 kb
DH5 alpha
NOR
1.1 (pMTY9ER)
_____________
*Common primers (first column) can be used to amplify the given target sequence;
however, for some sequences, internal primers (second column) result in stronger probe
signals. One explanation for the weaker signals is cross-probe hybridization at shared M13
vector sequences.
†Ampicillin
§NOR
(or carbenicillin) resistant, 100 µg/mL.
ladder produces a better signal than NOR 173, although both work well for most
applications.
Common primer sequences
T3
T7
M13
M13
5’-TAA CCC TCA CTA AAG
5’-TAA TAC GAC TCA CTA
forward
5’-CCC AGT CAC
reverse
5’-AGC GGA TAA
GGA-3’
TAG GG-3’
GAC GTT GTA AAA CG-3’
CAA TTT CAC ACA GG-3’
Specific primer sequences
EBH 1F*
BEH 2F*
Knob F
Knob R
MR77 F
MR77 R
5’-AGA
5’-AGG
5’-GGC
5’-GGC
5’-CCT
5’-CAC
ATT
ATC
CAC
CAT
CAA
TCA
CGG ATC CAA GCT TCT GGT TTG-3’
CGA ATT CAA CGT TGT CTT TG-3’
ACA ACC CCC ATT TTT G-3’
TGA TCA TCG ACC AGA-3’
ATG CCG TTT CCT AT-3’
CGC AAT TTG GCT AA-3’
*Reverse primer not required; same sequence on both ends
of insert.
Note: Error in PNAS (2004). EBH lF should not have a T at
the 3’end. Sequence is correct as written above.
47
Appendix B. M aize probe cocktail DNA
Fluorochrome
color
Target DNA
[Initial]
ng/µL
[Final]
ng/µL*
µL probe‡
Blue
Knob
200
40
1.0
4.0
Green
Green
Green
Green
4-12-1+
NOR 173
CentC
2-3-3
200
10
100
200
40
0.2
2
18
1.0
0.1
0.1
0.45
4.0
0.4
0.4
1.8
Red
Red
Red
Red
1.1 (pMTY9ER)+
Cent4
1-26-2
2-3-3
200
200
200
200
40
10
6
18
1.0
0.25
0.15
0.45
4.0
1.0
0.6
1.8
Far red
TR-1 (MR77)
200
20
0.5
2.0
_________________
Total volume
5 µL
20 µL
* from Kato et al. (2004)
‡ amount per slide followed by amount for four slides
+
small chromosomal targets; all others, large
Notes:
1. If 8-10 µL of total volume per slide is preferred, add 2x SSC, 1x TE to make up the
difference. Increase the concentrations of individual probes only if FISH signals are
weaker than desired.
2. If the full cocktail is not needed, add 2x SSC, 1x TE to adjust volume accordingly.
3. Amounts of individual probes will need to be adjusted to balance signal strength
among targets labeled with the same fluorochrome.
4. For untested probes, hybridize at a starting concentration of 40 ng/µL. If the probe
signal is weak relative to a fairly strong background signal, reducing the probe
concentration could yield better results.
5. For more complex cocktail mixes, it might be necessary to dissolve probe DNA
pellets at concentrations greater than 200 ng/µL.
Fluorochromes:
1. Blue, coumarin
2. Green, fluorescein or Alexa Fluor 488
3. Red, Texas Red
4. Far red, cyanine 5 (Cy5)
48
Appendix C. M inim ization of background
Metaphase spreads with little or no cytoplasmic background and in which all the
chromosomes are lying flat on the slide (same focal plane) and have good morphology (as
viewed with white light at 40x) are the best candidates for good hybridization results if the
probe itself is of good quality.
Increasing the concentration of cellulase will help minimize cytoplasmic background.
Using 100% acetic acid as a spreading agent may also help decrease background.
Probes made by random primer labeling tend to produce high background. Nick translation
labeling is recommended.
Probe DNA more than about 400-500 bp in size (after nick translation) produces higher
background. Though seldom necessary to do, the size can be checked by running probe DNA
on a mini-gel to ensure it is 50-350 bp in size (acquire probe image prior to staining gel with
ethidium bromide, which is needed to view DNA ladder bands but will obscure the probe
band).
Denaturation of the probe and chromosomal DNAs separately is better for the reduction of
background.
Some DNA strands with repeated sequences can produce double stranded DNA complexes
by self annealing. When these complexes are formed within chromatin during hybridization
and they are large enough, they will not be removed during the stringent wash and will
cause high background.
