SOUTHERN BLOTTING

M.PRASAD NAIDU
Msc Medical Biochemistry,
Ph.D Research scholar.

OUTLINE
DNA
SPECIMEN COLLECTION AND
STORAGE
PROCEDURE
WATCHPOINTS
USES

DNA
Each individuals unique genetic
blueprint is stored in material known
as DNA.
DNA is found in all cells containing a
nucleus.
DNA can be extracted for analysis
from hair, bones, saliva, sperm, skin,
organs, all body tissues and blood.
DNA
The deoxyribonucleic acid, DNA, is a
long chain of nucleotides which
consist of:
1. Deoxyribose(sugar with 5 carbons)
2. Phosphate groups
3. Organic(nitrogenous)bases

Nitrogenous Bases
Two classes:
Purines
Adenine
Guanine
Pyrimidines
Cytosine
Thymine

DNA
DNA molecules are arranged in a
double helix which resembles a tightly
coiled twisted ladder.
The sides of the ladder have
alternating units of phosphate and
deoxyribose sugar.
DNA
The rungs of the ladder are formed by
the nitrogenous base pairs.
Hydrogen bonds hold the strands
together.
The bases bind together in a
complementary fashion.
DNA
The base adenine (A) always pairs
with thymine (T).
The base guanine (G) always pairs
with cytosine (C).
DNA
Example
First strand GGGTTTAAACCC
Second strand CCCAAATTTGGG



DNA STORAGE AND
COLLECTION
I. Temperature Storage for
DNA
Purified DNA may be
refrigerated at 4C for up to 3
years.
Samples kept over 3 years
should be frozen at -70C.
DNA STORAGE AND
COLLECTION
II. Specimens used in DNA testing
Whole blood
Solid tissue
Serum and plasma
Urine
Bone marrow
and many others
DNA STORAGE AND
COLLECTION
III. Specimen Collection
Requirements
A. Blood and Bone Marrow
Collection tubes are EDTA or ACD
5-15 ml
Samples should not be frozen for
transport
4-25C
DNA STORAGE AND
COLLECTION
B. Serum
Collection tubes with no additives
100 l to 1 ml
Transported at 20-25C
DNA STORAGE AND
COLLECTION
Spin the samples to separate the
plasma, RBC, and buffy coat.
Extract the buffy coat
The buffy coat is used because the
WBC are nucleated and contain DNA.
DNA STORAGE AND
COLLECTION
C. Tissue
A sterile container with no formalin or
paraffin must be used for collection.
30 mg
Dry ice should be used for transport.
DNA STORAGE AND
COLLECTION
D. Urine
Urine container should be used for
collection.
At least 1 ml should be collected.
Transported at 4-25C
SOUTHERN BLOTTING
The technique was developed by E.M.
Southern in 1975.
The Southern blot is used to detect
the presence of a particular piece of
DNA in a sample.
The DNA detected can be a single
gene, or it can be part of a larger
piece of DNA such as a viral genome.
SOUTHERN BLOTTING
The key to this method is
hybridization.
Hybridization-process of forming a
double-stranded DNA molecule
between a single-stranded DNA probe
and a single-stranded target patient
DNA.

SOUTHERN BLOTTING
There are 2 important features of
hybridization:
The reactions are specific-the probes will
only bind to targets with a complementary
sequence.
The probe can find one molecule of target
in a mixture of millions of related but non-
complementary molecules.
























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Steps for hybridization
1. The mixture of molecules is separated.
2. The molecules are immobilized on a matrix.
3. The probe is added to the matrix to bind to the
molecules.
4. Any unbound probes are then removed.
5. The place where the probe is connected
corresponds to the location of the immobilized
target molecule.
SOUTHERN BLOTTING
I. DNA Purification
Isolate the DNA in question from the
rest of the cellular material in the
nucleus.
Incubate specimen with detergent to
promote cell lysis.
Lysis frees cellular proteins and
DNA.
SOUTHERN BLOTTING
Proteins are enzymatically degraded
by incubation with proteinase.
Organic or non-inorganic extraction
removes proteins.
DNA is purified from solution by
alcohol precipitation.
Visible DNA fibers are removed and
suspended in buffer.
SOUTHERN BLOTTING
II. DNA Fragmentation
Cut the DNA into different sized
pieces.
Use restriction endonucleases (RE)
Bacterial proteins
In vivo, they are involved in DNA
metabolism and repair or in bacterial
host defense.

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Nucleases hydrolyze the bonds
that connect bases within the
strand, resulting in cleavage of
the strand.
They cleave the double stranded
nucleic acid only at specific
points.


