Overview on southern blotting, procedure and applications

1. SOUTHERN BLOTTING
BHAVINI K and SNEHA P NAIR
I MSc BT, AIB
Amity University, Mumbai

2. INTRODUCTION & PRINCIPLE
• Blotting is the technique in which nucleic acids or proteins are immobilized onto a solid
support generally nylon or nitrocellulose membranes.
• Blotting of nucleic acid is the central technique for hybridization studies and are the
basis for a range of experimental techniques involving understanding of gene expression,
organization, etc.
• Southern blotting is a blotting and detection technique for the identification of
particular DNA sequence from mixture of similar molecules.
• This technique is named after its inventor, Edwin Southern.
• Southern blotting is based on the principle of separation of DNA fragments by gel
electrophoresis followed by the identification by labeled probe hybridization.

3. GENERAL
PROCEDURE
Extraction and purification of DNA from sample
Restriction digestion of DNA
Separation by Electrophoresis
Depurination
Denaturation
Blotting
Baking
Hybridization
Washing of unbound probes
Autoradiography

4. REQUIREMENTS
 Reagents and apparatus for DNA isolation and purification
 Reagents and apparatus for restriction digestion of DNA
 Reagents and buffers, apparatus for agarose gel electrophoresis
 Reagents and apparatus for southern blotting
1) Depurination solution – 0.2 N HCl
2) Denaturation solution – 0.5 N NaOH, 1.5 N NaCl
3) Neutralization solution - 0.5 M Tris-HCl with 1.5 M NaCl
4) Transfer buffer/ solution - 10X SSC (0.15 M trisodium citrate dihydrate, pH 7.0,
with 1.5 M NaCl)

5. 5) Pre-hybridization buffer – 6X SSPE stock solution
5x Denhardt's reagent
0.5% SDS
100 µg/ml Salmon Testes DNA
deionized formamide 50%
D.H 2 0
6) Labelled 32P probes
7) Hybridization solution- Prehybridization solution + dextran sulfate
8) Hybridization bottles, oven
9) Wash solutions- 1X SSC, 0.1% SDS and 0.25X SSC, O.1% SDS

6. • Blotting equipment-
1) Large plastic tray
2) Support
3) Tissues and paper towels
4) Whatman filter papers
5) Nitrocellulose sheet/ nylon membrane
6) Weight
• X ray film and other apparatus for autoradiography

7. APPLICATIONS
• To identify a specific part of DNA in a DNA sample.
• used in the identification and characterization of viral strains (Aubin. et al., 1991) and
certain bacterial strains.
• useful in the study of mutation (Mellars. & Gomez., 2011) and gene rearrangement, this
property is used to diagnose neonatal disease and genetic disease.
• Due to the precision in DNA identification this technique is used in phylogenetic
studies, paternity & maternity analysis, forensic studies (Thomson J., 1998) and personal
identification.
• The discovery of RFLPs by southern blotting has helped in the mapping of several
genomes that were crucial to be mapped. (Severson D.W., 1997)
• In the arena of immunology, the clonal rearrangements of the immunoglobulins as
well as the T Cell receptor genes that play an important role in eliciting an
immune response can be analysed by Southern blotting. (Moreau, E., Langerak, A., van Gastel-
Mol, E. et al., 1999)

8. Mutation Detection by Southern Blotting (Mellars. & Gomez., 2011)
This paper describes the Southern blotting technique used to detect inversions affecting
intron 22 in the F8 gene which are the commonest cause of severe haemophilia A.

9. RESULT

10. REFERENCES
• https://conductscience.com/southern-blotting-protocol/
• https://www.med.upenn.edu/robertsonlab/assets/user-
content/documents/Southern%20Blotting%20and%20Hybridization.pdf
• https://www.sigmaaldrich.com/IN/en/technical-documents/protocol/protein-biology/gel-electrophoresis/southern-and-
northern-blotting
• https://www.mybiosource.com/learn/southern-blotting/
1. Mellars., & Gomez., k. (2011). Mutation detection by Southern blotting. Methods Mol Biol, 688, 281-291.
2. J. Aubin., H. Collandre., D. Candotti., D. Ingrand., C. Rouzioux., M. Burgard., . . . Agut., H. (1991). Several groups
among human herpesvirus 6 strains can be distinguished by Southern blotting and polymerase chain reaction. J Clin
Microbiol, 29(2), 367-72.
3. Thomson J. (1998) Southern Blotting of Genomic DNA for DNA Profiling. In: Lincoln P.J., Thomson J. (eds) Forensic
DNA Profiling Protocols. Methods in Molecular Biology, vol 98. Humana Press. https://doi.org/10.1385/0-89603-443-
7:49
4. Severson D.W. (1997) RFLP analysis of insect genomes. In: Crampton J.M., Beard C.B., Louis C. (eds) The Molecular
Biology of Insect Disease Vectors. Springer, Dordrecht. https://doi.org/10.1007/978-94-009-1535-0_26
5. Moreau, E., Langerak, A., van Gastel-Mol, E. et al. (1999) Easy detection of all T cell receptor gamma (TCRG) gene
rearrangements by Southern blot analysis: recommendations for optimal results. Leukemia 13, 1620–1626.
https://doi.org/10.1038/sj.leu.2401540

11. THANK YOU