Some background is caused by addition of dextran sulfate (10%), a common ingredient in
hybridization solutions. This substance is added to increase the efficiency of a probe at low
concentrations. This substance also precipitates proteins and causes a high background. It
is necessary to remove all proteins from the probe solution if dextran sulfate is added. Also,
DNA greater than 1 kb precipitates and forms crystals in the cell when dextran sulfate is
used. Probe and salmon sperm DNA should be short (100-200 bp) if dextran sulfate is used.
The procedure presented here avoids this problem by using an undiluted, high
concentration probe solution applied directly to the metaphase spreads.
Unincorporated nucleotides of Texas Red-5-dUTP will produce high background.
49
Appendix D. Kits and reagents
DNA preparation
Plasmid DNA:
Wizard Plus Minipreps DNA purification system, Promega Cat. No. A7510
Qiagen Plasmid Maxi Kit, Cat. No. 12163; Mini prep kit, Cat. No. 27106
PCR/Gels:
JumpStart ReadyMix REDTaq DNA polymerase, Sigma-Aldrich Cat. No. P-0982
GoTaq Green Master Mix, Promega Cat. No. M7122
HotStarTaq DNA polymerase, Qiagen Cat. No. 203205
Primers from Integrated DNA Technologies, http://www.idtdna.com
10 mM dNTP Mix, Life Technologies Cat. No. 18427-013
Agarose, low EEO, electrophoresis grade, Fisher Cat. No. BP160-500
2-Log Ladder, New England Biolabs Cat. No. N3200
Gel and PCR clean up system, Promega Cat. No. A9282
T is s u e p r e p a r a t io n
Cellulase Onozuka R-10, Yakult Pharmaceutical Ind. Co., Cat. No. L0012
Pectolyase Y-23, Yakult (no cat No.) or MP Biomedicals Cat. No. 32095
L a b e lin g
Slide related:
Microscope slides, Gold Seal, Fisher Cat. No. 12-518-100A
Plastic cover slips, Fisher, Cat. No. 12-547, 22 mm
Glass cover slips, Fisher Cat. No. 12-545F (24 x 50 mm)
Tough-Tags, Diversified Biotech Cat. No. TTSW-1000, TTLW-1000
Tough-Spots, Diversified Biotech Cat. No. T-Spots, T-Spots-50
Vectashield Mounting medium, Vector Laboratories, Cat. No. H-1000
Vectashield Mounting medium with DAPI, 1.5 µg/mL; with PI, 1.5 µg/mL,
Vector Laboratories, Cat. Nos. H-1200 and H-1300, respectively
Nick translation and probe purification:
Chroma Tide Alexa Fluor 488-5-dUTP, Life Technologies, Cat. No. C-11397
Fluorescein-12-dUTP, Perkin Elmer, Cat. No. NEL413001EA
Texas Red-5-dCTP, Perkin Elmer, Cat. No. NEL426001EA
Cyanine 5-5-dUTP, Perkin Elmer, Cat. No. NEL579001EA
100 mM dNTP Set, Invitrogen Cat. No. 10297-018.
DNA Polymerase I, Life Technologies Cat. No. 18010-017 or 18010-025 (1000U)
DNase I recombinant grade I, Roche, Cat. No. 04536282001
Bio-Gel P-60, Bio-Rad Laboratories, Cat. No. 150-4160
Pasteur pipets for Bio-Gel columns, Fisher Cat. No. 13-678-20A
Glass wool, silane treated, Sigma-Aldrich Cat. No. 20411
50
Appendix E. Source information (partial listing)
Chroma Technology Corp., 10 Imtec Lane, Bellows Falls, VT 05101, Tel 800-824-7662
http://www.chroma.com
(microscope filters)
Fisher Scientific, 300 Industry Drive, Pittsburgh, PA 15275, 800-766-7000
http://www.fishersci.com (Pasteur pipets, slides, cover slips)
Integrated DNA Technologies, 1710 Commercial Park, Coralville, IA 52241
800-328-2661, http://www.idtdna.com
(primers, labeled oligos)
Life Technologies, 3175 Staley Rd, Grand Island, NY 14072, Tel 800-955-6288
http://www.lifetechnologies.com
(enzymes, fluorochromes)
MP Biomedicals (formerly ICN Biomedical, Inc.), 29525 Fountain Parkway, Solon, OH
44139, Tel 800-854-0530, http://www.mpbio.com
(pectolyase)
Perkin Elmer Life Sciences, 940 Winter Street, Waltham MA 02451, Tel 800-762-4000
http://www.perkinelmer.com
(fluorochromes)
Promega Corporation, 2800 Woods Hollow Road, Madison, WI 53711, 800-356-9526
(PCR clean-up kit, dNTPs)
http://www.promega.com
Qiagen Inc., 27220 Turnberry Lane, Suite 200, Valencia, CA 91355, Tel 800-426-8157,
http://www.qiagen.com
(plasmid prep)
Roche Applied Science, 9115 Hague Road, Indianapolis, IN 46256, 800-262-1640
http://www.lifescience.roche.com
(DNase)
Semrock, Inc., 3625 Buffalo Road, Suite 6, Rochester, NY 14624, 866-736-7625
http://www.semrock.com
(microscope filters)
Sigma-Aldrich Corporation, Box 14508, St. Louis, MO 63178, 800-325-3010,
http://www.sigmaaldrich.com
(primers, silanized glass wool, salmon sperm DNA)
Vector Laboratories, 30 Ingold Road, Burlingame, California 94010, 800-227-6666
http://www.vectorlabs.com/
(Vectashield mounting medium)
Yakult Pharmaceutical Ind. Co., LTD, Tokyo, Japan, [email protected]
http://www.yakult.co.jp/ypi/en/product/#enzyme, then select ‘For laboratory’ and email
for prices. Need to ask about pectolyase (not listed)
51
(cellulase, pectolyase)
Appendix F. Solutions frequently used in FISH
Scale down volumes according to needs.
