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5. SOUTHERN BLOTTING
Introduction
Localization of particular sequences with in genomic DNA is usually
accomplished by the transfer techniques described by Southern (1975).
Genomic DNA is digested with one or more restriction enzymes, and the
resulting fragments are separated according to size by electrophoresis
through an agarose gel. The DNA then denatured and transferred from gel to a
solid support (usually a nitrocellulose filter or nylon membrane). The
relative positions of the DNA fragments are preserved during their transfer
to the filter. The DNA attached to the filter is hybridized to radiolabeled
DNA or RNA, and autoradiography is used to locate the positions of bands
complementary to the probe.
Genomic DNA digestion
Genomic
DNA 70 µl (15 µg)
Assay
buffer 10 µl
XbaI
Enzyme 3 µl
Sterile
water 17 µl
Incubate the reaction at 37oC over night.
Southern blotting analysis
(Sambrook et al., 1989)
Preparation of solutions
20X
SSC
NaCl
175.3 g
Na3C6H5O7.2H2O
88.2 g
Adjust pH to 7.0 with 1N NaOH and final volume make up to 1 liter. Autoclave
and store at room temperature.
Denaturing solution
1.5 M NaCl
87.66 g
0.5 N NaOH
20.00 g
Make up to 1 liter, autoclave and store at room temperature.
Neutralizing solution
1 M Tris
pH 7.4 60.55 g
1.5 M NaCl
43.83 g
Make up to 500 ml, autoclave and store at room temperature.
0.2 N HCl.
Transfer of DNA from agarose gels to solid supports
There are three methods to transfer fragments of DNA from agarose
gels to solid supports (nitrocellulose filters or nylon membranes).
1) Capillary transfer
In this method 9Southern 1975), DNA fragments are carried from the gel in a
flow of liquid and deposited on the surface of the solid support. The liquid
is drawn through the gel by capillary action that is established and
maintained by a stack of dry, absorbent paper towels. The rate of transfer
of the DNA depends on the size of the DNA fragments and the concentration
of agarose gel. Small fragments of DNA are transferred with in 1 h. Larger
fragments are transferred slowly (18-24 h).
2)
Electrophoretic transfer
This method is not practical when nitrocellulose is used as the solid
support because of the high ionic strengths of the buffers that are required
to bind nucleic acids to these filters. It is necessary to use large volumes
to ensure that the buffering power of the system does not become depleted by
electrolysis.
3) Vacuum transfer
Nucleic acids can be transferred rapidly and quantitatively from
gels under vacuum. The gel is placed in contact with a nitrocellulose filter
or nylon membrane supported on a porous screen over a vacuum chamber. Buffer
drawn from an upper reservoir, elutes nucleic acids from the gel and
deposits them on the filter or membrane. It is very efficient than capillary
transfer and extremely rapid. But is expensive device, commercially
available.
Protocol for transferring of DNA on to nylon membrane by capillary method
1)
After electrophoresis, trim the gel neatly (unused areas) in a dish. Cut off
the left hand bottom of gel for orientation.
2)
Soak gel in 0.2N HCl for 10 min.
3)
Denature DNA by soaking the gel for 45 min. in Denatureation solution with
constant gentle agitation.
4)
Rinse the gel briefly in sterile water.
5)
Neutralize by soaking the gel in Neutralizing solution for 30 min.
6)
Change the solution and soak for further 15 min.
7)
While the gel is neutralizing solution wrap Whatman paper around a support
longer and wider than the gel. Place the support on a dish and fill the dish
with transfer buffer 10xSSC.
8)
Wet the Whatman paper on support and smooth out all air bubbles.
9)
Use gloves and cut as piece of Nylon membrane about 1mm larger than the gel
in both dimensions.
10)
Float the Nylon membrane in sterile water, wet both sides and then immerse
the filter in transfer buffer for 5 min. Cut a corner to match with the
corner cut from gel.
11)
Invert the gel and place on the support in the center and smooth out air
bubbles.
12)
Place parafilm along the sides of the gel.
13)
Place the wet Nylon membrane on top of the gel aligning the cut ends. Do not
move the filter once put. Make sure that there are no air bubbles between
the filter and the gel.
14)
Wet 2 pieces of 3mm whatman paper, cut exactly the same size as the gel, in
2X SSC and place on Nylon membrane.
15)
Place a stack of filter papers on the gel, put a 500 g. weight on the paper
stacks.
16)
Transfer for 8-24 h.
17)
Soak Nylon membrane in 6XSSC, dry at room temperature for 30 min.
18)
Fix the DNA by UV cross linking.
Radio labelling Probes
Probes are single-stranded DNA or RNA sequences used to identify
the DNA or RNA sequence based on their homology. The technique used is
nucleic acid hybridization. Any type of nucleic acid can be used as a probe
provided that it can be suitably labeled. The choice of probes depends on
the hybridization strategy, the availability of source of material for use
as probe and the degree to which it can be labeled.
Labeling of the DNA probe
For hybridization, the probe has to be labeled. An ideal probe is
easily attached to DNA, detectable at very low concentrations. There are two
types labeling methods. One is radio labeling and another is non-radio
labeling. There are different types of radio label probes.
Nick translation
Take DNA of the target sequence and treat it with a little DNaseI,
which will
“nick” the DNA making
single-stranded cuts in it. Then add DNA polymerase I and labeled-dNTPs, the
exonuclease of activity of polymerase I acts at the sites of the nicks and
removes nucleotides in the 5’-3’
direction. At the same time, using 3’-hydroxyl
group of the terminal nucleotide of the nick as primer, the polymerase
activity of polymerase I will replace the pre existing, unlabeled nucleotide
with labeled nucleotides. This labeled sequence can be used as the probe.
