Southern Blotting (SB) 4 jan 2015 final

SOUTHERN BLOTTING :
TECHNIQUE AND
APPLICATIONS
By
Dr Ichha Purak
University Professor
Department of Botany
Ranchi Women’s College,Ranchi
Southern Blotting: Technique and
Applications
101/04/15

SOUTHERN BLOTTING (HYBRIDIZATION )
Blotting techniques are used to transfer DNA or RNA fragments or
proteins from electrophoresis gel to a nitrocellulose sheet or nylon
membrane as blotting paper is used to blot ink.
Southern blotting is the transfer of DNA fragments from an
electrophoresis gel to a membranous support which results in
immobilization of DNA fragments. These immobilized single stranded
DNA fragments can then be subjected to hybridization with a labeled
probe.
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STEPS OF SOUTHERN BLOTTING
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Southern blotting was named after Edward M. Southern who developed
this procedure at Edinburgh University in the 1975.
It allows investigators to locate a particular sequence of DNA within a
complex mixture.
DNA (genomic or other source) is digested with a restriction enzyme and
separated by gel electrophoresis and transferred from an agarose gel onto
a Nitrocellulose sheet or Nylon membrane which is then incubated with a
single stranded DNA probe with known sequence. This probe is supposed
to form base pairs with its complementary DNA sequence and to form a
double-stranded DNA molecule.
The probe is labeled before hybridization either radioactively or is treated
enzymatically by alkaline phosphotase or horseradish peroxidase .
Finally, the location of hybridization with the probe is detected either by
directly exposing the membrane to X-ray film or by chemiluminescent
methods
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STEPS IN SOUTHERN/NORTHERN
BLOTTING
wells
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Sir Edwin Mellor Southern ,Fellow Royal Society and Trinity
won Albert Lasker Award (2005) in Clinical Medical Research
for the invention of the Southern Blot in 1975 when he was
working as Professor of Biochemistry at the University of
Edinburgh ,which is now a common molecular biology
procedure to identify DNA sequence
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Other blotting methods that employ similar principles but using RNA or
Protein are named as Northern Blot and Western Blot in reference to
original Southern Blot
In Northern hybridization (Blot ) RNAs are transferred from gel to DBM
( Diazobenzyl oxy methyl) paper or Nylon membrane and are fixed by
baking. Denaturation step is not needed and the probe used for
hybridization is single stranded DNA Since the base-pairing is in a
sense the reverse of a "southern" experiment, this technique is referred
to as a "northern blot”.
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The Western blot (Protein immunoblot) is widely used analytical technique to
detect specific proteins in the tissue homogenate or extract. Proteins after
extraction are separated by gel electrophoresis based on length of polypeptide.
Proteins are transferred to a membrane e.g. Nitrocellulose or Poly vinyl di
fluoride (PVDF) and are probed by using specific antibodies specific to target
protein or antigen. The technique is also called immunoblotting and is to identify
antigens during infection or disease.

SOUTHERN
BLOTTING
WESTERN
BLOTTING
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FOR PERFORMING SOUTHERN BLOTTING
FOLLOWING BROAD STEPS ARE TO BE TAKEN
• DNA ISOLATION AND PURIFICATION
• DIGESTION OF DNA BY RESTRICTION ENDONUCLEASES
• SEPARATE DNA FRAGMENTS BY GEL ELECTROPHOREIS
• DS STRANDED DNA FRAGMENTS ON GEL ARE DENATURED BY ALKALINE
TREATMENT TO GIVE SINGLE STRANDED DNA FRAGMENTS
 BLOTTING (TRANSFER OF DNA FRAGMENTS FROM GEL TO NITROCELLULOSE
SHEET /NYLON MEMBRANE)
 HYBRIDIZATION OF IMMOBILIZED DNA FRAGMENT WITH SINGLE STRANDED
RADIOACTIVELY LABELLED DNA PROBES
 DETECTION OF HYBRIDIZATION BY AUTORADIOGRAPHY OR
CHEMILUMNISCENT METHODS
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These steps shall be briefly discussed one by
one•DNA ISOLATION AND PURIFICATION
For DNA isolation various protocols have been designed for different type
of sources and many rapidly working kits are available
•DNA extraction from Blood
•DNA extraction from serum
•DNA extraction from Plasmids
•DNA extraction from animal tissue
•DNA extraction from plant tissue
DNA isolation is a routine procedure to procure DNA for various
molecular techniques such as RDT, DNA sequencing,restriction
mapping ,forensic analysis , construction of genomic library etc.
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DNA extraction protocols involve following common steps
1.Cell disruption or cell lysis by physical methods as blending or
grinding .Lysis frees cellular proteins ,DNA & RNA
2. Removing membrane lipids by detergents
3. Removing proteins by protease and RNA by Rnase enzymes
4. DNA precipitation by alcohols
Finally by repeated centrifugation DNA is obtained in the form of pellet.
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For genomic isolation of DNA from plants Virginia Walbot and other similar
methods are employed. As plants are rich in proteins, polysaccarides
and lipids the protocol involves extraction of DNA using Tris chloride
buffer (pH-8), Na2EDTA, Sodium Chloride and Sodium Dodicyl Sulphate
(SDS). Precipitation of DNA is done by Ammonium Acetate and Ethanol
.The extracted DNA by this method can be stored for long duration. A part
of extracted DNA as pellet can be subjected to confirmation by using
Diphenylamine (DPA) reagent.