5x Citric buffer
for 1000 mL
50 mM sodium citrate
14.7 g (dihydrate)
FW 294.1
50 mM disodium EDTA
18.6 g (dihydrate)
FW 372.24
Adjust to pH 5.5 by adding 1M citric acid. Autoclave.
6x Loading dye
for 10 mL
0.25% bromophenol blue
25 mg
0.25% xylene cyanol FF
25 mg
40% sucrose
4g
In TE buffer. Store at 4˚C. (or use 6x stock that comes with DNA ladder)
10x Nick translation buffer
for 100 mL
500 mM Tris base
6.05 g
50 mM MgCl2
476.0 mg
FW 121.14
FW
95.2
add HCl to pH 7.8
100 mM 2-mercaptoethanol
701.0 µL
(+ 100 µg/mL bovine serum albumin fraction V, Sigma-Aldrich A-9647, optional)
Does not store well, even at -20˚C. Better to make pH adjusted stock, sterilize and
freeze in aliquots of 993 µL. Thaw as needed and add 7 µL 2-mercaptoethanol.
When odor starts to diminish, make new.
Salmon sperm DNA (denatured)
Sigma-Aldrich D1626.
Dissolve DNA in 1x TE buffer pH 7.8 at a concentration of 10
µg/µL. Place on a rotary shaker (overnight, if necessary). The DNA must be completely
dissolved prior to autoclaving for 30 minutes. Run sample on gel to determine the size
of the DNA fragments. They should be about 100-300 bp in length. Store at -20˚C in
1-mL aliquots. Sheared salmon sperm DNA may also be purchased.
S o d i u m a c e t a t e 3 M , p H 5 .2
for 200 mL
3 M sodium acetate
49.2 g (anhydrous)
Adjust pH to 5.2 with glacial acetic acid. Autoclave.
52
FW 82.03
20x SSC
3 M NaCl
for 1000 mL
175.3 g
0.3 M sodium citrate
88.2 g (dihydrate)
FW 58.44
FW 294.1
(+ 20 mM EDTA, optional)
Adjust pH to 7.0 with a few drops of concentrated HCl. Pass through 0.45-µm filter
(optional). Autoclave. Store at room temperature.
Stop buffer
for 500 mL
0.5 M disodium EDTA
93.1 g (dihydrate)
FW 372.24
NaOH
20.0 g
FW
40.0
Adjust to pH 8.0. Won’t go into solution until pH close to 8. Autoclave. Store at room
temperature to avoid precipitation.
10x TE
for 1000 mL
100 mM Tris base
12.1 g
10 mM disodium EDTA
3.7 g (dihydrate)
FW 121.14
FW 372.24
Add HCl to pH 7.6 and pass through 0.45-µm filter. Autoclave.
50x TAE buffer (gels)
2 M Tris base
for 1000 mL
242.0 g
1 M glacial acetic acid
0.05 M EDTA
FW 121.14
57.1 mL
100.0 mL (0.5 M EDTA, pH 8.0)
53
Appendix G. Nitrous oxide gas cham ber
Gas chamber, fitted with a silicone rubber gasket and screw-on lid, used to expose roots
to nitrous oxide. The chamber can be used to simultaneously treat samples held in as
many as forty 0.6-mL microcentrifuge tubes, each containing from one to ten root tips
(holes are required in the lids to facilitate gas penetration). Scale bar = 2.54 cm.
[Figure 1 from Kato A, Albert PS, Vega JM, Birchler JA (2006)]
54
Alternative chamber design showing the attachment of a quick-disconnect hose coupling.
The tubing half of the coupling stands alone. Chamber height without the lid is 12.7 cm.
55