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There are many ways to detect polymorphism. In this study we use the microsatellite markers to detect the polymorphism for the salt tolerance. This method has been successfully conducted in Oryza sativa and Brassica juncea. The results are reproducible. In contrast to previous methods, our method is simple and quite accurate for detecting the polymorphic bands. In this study instead of using agarose gel and ethidium bromide staining, we used non-denaturing polyacrylamide gel and a low-cost improved method for silver staining when we compare it to 11 other methods for their ability to detect simple sequence repeat polymorphisms as small as 50 bp in denaturing polyacrylamide gels. All methods detected the same alleles and banding pattern. However, important differences in sensitivity, contrast, time consumption and background were observed.
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Simple and efcient way to detect small polymorphic bands in plants
Manu Kumar
a,
, Seong Ryong Kim
a
, Prabodh Chander Sharma
b
, Ashwani Pareek
c
a
Department of Life Science, Sogang University, Seoul 121-742, South Korea
b
Crop Improvement Division, Central Soil Salinity Research Institute, Karnal 132 001 (Haryana) India
c
Stress Physiology and Molecular Biology Laboratory, School of Life Science, Jawaharlal Nehru University, New Delhi 110067, India
abstractarticle info
Article history:
Received 9 April 2015
Received in revised form 2 June 2015
Accepted 3 June 2015
Available online 11 June 2015
Keywords:
Oryza sativa
Brassica juncea
Microsatellite
SSR
PAGE
Silver staining
There are many ways to detect polymorphism. In this study we use the microsatellite markers to detect the
polymorphism for the salt tolerance. This method has been successfully conducted in Oryza sativa and Brassica
juncea. The results are reproducible. In contrast to previous methods, our method is simple and quite accurate
for detecting the polymorphic bands. In this study instead of using agarose gel and ethidium bromide staining,
we used non-denaturing polyacrylamide gel and a low-cost improved method for silver staining when we
compare it to 11 other methods for their ability to detect simple sequence repeat polymorphisms as small as
50 bp in denaturing polyacrylamide gels. All methods detected the same alleles and banding pattern. However,
important differences in sensitivity, contrast, time consumption and background were observed.
© 2015 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license
(http://creativecommons.org/licenses/by/4.0/).
Introduction
In plant breeding, conservation and evolutionary studies, molecular
markers such as simple sequence repeats (SSRs) or sequence-tagged
microsatellite sites (STMS), are widely used (e.g., genome mapping,
marker assisted selection). Efcient methodology is required for mass
genotyping [1]. Uses of the radioactive or uorescent labelled nucleo-
tides are the most efcient way to visualize single strand DNA in poly-
acrylamide gels. These procedures are expensive, time consuming and
require special facilities and it makes them impracticable for most of
the tropical countries where sophisticated infrastructures are lacking
[2].
The silver-staining of proteins has been used for a wide variety of
physical and biological analyses in polyacrylamide gels. In last decade,
it has been applied in polyacrylamide gels to detect nucleic acid as silver
ions bind to the bases and then under alkaline conditions it can be selec-
tively reduced by formaldehyde. For detecting DNA in PCR-single strand
conformation polymorphism analysis, silver staining of nucleic acid
is widely used. Many other alternative methods to silver staining for
DNA detection have been described previously. However, most of
them are not rapid enough [3] becauseoftimeconsumingstepsandin-
volve the changing of solutionsrepeatedly. It is hard to establish a highly
output staining method. Although many simplied methods have been
reported before [414], they still lack in sensitivity and/or efciency.
The main objective of this study was to evaluate and standardize a
new low-cost method for detecting polymorphism usingsilver staining
using rice (Oryza sativa) and Indian mustard (Brassica juncea)asa
model system. We compared sensitivity of this procedure to the 11
other commonly used procedures [414] and optimized the reaction
conditions for detection of polymorphism by using denaturing poly-
acrylamide gels.
Materials
Reagents and equipment
Genomic DNA isolation
CTAB buffer, Microfuge tubes, Mortar and Pestle, Liquid nitrogen,
Microfuge, 70% Ethanol (ice cold), Isopropanol, 60 °C water bath, Chlo-
roform: Iso-amyl alcohol (24:1), Water (sterile), Agarose, 6× Loading
buffer, 1× TBE solution, Agarose gel electrophoresis system, Ethidium
bromide solution.