For extraction of DNA from blood many protocols are in practice . Some
protocols are quite rapid taking only 15-30 minutes employing simple lysis
with proteinase K enzyme.
Protocol for extraction of genomic DNA from whole blood
Reagents
Buffer A (Red blood cell lysis buffer) composition
•0.32 M sucrose
•10 mM Tris HCl
•5 mM MgCl2
•0.75% Triton-X-100 Adjust pH to 7.6
Buffer B (Proteinase K buffer) composition
•20 mM Tris-HCl
•4 mM Na2
EDTA
•100 mM NaCl Adjust pH to 7.4
All solutions should be sterile. Buffer A should be autoclaved prior to
addition of Triton-X-100. Sterile filtering of solutions instead of autoclaving
is a better option. Southern Blotting: Technique and
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Procedure
1.2 ml of buffer A is added to 2 ml of blood and 4 ml of cold, sterile,
distilled, deionised water. The mixture is poured in centrifuge tubes.
Tubes are spinned gently and left on ice for 2-3 minutes for incubation.
2. Tubes are further spinned for 15 minutes at 3500 rpm at 4o
C.
Supernatant is discarded and pellet is resuspended in 2ml of buffer A
and 6 ml of water. The tubes are again spinned for 15 minutes at 3500
rpm at 4o
C. The pellet should be white or cream in colour if red it is
washed again.
3. 5ml of Buffer B and 500 µl of 10% SDS is added to the pellet and
spinned for 30-60 seconds. Then 50 µl of freshly prepared Proteinase
K solution (20mg/ml) is added.
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4. Left for incubation for two hours at 55o
C in a water bath. After that
left to cool at room temperature. Then 4 ml of 5.3 M NaCl solution is
added and spinned at 4500 rpm for 15-20 minutes at 4o
C.
5. Supernatant is poured off into a fresh tube .Care should be taken
not to dislodge pellet. Equal volume of isopropyle alcohol stored at
20o
C is added and tubes are shaked 5-6 times to precipitate DNA.
6. Pellet is transferred to microfuge tube and washed with 1 ml of
70% ethanol. DNA is left to dry for 15-20 minutes at 37o
C. DNA is
resuspended in 300-400 µl of Tris HCL (ph 8.5). Left overnight to
dissolve at room temperature and can be refrigerated and stored for
up to a year in ethanol.
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1401/04/15