SOUTHERN BLOTTING
This allows for specific sequences
to be identified more readily.
Fragments are now easily
separated by gel electrophoresis.
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III. Gel Electrophoresis
Sorts the DNA pieces by size
Gels are solid with microscopic
pores
Agarose or polyacrimide
Gel is soaked in a buffer which
controls the size of the pores
Standards should also be run
SOUTHERN BLOTTING
Nucleic acids have a net negative charge and will
move from the left to the right. The larger molecules
are held up while the smaller ones move faster. This
results in a separation by size.
SOUTHERN BLOTTING
Gels can be stained with ethidium
bromide.
This causes DNA to fluoresce under
UV light which permits photography of
the gel.
You can tell the exact migration of
DNA standards and the quality of the
RE digestion of the test DNA.
SOUTHERN BLOTTING
High quality intact DNA should give
the appearance of a single band.
Degraded material will smear
downwards.
Only a small amount of degradation is
tolerable.
SOUTHERN BLOTTING
IV. Blotting
Transfer the DNA from the gel to a
solid support.
The blot is usually done on a sheet
of nitrocellulose paper or nylon.

SOUTHERN BLOTTING
DNA is partially depurinated with dilute
HCL which promotes higher efficiency
transfer by breaking down fragments
into smaller pieces.
DNA is then denatured with an
alkaline solution such as NAOH.
This causes the double stranded to
become single-stranded.

SOUTHERN BLOTTING
DNA is then neutralized with NaCl
to prevent re-hybridization before
adding the probe.
Transferred by either electrophoresis
or capillary blotting.

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1) Electrophoresis- takes advantage of the
molecules negative charge.
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2) Capillary blotting-fragments are eluted from the gel
and deposited onto the membrane by buffer that is
drawn through the gel by capillary action.
SOUTHERN BLOTTING
The blot is made permanent by:
Drying at ~80C
Exposing to UV irradiation
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V. Blocking
Buffer binds to areas on the blot not
occupied by patient DNA.
Blocks the empty sites from being
bound during hybridization.

SOUTHERN BLOTTING
VI. Preparing the probe
Small piece of DNA used to find
another piece of DNA
Must be labeled to be visualized
Usually prepared by making a
radioactive copy of a DNA fragment.
SOUTHERN BLOTTING
The DNA fragment is labeled by the
Random Hexamer Labeling Process:
1. The template DNA is denatured by
boiling.
2. A mixture of hexamers (6
nucleotides) containing all possible
sequences is added and allow to
base pair.



SOUTHERN BLOTTING
3. DNA polymerase is added
with radioactive nucleotides.
4. The mixture is boiled to
separate the strands and is
ready for hybridization.
SOUTHERN BLOTTING
The Random Hexamer Labeling
Process produces a radioactive
single-stranded DNA copy of both
strands of the template for use as
a probe.

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VII. Hybridization
The labeled probe is added to the
blocked membrane in buffer and
incubated for several hours to allow
the probe molecules to find their
targets.
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VIII. Washing
Excess probe will have bound
nonspecifically to the membrane despite
the blocking reagents.
Blot is incubated with wash buffers
containing NaCl and detergent to wash
away excess probe and reduce
background.
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IX. Detection
Radioactive probes enable
autoradiographic detection.
SOUTHERN BLOTTING
If the probe is radioactive, the particles
it emits will expose X-ray film.
By pressing the filter and film, the film
will become exposed wherever probe
is bound to the filter.
After development, there will be dark
spots on the film wherever the probe
bound.
SOUTHERN BLOTTING
Summary of procedure
1. Extract and purify DNA from cells
2. DNA is restricted with enzymes
3. Sort by electrophoresis
4. Denature DNA
5. Transfer to nitrocellulose paper
6. Block with excess DNA
7. Wash off unbound probe
8. Autoradiograph
Watch points
Using too little DNA-compromise the
sensitivity of the test
Using too much DNA- poor restriction
enzyme digestion
Using too high voltage setting for
electrophoresis- gel to melt or
appearance of artifacts
Watch points
Improper blocking-high background
and uninterpretable results.
Insufficient washing-high background
and uninterpretable results.
Excess washing- dissociate the
specific hybrids.

USES
Identify mutations, deletions, and gene
rearrangements
Used in prognosis of cancer and in
prenatal diagnosis of genetic diseases
Leukemias
Diagnosis of HIV-1 and infectious
disease
USES
Every person has repeated sequences
of base pairs which are called Variable
Number Tandem Repeats (VNTRs)
To find a particular VNTR we use a
radioactive version of the one in
question.
This pattern is known as a DNA
fingerprint.
USES
Applications of DNA fingerprinting
include:
Paternity and Maternity Testing
Criminal Identification and Forensics
Personal Identification
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YOU