Southern Blotting Technique

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

• DNA
• SPECIMEN COLLECTION AND STORAGE
• PROCEDURE
• WATCHPOINTS
• USES

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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.

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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

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Nitrogenous Bases

• Two classes
• Purines
• Adenine
• Guanine
• Pyrimidines
• Cytosine
• Thymine

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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.

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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.

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DNA

• The base adenine (A) always pairs with thymine
  (T).
• The base guanine (G) always pairs with cytosine
  (C).

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DNA

• Example
• First strand GGGTTTAAACCC
• Second strand CCCAAATTTGGG

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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.

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DNA STORAGE AND COLLECTION

• II. Specimens used in DNA testing
• Whole blood
• Solid tissue
• Serum and plasma
• Urine
• Bone marrow
• and many others

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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

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DNA STORAGE AND COLLECTION

• B. Serum
• Collection tubes with no additives
• 100 µl to 1 ml
• Transported at 20-25C

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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.

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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.

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DNA STORAGE AND COLLECTION

• D. Urine
• Urine container should be used for collection.
• At least 1 ml should be collected.
• Transported at 4-25C

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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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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.

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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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SOUTHERN BLOTTING
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SOUTHERN BLOTTING

• 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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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.

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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.

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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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SOUTHERN BLOTTING

• 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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SOUTHERN BLOTTING

• This allows for specific sequences to be
  identified more readily.
• Fragments are now easily separated by gel
  electrophoresis.

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SOUTHERN BLOTTING

• 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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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.

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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.

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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.

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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.

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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.

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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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SOUTHERN BLOTTING

• 1) Electrophoresis- takes advantage of the
  molecules negative charge.

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SOUTHERN BLOTTING

• 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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SOUTHERN BLOTTING

• The blot is made permanent by
• Drying at 80C
• Exposing to UV irradiation

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SOUTHERN BLOTTING

• 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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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.

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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.

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SOUTHERN BLOTTING

• 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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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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SOUTHERN BLOTTING
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SOUTHERN BLOTTING

• 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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SOUTHERN BLOTTING

• 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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SOUTHERN BLOTTING

• IX. Detection
• Radioactive probes enable autoradiographic
  detection.

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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.

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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

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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

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Watch points

• Improper blocking-high background and
  uninterpretable results.
• Insufficient washing-high background and
  uninterpretable results.
• Excess washing- dissociate the specific hybrids.

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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

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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.

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USES

• Applications of DNA fingerprinting include
• Paternity and Maternity Testing
• Criminal Identification and Forensics
• Personal Identification

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• THANK YOU