•DIGESTION OF DNA BY RESTRICTION ENDONUCLEASE
Next step is digesting DNA into several pieces of different sizes. This is
done using one or more restriction endonucleases.
Restriction enzymes are isolated from bacteria that recognize specific
sequences in DNA and then cut the DNA to produce fragments, called
restriction fragments or RFLP (Restriction Fragment Length
Polymorphism ) with either blunt or cohesive ends. A unique feature of
these enzymes is that they cleave the DNA at palindromic sequences
that are 4-8 base pairs in length. Thus the enzyme EcoRI cleaves the
DNA wherever the sequence,
Is found. Now more than 60 of these enzymes are known, and by a
suitable choice of restriction enzymes, the DNA can be cut at
appropriate positions .
.
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•SEPARATION OF DNA FRAGMENTS BY GEL
ELECTROPHOREIS
Electrophoresis is a common lab technique used to identify, quantify, and
purify nucleic acid fragments. DNA samples cut by restriction
endonuclease are loaded into wells of an agarose or acrylamide gel and
subjected to an electric field, causing the negatively charged nucleic
acids to move toward the positive electrode. Shorter DNA fragments will
travel more rapidly, whereas the longest fragments will remain closest to
the origin of the gel (near well) , resulting in separation of DNA
fragments based on size.
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An Agarose Gel after electrophoresis showing DNA
fragments separated by size
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Agarose remains the most widely used gel matrix for separating nucleic
acid fragments because it is nontoxic, easy to use, and offers a broad
separation range. Varying the agarose concentration controls the gel
pore size, facilitating the separation of a wide range of different-size
nucleic acids. The migration of nucleic acids in agarose gels is also
affected by the choice of buffer and applied voltage
Polyacrylamide is a cross-linked polymer that provides very high
resolution of DNA molecules in the 10–3,000 bp size range. Under the
appropriate conditions, DNA molecules differing in size by a single base
pair can be resolved
DNA is loaded by micropipette in the wells casted on the gel plate with
the help of comb. Ethidium bromide is added to DNA sample before
loading or in the electrophoretic buffer.
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Fragmented DNA is typically electrohoresed on an agarose gel to
separate the fragments according to their molecular weights.
Acrylamide gels can alternatively be used for good resolution of smaller
DNA fragments (<800 bp).
Agarose gel electrophoresis is ideal for rapid, high-resolution
electrophoresis of restriction digests, PCR reactions.
Because there are so many different restriction fragments on the gel, it
usually appears as a smear rather than discrete bands.
For determination of DNA size, a wide range of DNA ladders are
available for accurate size and mass estimations, including 100 bp
ladders and 1 Kb Plus ladders which can be loaded in 1-2 wells.
Gel loading buffers include one or two tracking dyes as bromophenol
blue or xylene cyanol to monitor the progress of electrophoresis by
migration of the dye
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DS STRANDED DNA FRAGMENTS ON GEL ARE
DENATURED BY ALKALINE TREATMENT TO GIVE SINGLE
STRANDED DNA FRAGMENTS
The DNA is denatured into single strands by incubation with 0.5 M
NaOH so as to obtain single stranded DNA . These negatively charged
SS DNA can bind to positively charged membrane. This step also
destroys any residual RNA that may still be present with the DNA or
denaturation may be caused by high temperature treatment.
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A depurination step is optional. Fragments greater than 15 kb are hard to
transfer to the blotting membrane. Keeping this in view the gel may be
treated with dilute HCL for about 15 minutes which can depurinate DNA
fragments ,breaking the DNA into smaller fragments.
A neutralization step by Nacl is required after alkali or acid treatment to
prevent base pairng before hybridization with probe

BLOTTING (TRANSFER OF DNA FRAGMENTS FROM GEL TO
NITROCELLULOSE SHEET /NYLON MEMBRANE)
To transfer denatured single stranded DNA from gel to a solid
support a sheet of nitrocellulose or nylon membrane is placed on the
top of the gel.
Above the gel stacks of paper towels are placed . A glass can be placed on
the paper towels on which a weight is placed to ensure good and even
contact between gel and membrane. A wick of whatman filter paper is used
for buffer transfer from a region of high water potential to a region of low
water potential by inducing capillary action. In place of paper wick sponge
sheet can also be used
With this buffer transfer DNA strands are transferred from gel to membrane.
Ion exchange interaction binds the DNA to the membrane due to negative
charge of DNA and positive charge of membrane.
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Many scientists feel nylon is better since it is less fragile.
Transfer is usually done by capillary action, which takes several hours.
Capillary action transfer draws the buffer up through the gel onto the
membrane, ssDNA fragments are drawn along with buffer.
A vacuum blot apparatus can be used instead of capillary action. In this
procedure, a vacuum sucks SSDNAs through the membrane.
The membrane is then baked at about 80ᵒ
C for about two hours to
permanently attach the transferred DNA to the membrane or the
membrane is exposed to ultraviolet radiation.
Care should be taken while baking the Nitrocellulose sheet at high
temperature as it is highly cumbustible
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TRANSFER OF FRAGMENTS FROM GEL TO MEMBRANE
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BLOTTING ARRANEMENT
Weight