Elution buffer 100 ml. 2.0 g CTAB (Hexadecyl trimethyl-ammonium bro-
mide), 28.0 ml 5 M NaCl, 4.0ml 0.5 M EDTA pH (8.0), 10.0 ml 1 M Tris-cl
(pH 8.0), 1% (v/v) 2-Mercaptoethanol, 1% (w/v) polyvinyl-pyrrolidone
Genomics Data 5 (2015) 218222
Abbreviations:SSR, simple sequence repeat;TBE, tris borateethylenediaminetetracetic
acid; TEMED, tetramethylethylenediamine.
Corresponding author at: Department of Life Science, Sogang University, Seoul
121-742, South Korea.
E-mail addresses: manukumar007@gmail.com,manukumar7@sogang.ac.kr
(M. Kumar).
http://dx.doi.org/10.1016/j.gdata.2015.06.006
2213-5960/© 2015 The Authors. Published by Elsevier Inc. This is an open access article under the CCBY license (http://creativecommons.org/licenses/by/4.0/).
Contents lists available at ScienceDirect
Genomics Data
journal homepage: http://www.journals.elsevier.com/genomics-data/
(PVP, Mw 10,000). Adjust all to pH 5.0 with HCL and make up to 100 ml
with H
2
O.
1 M Tris pH 8.0. Dissolve 121.1g of Tris base in 800 ml of H
2
O. Adjust pH
to 8.0 by adding 42 ml of concentrated HCL. Allow the solution to cool to
room temperature before making the nal adjustments to the pH.
Adjustthevolumeto1lwithH
2
O. Sterilize using an autoclave.
Polymerase chain reaction using microsatellite markers
10 mM TrisHCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl
2
,200μMdNTP,
0.4 μM 20 mer primer, 1 unit Taq DNA polymerase, 20 ng template DNA.
Non-denaturing polyacrylamide gel electrophoresis for the separation of
the bands
Buffer and solutions. Acrylamide: bisacrylamide (29:1) (% w/v), Ammo-
nium persulfate (10% w/v), Ethanol, KOH/methanol, 6× Gel-loading
buffer, 5× TBE Electrophoresis buffer, TEMED.
Special equipment. Electrophoresis apparatus, glass plates, combs and
spacers, gel-sealing tape, micropipette with drawn-out plastic tip,
petroleum jelly and syringe.
Detection of the polymorphism in non-denatured polyacrylamide gel by
silver staining
Acetic acid (3% v/v), Developer Dissolve 30 g of sodium carbonate
in a nal volume of 1 l of distilled H
2
O. Ethanol (10% v/v), Formaldehyde
(37% v/v), Nitric acid (0.7% v/v), Silver nitrate (0.2% w/v) freshly
prepared and Gel scanner.
Procedure
Legend
The study comprised of four main steps (Fig. 2):
1. Genomic DNA isolation.
2. Polymerase chain reaction using microsatellite markers.
3. Non-denaturing polyacrylamide gel electrophoresis for the se paration
of the bands.
4. Detection of the polymorphism in polyacrylamide by silver staining.
Genomic DNA isolation
Extraction of DNA from 10 DAG seedlings was isolated from salt
tolerant and salt sensitive wild type variety of O. sativa and B. juncea
as described by [1516].
Polymerase chain reaction using microsatellite markers
:Annealing temperature (Tm)can be adjusted according to
primer set.
Amplication reactions were carried out in a volume of 20 μl. Reac-
tion mixtures contained 10 mM TrisHCl (pH 8.3), 50 mM KCl, 1.5 mM
MgCl
2
, 200 μMeachdNTP,0.4μM of 20-mer primer, 1 unit Taq DNA po-
lymerase and approximately 20 ng of template DNA. The amplication
was carried out using a thermocycler. The ther mal cycler was programmed
to one cycle of 4 min at 94 °C for the initial strand separation, followed
Fig. 2. Flowdiagram to illustratethe major stepsof the way to assess the smallpolymorphic
bands. Each step, which is shown in parentheses, corresponds to thestep in the Procedure
section.
Fig. 1. PAGE of SSR markers of salt tolerant mutant lines of Oryza sativa and Brassica
juncea. (a). PAGE of mutant lines(110) of rice amplied with specicSSRmarker
(details not given) at Tm 64 °C. Pokkali is a wild type salt tolerant cultivar. (b).
PAGE of mutant lines (15) of Brassicajuncea amplied with specic SSR marker (details
not given) at Tm 66 °C. Lines 1, 2, 4 and 5 are salt tolerant lines. Red arrow indicates
polymorphism.
