SOUTHERN HYBRIDIZATION

Presented by
Amrutha S
BLOTTING
• A blot, in molecular biology and genetics,
is a method of transferring proteins, DNA
or RNA onto a carrier.
• The term blotting refers to the transfer of
biological samples from a gel to a
membrane and their subsequent
detection on the surface of the
membrane.
• Technique for transferring DNA, RNA and
proteins onto a carrier so they can be
separated and often follows the use of a
gel electrophoresis.
 TYPES OF
BLOTTING

SOUTHERN NORTHERN WESTERN

BLOTTING BLOTTING BLOTTING
Southern Blotting
• It is a method used in molecular
biology for DNA analysis.
• The method is named after its
inventor, the British biologist Edwin
Mellor Southern in 1975.
• This method is also known as DNA
blotting/Southern hybridization.
PRINCIPLE
• Southern blotting is an example of RFLP (restriction fragment length
polymorphism).
• Southern blotting is a hybridization technique for identification of
particular size of DNA from the mixture of other similar molecules.
• This technique is based on the principle of separation of DNA
fragments by gel electrophoresis and identified by labelled probe
hybridization.
HYBRIDIZATION
• A technique which has the ability of individual single stranded nucleic acid
molecules to form double stranded molecules.
• The principle of hybridization is the addition of a probe to a complex mixture of
target DNA.
• The mixture is incubated under conditions that promote the formation of
hydrogen bonds between complementary strands.
• A hybridization probe is a short (100-500bp), single stranded DNA. The probes
are labeled with a marker so that they can be detected after hybridization.
STEPS INVOLVED
1. DNA isolation
2. DNA digestion
3. Gel electrophoresis
4. Denaturation(Pre treatment of DNA)
5. Blotting
6. Immobilization of DNA
7. Pre hybridization
8. Hybridization & Post hybridization washing
9. Visualization
DNA ISOLATION
• The DNA to be studied is isolated from various tissues.
• The most suitable source of DNA is blood tissue.
• Specimen is incubated with detergent to promote cell lysis.
• Lysis frees cellular proteins and DNA.
• Proteins are enzymatically degraded by incubation with proteinase.
• Organic or inorganic extraction removes proteins.
• DNA is purified from solution by alcohol precipitation.
• Visible DNA fibers are removed and suspended in buffer.
DNA DIGESTION
• DNA has been treated with one or more restriction endonucleases.
• This is done by adding the desired amount of DNA with the restriction enzyme, enzyme buffer
and purified water then incubate the reaction at 37 °C overnight.
• The DNA is extensively fragmented, so as to obtain specific restriction fragments of 20 kb and
more.
Types of DNA fragments used:
• Genomic DNA
• cDNA
• Probe DNA
• Control DNA
GEL ELECTROPHORESIS
• The DNA fragments are then electrophoresed
on an agarose gel to separate them by size.
• If some of the DNA fragments are larger than
15 kb, then before blotting, the gel may be
treated with an acid, such as dilute HCl.
• This depurinates the DNA fragments, breaking
the DNA into smaller pieces, thereby allowing
more efficient transfer from the gel to
membrane.
 DENATURATION(PRETREATMENT OF DNA)
• The restriction fragments present in the gel are denatured with alkali.
• This causes the double-stranded to become single-stranded.
• DNA is then neutralized with NaCl to prevent re-hybridization before adding the
probe.
• The pretreatment has two objectives:
• First, it is desirable to break the DNA molecules in individual bands within the gel into smaller
fragments, because smaller fragments transfer more quickly than larger ones.
• The second pretreatment is with an alkaline solution that denatures the double-stranded DNA
molecules by breakage of their hydrogen bonds, so the molecules become single-stranded.
• If a nitrocellulose membrane is being used then the alkali pretreatment is
followed by neutralization of the gel by soaking in a Tris-salt buffer, this step
being essential because DNA does not bind to nitrocellulose at a pH of greater
than 9.0.
 BLOTTING
• Traditionally, a nitrocellulose membrane is used, although nylon or a
positively charged nylon membrane may be used.
• Nitrocellulose typically has a binding capacity of about 100μg/cm,
while nylon has a binding capacity of about 500 μg/cm.
• Many scientists feel nylon is better since it binds more and is less
fragile. Transfer is usually done by capillary action, which takes several
hours.
• Capillary action transfer draws the buffer up by capillary action
through the gel an into the membrane, which will bind ssDNA.
• Vacuum blot apparatus may be used instead of capillary action. In this
procedure, a vacuum sucks SSC(Saline-Sodium Citrate buffer) through
the membrane.
• This works similarly to capillary action, except more SSC goes through
the gel and membrane, so it is faster (about an hour).
IMMOBILIZATION OF DNA
• After blotting, the transfer setup is dismantled and the membrane rinsed in 2 X SSC and left to
dry.
• If the blot has been made onto a nitrocellulose or uncharged nylon membrane, then the DNA is
only loosely bound to the membrane at this stage.
• After transfer of DNA to the membrane (nylon), it is treated with UV light - This help to cross links
(via covalent bonds) the DNA to the membrane.
• Membrane can be baked (nitrocellulose) at about 80ᵒC for a couple of hours, but be aware that it
is very combustible (noncovalent but semi-permanent attachment of DNA to a nitrocellulose
membrane).
PRE-HYBRIDIZATION
• To block the unused DNA binding sites on the membrane, the prehybridization step is necessary.
• If this step is omitted, then the probe will bind nonspecifically to the surface of the membrane
and the signal resulting from hybridization to the specific restriction fragment will be difficult if
not impossible to identify.
• To prehybridize, non-specific ssDNA is added to the membrane. Sonicated salmon sperm DNA is
commonly used (unrelated to the one whose DNA has been blotted).
• Denhardt's solution containing Ficoll (biological polymers, a carbohydrate-based compound) and
Polyvinylpyrrolidone (nonbiological polymeric compounds) and BSA (bovine serum albumen, a
non-specific protein), SDS (sodium dodecyl sulfate), and formamide (destabilizing agent) are
added during prehybridization process.
• Prehybridization takes between 15 min and 3 h at 68ᵒC, depending on the type of membrane.
HYBRIDIZATION & POST
HYBRIDIZATION WASHING
• A hybridization probe is a short, single
stranded DNA. The probes are labeled with a
marker so that they can be detected after
hybridization.
• This process relies on the ssDNA hybridizing
(annealing) to the DNA on the membrane due
to the binding of complementary strands.
• Hybridization analysis is carried out by
soaking the Southern blot in a buffer
containing the hybridization probe, usually in
a tube that is constantly rotated so all parts of
the membrane are exposed to the probe, or
alternatively in a sealed plastic bag that is
placed on a shaker.
• Probing is often done with 32P labeled ATP, biotin/streptavidin or a bioluminescent probe.
• There are two methods of hybridization:
• Radioactive method
i. Probe with 32P labeled ATP (Nick Translation)
ii. Random oligo primed synthesis
iii. End labelling
• Nonradioactive method
i. Colorimetric detection
ii. Fluorescent detection
iii. Chemiluminescence
Disadvantages of 32P:
i. Short half-life (about 2 weeks) means probes must be used immediately, and the labeling reagent cannot be
stored for long.
ii. Contamination problems: all materials and equipment must be dedicated to radioactive work only. Regular
lab-wide testing for contamination is required. Expense of disposal of radioactive waste.
iii. Must have access to a dark room to set up and develop films.
VISUALIZATION
• After hybridization, excess probe is washed from the membrane (typically using SSC buffer).
• The membrane bound DNA labelled with probe can be visualized under autoradiogram which give
pattern of bands.
• There are various methods of visualization:
1. Autoradiography
Radioactively labeled probes generate a signal that can be visualized on an
autoradiograph.
2. Chemiluminescence
Labeled probes produce a light signal that can be detected using
specialized detection systems.
3. Fluorescence
Fluorescently labeled probes emit light at specific wavelengths upon
binding to the target DNA fragments.
• Biotin/streptavidin detection is done by colorimetric methods, and bioluminescent visualization
uses luminescence.
APPLICATIONS
• Southern blotting technique is used to detect specific DNA in given sample.
• Used during DNA finger printing.
• To identify clone.
• Used for paternity testing, criminal identification, victim identification.
• To isolate and identify desire gene of interest.
• Used in restriction fragment length polymorphism.
• To identify mutation or gene rearrangement in the sequence of DNA.
• Used in diagnosis of disease caused by genetic defects.
• Used to identify infectious agents.
ADVANTAGES
• Effective way to detect a specific DNA sequence in a large complex of DNA.
• Can be used to quantify the amount of the present DNA.
• Cheaper than DNA sequencing
• Allows for determination of gene copy number or expression level.

