Southern blotting

1. SOUTHERN BLOTTING
MERIN ALICE GEORGE

2. BLOTTING
 is a method of transferring proteins, DNA or RNA onto a carrier (for
example, a nitrocellulose or nylon membrane.
 Types
1. Southern Blotting (DNA)
2. Northern Blotting (RNA)
3. Western Blotting (Proteins)
4. Eastern Blotting (Post translational modifications of proteins)

3. SOUTHERN BLOTTING
 Discovered by Edwin Southern , 1975
Purpose:
1. Used for detection of a specific DNA sequence in DNA samples.
2. Southern blotting technique is used to isolate a particular DNA
3. To study mutation and gene rearrangement.
4. This technique is used in phylogenetic studies, paternity & maternity
analysis and forensic studies.

4. Principle:
KEY FEATURE: Hybridization
Hybridization: It is the process of forming a double stranded
DNA molecule between a single-stranded DNA probe and a single-stranded
target DNA.
 Separation of DNA fragments by gel electrophoresis
 followed by the identification by labeled probe hybridization.
 The DNA fragments are separated based on their size and charge during
electrophoresis

5. 1.Extract and Purify Dna
2.Digest the DNA by restriction enzyme-
fragments formed
3.Separation of fragments by Gel
Electrophorsis
4.Denature the DNA by Alkali treatment -
single stranded Dna
5.Transfer the denatured DNA to the
membrane..

6. 6.Add labeled probe for hybridization to
take
7. Wash off unbound probe;
8. Autoradiograph

7. STEPS
1.Extract and purify DNA from cells
• Isolate the DNA from the rest of the cellular material in the
nucleus.
• Isolated DNA is purified from solution by alcohol precipitation.
2. DNA is restricted with enzymes.

8. 3.Separated by electrophoresis.
Restriction fragments are gel electrophoresed in an Agarose gel and the
fragments are separated based on their size.

9. 4. Denature DNA
 DNA bands in the gel are denatured into single stranded form by
alkali treatment (0.5 M NaOH).
5.Transfer to nitrocellulose paper:
 Place the gel on top of a buffer saturated filter paper
 Lay the Nitrocellulose filter membrane on the top of the gel
 Place some dry filter paper on top of this membrane. From the bottom
filter paper , buffer move by capillary action through the gel , carrying
with it the denatured DNA present in the gel .
 DNA get trapped in the nitrocellulose membrane as the buffer passes
through it because of its greater affinity → replica pattern.

10.  Bake nitrocellulose membrane with ssDNA bands at 80 ⁰ C for 2-3 hours-
to fix the DNA permanently on the membrane.
 Nitrocellulose membrane with the replica of DNA bands from the Agarose
gel is used for hybridization with labelled DNA probe –

11. 6. Add labeled probe for hybridization:
 Nitrocellulose sheet is moistened with a minimal quantity of solution
containing a ³²P labelled ssDNA probe that is complementary in sequence
to the DNA of interest
 The probe hybridizes with the complementary Dna.
A hybridization probe is a fragment of DNA or RNA of variable length
(usually 100–10000 bases long) which can be radioactively or fluorescently
labeled.
It is used in DNA or RNA samples to detect the presence of nucleotide
substances that are complementary to the sequence in the probe

12. 7. Wash the unbound probe.
8. Autoradiograph:
Place it over a sheet of X –ray film – the positions of the molecules that
are complementary to the radioactive probe are indicated by a
darkening of the developed film.