219M. Kumar et al. / Genomics Data 5 (2015) 218222
by 35 cycles of 30 s at 94 °C for denaturation, 1 min at 60 °C and 72 °C for
primer extension. Finally, one cycle of 10 min at 72 °C was used for the
nal extension, followed by storing at 4 °C.
:Percentage of the polyacrylamide gel depends on the size of
the fragment of DNA.
Non-denaturing polyacrylamide gel electrophoresis for the separation of
the bands
1. Wash the glass plates and spacers well rst in tap water then in
deionized water. Hold the plates from the edges to save the surface
of the glasses. Rinse the plates with ethanol and let them dry.
2. Assemble the glass plate with spacer.
:Apply the agarose gel or wrap the tape around it
except on the top so that gel liquid does not leak out.
3. Taking into account the size of polymorphic bands, the size of glass
plate and the thickness of the spacers 6% of PAGE (50 ml) were made
by using 10 ml 30% acrylamide +1% N,N-Methylenebisacrylamide,
29.965 ml H
2
O, 10 ml 5× TBE and 350 μl of 10% ammonium persul-
fate.
4. Add 17.5 μl TEMED for 50 ml of acrylamide: bis solution to polymer-
ize the gel.
5. Pour the casting solution into the glass plate carefully.
:Be careful,air bubble under the teeth of the comb
should be avoided,immediately insert the comb into the gel. Make sure
the gel does not leak.
6. Allow the acrylamide to polymerize for 30 min at room temperature.
7. After polymerization, remove combs very carefully and rinse the
wells with 1× TBE.
8. Attach the gel to electrophoretic tank using the clamps built in the
apparatus. Fill the electrophoretic tank with the 5× TBE. Use pipette
to ush out wells once more.
:Make sure that the wells you are using are absolutely
clean from gel pieces and the well should be intact to get a better result.
9. 6 × gel loading buffer was made by 0.25% bromophenol blue,
0.25% xylene cyanol FF and 30% glycerol in water.
10. 20 μl PCR products were used for loading with 3 μl of 6× dye.
11. Run the gel on 50 V till dye comes closer to bottom.
:Running the gel to lower voltage is very important for
the bands to be linear.
Detection of the polymorphism in non-denatured polyacrylamide gel by
silver staining
:Be neat and clean,silver nitrate will stain skin,cloths,walls,
oors and anything that comes in contact with it. Powder free gloves should
be used. Container for gel should be large enough so that gels can oat
freely.
12. After electrophoresis, place the gel very carefully (still attached to
one plate) in a plastic tray reserved for silver staining.
:Do not touch the surface of the gel at any time it can
cause background staining from ngerprints,use gloves.
13. Rinse the gel twice with distilled H
2
O to remove electrophoresis
buffer and gel pieces. During rinsing, the gel will oat freely; glass
plate can then be removed.
14. Fix the gel in 10% ethanol by shaking gently for 10 min on theshak-
er. Remove the 10% ethanol by suction and repeat the process 3
times.
:3 times washing is very important to make back-
ground around the gel clear.
:Gel can be left at this stage for several hours in 10% ethanol.
15. Add just enough 0.7% nitric acid to cover the gel. Shake the gel gently
on the shaker for 6 min. Remove the nitric acid by suction and rinse
the gel 2 times with distilled H
2
O.
16. Add just enough 0.2% silver nitrate to cover the gel. Shake the gel
gently for 30 min.
:Make sure staining should be in dark.
17. Rinse the gel and the staining tray 3 times with distilled water.
:3 times washing is very important to remove all silver
nitrate solution.
18. To the 100 ml of developer add 125 μl of formaldehyde solution.
Transfer the developer/formaldehyde solution to the staining tray
and shake the tray gently in an indirect light.
:Cover the container in aluminium foil.
When the solution turns yellow to dark black precipitate become
noticeable; replace the developer/formaldehyde solution with a second
batch of 100 ml of the same solution. Continue to shake the gel in indirect
light. Monitor the appearance of bands and background. When the ratio
of the signal to the noise is high, remove the second batch of developer/
formaldehyde solution.
:Timing of adding fresh developer is very important,
delay can cause the black background of the gel.