LIMITATIONS
• Time-consuming and labor-intensive
• Can be expensive due to the need for labeled probes
• High quality and large amounts of DNA are needed
• It cannot be used to detect mutations at base-pair level
NORTHERN BLOTTING
• Northern blotting was developed by James
Alwine, George Stark and David Kemp (1997).
• In this technique, RNA is being analysed
instead of DNA.
• It is a technique by which RNA fragments are
separated by electrophoresis and immobilized
on a membrane.
• The identification of specific RNA is done by
using nucleic acid probes.
• It helps to study the gene expression by
detection of RNA.
APPLICATIONS
• To study the gene expression of various tissues, organs, development stages, pathogen infections.
• Study mRNA splicing, RNA degradation and half-life.
• Detection of mRNA transcript size.
• Identification of transferred genes in transgenic individuals.

LIMITATIONS
• The standard Northern Blot method is relatively less sensitive than nuclease protection assays and
RT-PCR.
• Detection with multiple probes is a problem.
• If RNA samples are even slightly degraded by RNAses, the quality of the result and quantification
of expression is quite negatively affected.
WESTERN BLOTTING
• The western blot (alternatively, immunoblot)
is used to detect specific proteins in a given
sample of tissue homogenate or extract.
• The method originated from the laboratory of
George Stark at Stanford.
• The name western blot was given to the
technique by W. Neal Burnette.
• Western blots are used to determine the
identity, size, and abundance of specific
proteins within a sample.
APPLICATIONS
• The confirmatory HIV test
• Western blot is also used as the definitive test for Bovine spongiform encephalopathy(BSE)
• Some forms of Lyme disease testing employ Western blotting
• Confirmatory test for Hepatitis B infection and HSV-2 infection
• Study different properties of protein based on molecular weight
• Investigations of mitochondrial uniporter selective calcium channels in the organelle’s inner
membrane
• Also used in Blood Doping to detect drug abuse in sports.