Southern blotting is a technique used to detect specific DNA sequences in a DNA sample. It involves separating DNA fragments by size via gel electrophoresis, transferring them to a membrane, and using a labeled probe to identify the DNA fragment of interest through hybridization and autoradiography. Key steps include restriction enzyme digestion of DNA, gel electrophoresis, transferring fragments to a membrane, hybridizing with a probe, washing unbound probe, and detecting the bound probe fragment. This allows researchers to identify specific DNA sequences and analyze DNA samples.

1. SOUTHERN
BLOTTING
M.Meenakshi,
Assistant Professor,
Department of Microbiology,
Sri Ramakrishna college of Arts & Science for Women, Coimbatore

2. Southern Blotting
• The analytical technique that involves the transfer of a specific dna, rna or a protein separated on gel to a
carrier membrane, for their detection or identification is termed as blotting.
• The process of transfer of the denatured fragments out of the gel and onto a carrier membrane makes it
accessible for analysis using a probe or antibody.
• Depending upon the substance to be separated, blotting techniques may be – southern blot, northern blot
or western blot which separates dna, rna and proteins respectively.
• Southern blot is the analytical technique used in molecular biology, immunogenetics and other molecular
methods to detect or identify dna of interest from a mixture of dna sample or a specific base sequence
within a strand of dna.
• The technique was developed by a molecular biologist E.M. Southern in 1975 for analysing the related
genes in a dna restriction fragment and thus named as southern blotting in his honour.

4. Principle of Southern BloT
• The process involves the transfer of electrophoresis-separated DNA
fragments to a carrier membrane which is usually nitrocellulose and the
subsequent detection of the target DNA fragment by probe hybridization.
• Hybridization refers to the process of forming a double-stranded DNA
molecule between a single-stranded DNA probe and a single-stranded target
DNA.
• Since the probe and target DNA are complementary to each other, the
reaction is specific which aids in the detection of the specific DNA
fragment.

6. Steps involved in Southern blotting

8. Steps involved in Southern blotting
• Extraction and purification of DNA from cells
• DNA is first separated from target cells following standard methods of
genomic DNA extraction and then purified.
• Restriction Digestion or DNA Fragmentation
• Restriction endonucleases are used to cut high-molecular-weight DNA
strands into smaller fragments. One or more restriction enzymes can be used
to achieve such fragment

9. Steps involved in Southern blotting
• Separation by Electrophoresis
• The separation may be done by agarose gel electrophoresis in which the negatively charged DNA
fragments move towards the positively charged anode, the distance moved depending upon its size.
• Depurination
• Partial depurination is done by the use of dilute HCl which promotes higher efficiency transfer of
DNA fragments by it breaking down into smaller pieces.
• Denaturation
• DNA is then denatured with a mild alkali such as an alkaline solution of NaOH. This causes the
double stranded DNA to become single-stranded, making them suitable for hybridization. DNA is
then neutralized with NaCl to prevent re-hybridization before addition of the probe.

10. Steps involved in Southern blotting
• Blotting
• The denatured fragments are then transferred onto a nylon or nitrocellulose filter membrane
which is done by placing the gel on top of a buffer saturated filter paper, then laying
nitrocellulose filter membrane on the top of gel. Finally some dry filter papers are placed on
top of the membrane. Fragments are pulled towards the nitrocellulose filter membrane by
capillary action and result in the contact print of the gel.
• Baking
• The nitrocellulose membrane is removed from the blotting stack, and the membrane with
single stranded DNA bands attached on to it is baked in a vacuum or regular oven at 80 °C
for 2-3 hours or exposed to ultraviolet radiation to permanently attach the transferred DNA
onto the membrane.

11. Steps involved in Southern blotting
• Hybridization
• The membrane is then exposed to a hybridization probe which is a single DNA
fragment with a specific sequence whose presence in the target DNA is to be
determined. The probe DNA is labeled so that it can be detected, usually by
incorporating radioactivity or tagging the molecule with a fluorescent or
chromogenic dye.
• Washing of unbound probes
• After hybridization, the membrane is thoroughly washed with a buffer to remove
the probe that is bound nonspecifically or any unbound probes present.

12. Steps involved in Southern blotting
• Autoradiograph
• The hybridized regions are detected autoradiographically by placing the
nitrocellulose membrane in contact with a photographic film which shows
the hybridized DNA molecules. The pattern of hybridization is visualized on
X-ray film by autoradiography in case of a radioactive or fluorescent probe is
used or by the development of color on the membrane if a chromogenic
detection method is used.

14. Summary
• Southern blotting is a technique that can be used to separate and detect DNA
molecules of interest while Northern blotting can be used to isolate and detect
RNA molecules of interest. Both techniques work in the same way. The nucleic acid
is broken down into many smaller fragments by the action of restriction enzymes.
The fragments are then denatured and run under gel electrophoresis conditions.
The results of the electrophoresis are transferred onto a polymer sheet and a nucleic
acid probe is added. The radioactively-labeled probe is built so that its sequence is
complementary to the sequence of the fragment to be isolated. Once the probe
hybridizes with the fragment of interest, autoradiography can then be used to detect
the band that contains the fragment of interest

15. Applications of Southern Blot
• Identifying specific DNA in a DNA sample.
• Preparation of RFLP (Restriction Fragment Length Polymorphism) maps
• Detection of mutations, deletions or gene rearrangements in DNA
• For criminal identification and DNA fingerprinting (VNTR)
• Detection and identification of trans gene in transgenic individual
• Mapping of restriction sites
• For diagnosis of infectious diseases
• Prognosis of cancer and prenatal diagnosis of genetic diseases
• Determination of the molecular weight of a restriction fragment and to measure relative amounts in
different samples.

16. References
• ^ Southern, Edwin Mellor (5 November 1975). "Detection of specific sequences among DNA fragments
separated by gel electrophoresis". Journal of Molecular Biology. 98 (3): 503–517. doi:10.1016/S0022-
2836(75)80083-0. ISSN 0022-2836. PMID 1195397.
• ^ Towbin; Staehelin, T; Gordon, J; et al. (1979). "Electrophoretic transfer of proteins from polyacrylamide
gels to nitrocellulose sheets: procedure and some applications". PNAS. 76 (9): 4350–
4. doi:10.1073/pnas.76.9.4350. PMC 411572. PMID 388439.
• ^ Burnette, W. Neal (April 1981). "Western Blotting: Electrophoretic Transfer of Proteins from Sodium
Dodecyl Sulfate-Polyacrylamide Gels to Unmodified Nitrocellulose and Radiographic Detection with
Antibody and Radioiodinated Protein A". Analytical Biochemistry. 112 (2): 195–
203. doi:10.1016/0003-2697(81)90281-5. ISSN 0003-2697. PMID 6266278.
• ^ Biochemistry 3rd Edition, Matthews, Van Holde et al, Addison Wesley Publishing, pg 977