Random priming/Random labeling
Take target DNA and denature it (by boiling), then add random
primers and DNA polymerase with labeled dNTPs, and synthesize labeled ssDNA
that is complementary to the target DNA.
cDNAs as probes
Take mRNA of interest and use reverse transcriptase with a labeled oligo-dT
primer and/or labeled dNTPs, and the obtained cDNA will be labeled and
should anneal to any sequence encoding a portion of the mRNA.
PCR product as probe
If any cloned gene or cDNA is there and looking the genomic sequence
encoding the mRNA, or for the gene in a related species, just use PCR to
amplify a specific region of the gene or cDNA and use that as a probe, use
labeled dNTPs during the polymerization. Although amplifying a subset of the
cDNA or gene requires sequence knowledge to design appropriate primers,
entire cloned pieces can be used as probes without sequence knowledge by
just amplifying the entire sequence with PCR, using primers for within the
vector flanking the insert.
Random radio labeling of probe
1)
Take 1 µl of 25 ng/µl DNA into a clean autoclaved eppendorf tube.
2)
Add 9 µl of sterile water.
3)
Boil the contents at least for 5 min. in a boiling water bath.
4)
Chill the denatured sample for 5-10 min.
5)
Spin at 8000 rpm for 30 sec.
6)
Add 2.5 µl of 10X labeling buffer.
7)
Add 1 µl of 100ng/µl random primer.
8)
Add 2.5 µl of 20mM DTT solution.
9)
Add 2 µl of dATP, dGTP and dTTP mix.
10)
Add 3 µl of p32 dCTP.
11)
Add 3 µl of autoclaved distilled water.
12)
Add 1 µl of 3 u/µl klenow fragment and mix the reaction gently.
13)
Incubate at room temperature at least for 2 h.
Hybridization of the blotted membrane
Preparation of solutions
50X Denhard’s
reagent
Ficoll
1 g
PVP
1 g
BSA
1 g
Dissolve in 100 ml sterile water and store at -200C.
Denatured Salmon sperm DNA
The DNA was dissolved in sterile water to concentration of 10
mg/ml by stirring on a magnetic stirrer for 2-4 h. DNA was sheared by
passing it several times through an 18-guage hypodermic needle, boiled for
10 min. made into aliquots and stored at -200C. Just before use,
the DNA was heated for 5 min. in a boiling water bath and chilled quickly on
ice.
Pre-hybridization solution
6X SSC
30 ml form 20XSSC
5X Denhard’s
reagent 10 ml from 50X Denhard’s
0.5% SDS
500 mg
100
mg/ml
Sperm DNA 1 ml
Dissolve, and make up to 100 ml.
Hybridization solution
6X SSC
30 ml from 20XSSC
0.5% SDS
500 mg
100
mg/ml
Sperm DNA 1 ml
Dissolve and make up to 100 ml.
Protocol
1)
Float the Nylon membrane containing the target DNA on the surface of a tray
of 6XSSC until it becomes thoroughly wetted from beneath. Submerge the
membrane for 2 min.
2)
Slip the wet membrane into heat sealable bag or hybridization bottle. Add
50-100 ml of pre-hybridization solution.
3)
Incubate the bag or bottle for 1-2 h. submerged at 65oC.
4)
Remove the pre-hybridization solution, add 50-100 ml hybridization solution
and radio labeled probe, seal it.
5)
Incubate bag or bottle submerged in a water bath set at 65oC over
night.
Washings
1)
Removed the Nylon membrane and transfer to a tray containing 2X SSC, 0.5%
SDS, wash at room temperature for 5 min.
2)
Remove the wash solution and add 2X SSC, 0.5% SDS, wash for 15 min. with
gentle agitation at room temperature.
3)
Again wash the membrane at 370C for 30 min in 0.1%X SSC, 0.5%
SDS and once at 650C for 1 h. in 0.1%X SSC, 0.5% SDS.
4)
Finally wash the membrane briefly in 0.1%X SSC at room temperature.
5)
Remove most of the liquid by placing the membrane on paper towels.
6)
Wrap the membrane in saran wrap and expose for 24 h. to hyperfilm with
intensifying screens at -700C.
Autoradiography
Autoradiography is used to detect radioisotopiccally-labeled materials. This
technique is based on film imaging. When an X-ray film is directly exposed
to radioactive compounds present in electrophoresis gels or nitrocellulose
filters or nylon membranes radioactive decay products strike the film
interacting with the silverhalide contain in the emulsion, there by
producing a latent image. P32 is a high energy gamma-emmiter and
can be directed by this technique.
All steps in autoradiography have to be carried out in a dark
room. Safety lights with red filters are suitable to work with X-ray film.
Safety lamp is red light.
Protocol
1)
Switch on the safety light.
2)
After wearing gloves, take out one sheet of X-ray film, cut it to the size
of the nylon or nitrocellulose filter.
3)
Cut one corner of the film to mark the orientation of the film to that of
the filter.
4)
Open a X-ray film exposure cassette, place the film over the polythene
sheet. Place the hybridized membrane over the film according to the
orientation marks. Close the cassette and keep at -70oC over
night.
Developing the X-ray film
This procedure should be carried out in a dark room with safety
lights on.
1)
Wash the film for 3 min in developer
2)
Wash the film in water for 2 min.
3)
Wash for 3 min in fixer solution.
4)
Rinse the film in running water for 5 min and hang the film to dry at room
temperature.
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