SOUTHERN BLOTTING

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
DNA STORAGE AND COLLECTION

 I. Temperature Storage for DNA

 PurifiedDNA may be refrigerated
at 4°C for up to 3 years.
 Samples kept over 3 years should
be frozen at -70°C.
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
 4-25°C
DNA STORAGE AND COLLECTION

 B. Serum
 Collection tubes with no additives
 100 µl to 1 ml

 Transported at 20-25°C
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 4-25°C
 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.
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 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.
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 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.
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 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.
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 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.
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 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.
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 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
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 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.
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 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.
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 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.
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 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.
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 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.
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 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.
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 The blot is made permanent by:
 Drying at ~80°C
 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.
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 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.
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 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.
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 3. DNA polymerase is added with
radioactive nucleotides.
 4. The mixture is boiled to
separate the strands and is ready
for hybridization.
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 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.
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 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.
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 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
 USES
 Applications of DNA fingerprinting
include:
 Paternity and Maternity Testing
 Criminal Identification and Forensics

 Personal Identification