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
mujahid hussain, Department of Botany, University of Sargodha, Sargodha, Punjab, Pakistan
2. 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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3. Steps
• Extract and purify DNA from the cell
• DNA is restricted with enzyme
• Separated by gel electrophoresis
• Denatured the DNA
• transfer to the Nitrocellulose membrane paper
(Blotting)
• Add radio labelled probe for hybridization to take
place
• Wash off unbound probe
• Autoradiograph
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4. Isolation of DNA
• Isolate the DNA from the rest of the cellular
material in the nucleus.
• Incubate specimen with detergent to promote cell
lysis.
• A buffer containing a detergent such as sodium
dodecyl sulfate (SDS) is usually sufficient to
disrupt the cell membranes and release high-
molecular weight DNA.
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5. Isolation of DNA
• Lysis frees cellular proteins and DNA.
• Proteins are enzymatically degraded by incubation
with proteinase.
• Digestion of RNA with ribonuclease.
• A final treatment with ethanol, which precipitates the
remaining nucleic acid polymers, enabling
ribonucleotides and other low-molecular weight
contaminants to be removed.
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6. Restriction Digestion
• Cut the DNA into different sized pieces.
• Use restriction endonucleases (RE)
• Example, Eco R1
• For genomic DNA, overnight digestion is usually
required.
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7. Gel electrophoresis
• Nucleic acid have a net negatively charge and will
move from left to right.
• The larger molecule are hold up while the smaller
ones moves faster.
• This results in separation by size.
• Agarose gels have microscopic pores
• Gel is soaked in a buffer which controls the size of
pores.
• Gel can be stained with ethidium bromide.
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8. Gel electrophoresis
• EtBr causes the DNA to fluoresce under UV light.
• This will help to know the exact migration of DNA.
Denaturation and blotting .
• DNA is then denature with an alkaline solution e.g. NaOH.
• This causes the double stranded of DNA to single stranded.
• The process of transferring the DNA from the gel to membrane is called as blotting.
• The blot is done on the sheet of nitrocellulose membrane or nylon.
• DNA is neutralize with NaCl to prevent re-hybridization before adding the probe.
• Transfer is by capillary blotting.
• The blot is made permanent either by
• 1) Drying at 80~ C
• 2)Exposing to UV irradiation.
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10. Hybridization
• It is a process of forming a double stranded DNA
molecule between a single stranded DNA probe and a
single stranded target DNA.
• The labelled probe is add to the membrane in a buffer
and incubated for several hours to allow the probe
molecule to find their targets.
• Probe
• Small piece of labelled DNA used to find another
piece of DNA.
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11. Wash and autoradiography
• Wash excess probe that are bound non-specifically
with the membrane.
• Blot is incubate with wash buffer containing NaCl
and detergent to wash away excess probe.
• Radioactive probes enable autoradiography
detection.
• The probe is radioactive, the particle it emits will
expose X-ray film.
• After development, there will the dark spots on the
film where the probe bound.
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13. Advantages
• Examine entire genome
• Easier
• Detect multiple product
• Disadvantages
• Costly
• Greater quantity of DNA required
• Labor intensive
• Time consuming
• This technique requires fresh or frozen specimens
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14. Application
• To identify specific DNA in the sample
• To isolate desired DNA for making of rDNA
• Used to prognosis cancer and in prenatal diagnosis of
genetic disease
• Used in phylogenetic analysis
• Diagnosis of HIV-I and infectious disease
• And application DNA finger printing
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