HYBRIDIZATION OF IMMOBILIZED DNA FRAGMENT
WITH SINGLE STRANDED DNA PROBES.
For hybridization a nucleic acid probe ( DNA or RNA ) with sequence
homologous to the target sequence is labeled with radioactivity,
fluorescent dye, or an enzyme that can generate a chemiluminescent
signal when incubated with the appropriate substrate. The choice of
the label depends on several factors such as the nature of probe or
probe template, sensitivity needed, quantification requirements, ease
of use, and experimental time. For this the Nitrocellulose sheet bearing
ssDNA fragments is placed in a bag containing a solution of
radioactively labeled single stranded DNA probe with known
sequence.
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32
P ATP is used to label the probe radioactively. After hybridization,
excess or unhybrized probe is washed from the membrane in
several changes of buffer The wash can be with low or high
stringency which can remove hybridization solution and also unused
probes
The result is that only fully hybridized labeled probe molecules, with
complementary sequence to the region of interest, remain bound.
A pre-hybrdization step is required before hybridization to block
non-specific sites, to prevent single-stranded probe binding just
anywhere on the membrane.
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DETECTION OF HYBRIDIZATION BY AUTORADIOGRAPHY OR
CHEMILUMNISCENT METHODS
In the detection step, the bound, labeled probe is detected using the
method required for the particular label used. For example, radiolabeled
probes may be detected using X-ray film or a phosphorescense imaging
instrument, and enzymatically labeled probes are typicallly detected by
incubating with a chemiluminescent substrate and exposing the blot to
X-ray film.
.
The transfer step of the DNA from the electrophoresis gel to a
membrane permits easy binding of the labeled hybridization probe to the
size-fractionated DNA. It also allows for the fixation of the target-probe
hybrids, required for analysis by autoradiography or other detection
methods.
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In order to analyze a Southern Blot, a radioactive genetic probe is used
in a hybridization reaction with the DNA . X-ray is taken of the Southern
Blot after a radioactive probe has been allowed to bind with the
denatured DNA on the paper, only the areas where the radioactive probe
binds will show up on the film. This allows researchers to identify, in a
particular person's DNA, the occurrence and frequency of the particular
genetic pattern contained in the probe.
Hybridization of the probe to a specific DNA fragment on the filter
membrane indicates that this fragment contains DNA sequence that is
complementary to the probe
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a
b
d c
a) DNA after digestion by RE ,Fragments separated on Agarose Gel
b) Denatured DNA fragments are transferred from Gel to Nitrocellulose Sheet (Blotting)
c) The Nitrocellulose sheet is incubated with specific single stranded DNA probe
d) The location of the DNA fragment that hybridizes with the probe can be displayed by
autoradiography.

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3001/04/15

The Southern blot is used to detect the presence of a particular
bit of DNA in a sample
Extracted and
purified DNA
DNA digested with
restriction endonuclease Restriction fragments
DNA is loaded into
wells of the gel
DNA fragments
move on gel as
per size
SSDNA fragments
transferred to NC
sheet
Development
of Blot
denaturationElectric current
DNA probe added
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01/04/15
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32
APPLICATIONS OF SOUTHERN BLOTTING
Suthern Blotting is a very useful techniquae
It can be used for identification of DNA fragment in a DNA sample
It is significantly useful in identification of criminals and is a tool in
forensics
Diagnosis of Infectious disease and HIV-1
Used in personal Identification
Used in phyllogenetic analysis
Used in paternity and maternity tests
It can be used to map the restriction sites and make RFLP maps.
Used to identify mutations,deletions and gene rearrangements.
Used in prenatal diagnosis of genetic diseases (Sickle cell anaemia)

Southern blots performed with restriction enzyme-digested
genomic DNA may be used to determine the number of sequences
(e.g., gene copies) in a genome .
It is used for detection and identification of the transferred genes
in transgenic individuals.
In regards to genetically modified organisms, Southern blotting is
used as a definitive test to ensure that a particular section of DNA
of known genetic sequence has been successfully incorporated
into the genome of the host organism
It can be used to map the restriction sites and make RFLP maps.
Southern Blotting can also be used to follow the inheritance of
selected genes
Southern Blotting and DNA
fingerprinting
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Mutations within restriction sites change the sizes of restriction
fragments and as a result, the positions of bands in the Southern-blotting
analysis and autoradiography also change.
This change in position can later be compared to normal blot-analyses in
order to reveal where the possible change has occurred. The existence of
genetic diversity created by these mutations in a population, is termed
polymorphism.
The detected mutation in turn may have different effects. It may cause
disease . Some examples of such diseases include sickle-cell anemia,
cystic fibrosis, and Huntington chorea.
Polymorphism refers to the DNA sequence variation between
individuals of a species. If the sequence variation occurs at the restriction
sites, it could result in RFLP. The most well known example is the RFLP
due to globin gene mutation.β
Southern Blotting and DNA
fingerprinting
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Applications
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All of these mutations can be detected by comparing the restriction-
fragment-length polymorphisms with normal fragments of DNA .
It is also used to determine the molecular weight of a restriction
fragment and to measure relative amounts in different samples. Under
optimal conditions, Southern blotting detects ~ 0.1 pg of the DNA of
interest
.

Detection of the sickle-cell globin gene by Southern blotting. The base
change (A T) that causes sickle cell anaemia a → MstII target site that is
present in the normal -globin gene. This difference can be detected byβ
Southern blotting.
Use of Southern Blotting to detect sickle cell globin gene

RFLP resulting from -globin gene mutation.β
In the normal cell, the sequence corresponding to 5th to 7th amino acids of
the -globin peptide is CCTGAGGAG, which can be recognized by theβ
restriction enzyme MstII.
In the sickle cell, one base is mutated from A to T, making the site
unrecognizable byMstII. Thus, MstII will generate 0.2 kb and 1.2 kb
fragments in the normal cell, but generate 1.4 kb fragment in the sickle
cell. These different fragments can be detected by the southern blotting

SOUTHERN BLOTTING
(A-E)
END OF PRESENTATION
THANKS
01/04/15 38
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