19. Add 250 ml of 3% acetic acid to the staining tray immediately. Shake
the gel gently for 5 min. Remove the 3% acetic acid and wash the gel
with 10% ethanol. Remove the ethanol and store the gel for 2 min in a
fresh batch of 10% ethanol. You can clearly see the bright polymor-
phic bands in a white background.
:Timing of adding acetic acid is also very important,
delay can cause the black background of the gel.
:Gel can be stored at this stage for several weeks in 10% ethanol.
20. Scan the gel in gel scanner.
Timing
1. Genomic DNA isolation (23h).
2. Polymerase chain reaction using microsatellite markers (34h).
3. Non-denaturing polyacrylamide gel electrophoresis for the separa-
tion of the bands, steps 120 (56h).
Troubleshooting
Troubleshooting advice can be found in Table 2.
Advantage of silver staining of DNA over other staining methods
1. Silver staining avoids radioactive handling, delays from development
times and waste disposal issues and offers similar sensitivity to auto-
radiography.
2. Under normal light image development and visualization are done.
Therefore, the procedure can be performed entirely at the lab
bench without the need for UV illumination facilities and darkroom.
3. Because silver is deposited directly on the molecules within the
transparent gel matrix, the image is resolved with the best possible
sensitivity and detail.
4. For creating a permanent record of the original material, silver
stained gels can be dried onto a semi-rigid plastic backing lm.
They can also be stored for a long time without breaking, removing
the need and added expense of printing and photography. In
220 M. Kumar et al. / Genomics Data 5 (2015) 218222
Table 1
Comparison of silver staining protocols from this method with others.
Step Method 1
Morrissey [4]
Method 2
Bassam et al.
[5]
Method 3
Sanguinetti
et al. [6]
Method 4
Creste et al. [7]
Method 5Qu
et al. [8]
Method 6
Modication of
Benbouza et al. [9]
Method 7
Ji et al. [10]
Method 8
Zhang et al.
[11]
Method 9 Han
et al. [12]
Method 10
Byun et al. [13]
Method 11 An
et al. [14]
Method 12
Our procedure
Prex 50% methanol, 10% acetic
acid (10 min)
5% methanol, 7% acetic
acid (30 min)
––1.5% nitric acid (3
min)
–– – –
Fixation 10% glutaraldehyde
(30 min)
10% acetic
acid(20 min)
10% acetic
acid
0.5% acetic
acid(3 min)
10% ethanol, 1%
acetic acid (10 min)
10% absolute
ethanol
0.5% acetic acid
(5 min)
–– –
Rinse H
2
O (overnight)
H
2
O (30 min)
H
2
O(2h)
H
2
O (2 min)
3 times
H
2
O (1 min) –– –H
2
O (10 s)
Two times
–– –H
2
O (5 s) 2 times
Soaking 5g/ml dithiothreitol (30
min)
–– – – – –
Impregnation 0.1% AgNO
3
(30 min) 0.1% AgNO
3
1.5 ml
37% HCOH
(30 min)
2% AgNO
3
(5
min)
2% AgNO
3
(20 min) 25% ethanol,
1% nitric acid,
2% AgNO
3
(510)
1.5% AgNO
3
,
1.5 ml
37% HCOH (2224
°C)
(67 min)
0.1% AgNO
3
(1015
min)
0.1% AgNO
3
(4 °C)
(15 min)
1% nitric acid
0.1% AgNO
3
(5 min)
10% ethanol,
0.5%acetic acid,
0.2% AgNO
3
(320 min)
5% ethanol,
1% nitric acid,
0.1% AgNO
3
(5 min)
10% ethanol
0.7% nitric acid
0.22% AgNO
3
,
(Room temp.)
(530 min)
Rinse H
2
O One time H
2
O, 20 s,
optional
Distilled H
2
O,
1s
H
2
O (30 s)
Two times
H
2
O (3 min) Distilled H
2
O, (10 s)
3 times
H
2
O(5s)
Two times
2% NaOH,
0.01%
HCOH, (10
s)
H
2
O(5s)
Two times
H
2
O(5s)
One times
H
2
O (10 s)
Development 50 l 37% formaldehyde
+100 ml 3% Na
2
CO
3
35 min
3% Na
2
CO
3
,
1.5 ml
37% HCOH;
2mg
Na
2
SO
3
.5H
2
O
(10 °C)
(25 min)
1.5% NaOH,
2ml
37% HCOH
(5 min)
3% Na
2
CO
3
, 1.5 ml
0.54 ml 37% HCOH
(4-7 min)
3% Na
2
CO
3
,
0.2% HCOH,
(25 min)
1.5% NaOH,
2ml
37% HCOH (2224
°C)
(35 min)
0.04%
Na
2
CO
3
,
0.2% HCOH,
(56 min)
2% NaOH,
0.01%
HCOH,
(2-3 min)
2% NaOH,
0.04% NaCO
3
,
0.0025% EBT,
0.15% HCOH
(5 min)
3% NaOH,
0.1% HCOH (55
°C)
(510 min)
1.3% NaOH,
0.65% NaCO
3
,
0.4% HCOH;
(23 min)
Per liter:
22.9 g Na
2
CO
3
,
1.25 ml
37% HCOH,
2mgNa
2
SO
3
.5
H
2
O (4 °C)
(35 min)
Stop 5 ml of 2.3 M citric acid 10% acetic
acid(10 °C)
(5 min)
5% acetic acid
(5 min)
10% acetic
acid,(25 min)
0.5% acetic acid
10% absolute
ethanol
(2 min)
H
2
O(5s)
Two times
H
2
O(5s)
Two times
2.5%
ampicillin
(5 s)Two
times
10% ethanol,
0.5%acetic acid
(1 min)
5% ethanol,
1% nitric acid
(1 min)
250 ml of 3%
acetic acid (4 °C)
10% ethanol
5 min
221M. Kumar et al. / Genomics Data 5 (2015) 218222
addition, the preserved gel contained real stained DNA bands that
can be extracted, amplied, cloned and DNA-sequenced.
Anticipated results
To check the polymorphic bands for salinity tolerance by silver
staining method, PAGE gels were run as described in the procedure
(steps 120). In case of O. sativa, gel was loaded with thirteen sets of
lanes consisting of a DNA size marker and twelve lanes of amplication
products from a SSR primer already tested as polymorphic for the
salinity tolerant Pokkali parent line (Fig. 1a). In case of B. juncea,gel
was loaded with six sets of lanes consisting of a DNA size marker and
ve lanes of amplication products from a SSR primer already tested
as polymorphic for the salinity tolerant B. juncea parent line (Fig. 1b).
Gel development was stopped when the image reached optimal image
contrast (as judged by the eye).
Application
A valuableapplication of this procedure has on the detection of small
nucleic acids (2050 nucleotide length) that are polymorphic in nature
for various phenotypic variations (in this case it was salinity stress
tolerance). When compared with the 11 alternative treatment, as listed
in Table 1, the results obtained by our method were less time consum-
ing and easy to implement on normal basic facilities available.
Here we present an updated and optimized simple and efcient way
to detect small polymorphic bands in plants.
Competing interests
The authors declare that they have no competing interest.
Acknowledgments
I would liketo thank JawaharlalNehru University New Delhi, Central
Soil Salinity Research Institute Karnal and Sogang University Seoul for
providing logistics support and spaceto work. This work was supported
by a grant from IAEA, Vienna (No. 1(60)/E-1) and Rural Development
Administration (No. PJ008197) Republic of Korea. We apologize to col-
leagues whose work was not included in this method owing to space
constraints.
References
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Electrophoresis 19 (1998) 152157.
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Table 2
Troubleshooting.
Problems Cause Solutions
No amplication in PCR Annealing temperature
problem
Varies the Tm from
56°66°
No polymorphic bands Primers are not
polymorphic
Use different sets of
markers
No bands at all Using acetic acid as a
xative
Avoid using acetic acid
as a xative given in
other methods as it
stops the silver nitrate
activity.
Bent band High voltage and less
buffer
Regularly add buffer to
the top of gel and run
gel as low voltage as
possible
Broken bands Gel pieces in the well Clean the well properly
before running the gel
Black background Touching of the gel by
naked hand and not
washing gel properly
as remaining pieces of
gel can cause that.
Use cloves during silver
staining, clean the gel
properly.
222 M. Kumar et al. / Genomics Data 5 (2015) 218222
... The obtained gels were stained using the silver nitrate method ( Benbouza et al., 2006). The silver-staining method for microsatellite studies was previously validated by Creste et al. (2001), Benbouza et al. (2006), Rezaei et al. (2011) and Kumar et al. (2015). After recording the gel images using a gel documentation system (Gel Doc XR, Bio-Rad, USA), microsatellite allelic size was determined using Gel-Pro Analyzer 3.9 software (Media Cybernetics, Silver Spring, MD, USA). ...
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