1. SOUTHERN BLOTTING
Ali Zaeri
Medical Genetics and diagnostic lab
Lab 5

2. Southern Blot Lab Southern, Northern & Western Blotting Background
Overview
Detailed Protocol

3. Who is this guy?

4. 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.

5. SOUTHERN BLOTTING Probe- The key to this method is hybridization.
IT IS process of forming a double-stranded DNA molecule between a single-stranded DNA probe and a single-stranded target patient DNA.
Probe-
It is a small piece of labelled DNA used to find another piece of DNA. It is usually labelled by radioactive copy of DNA

6. 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.

7. Characterization: Southern blot hybridization
-transfer of DNA from a gel to a membrane (e.g., nitrocellulose, nylon)
-developed by Edwin Southern

8. Characterization: Northern blot hybridization
X RNA X x
salt X
RNA
-transfer of RNA from a gel to a membrane (e.g., nitrocellulose, nylon)
-reveals mRNA size (and approximate protein size), tissue- and organ-
specific expression, and kinetic patterns of expression

9. Characterization: Western blotting
X Protein
Enzyme
reaction or X
React with
Antibody X x
Buffer; requires electric current X
-transfer of protein from a gel to a membrane (e.g., nitrocellulose, nylon)
-requires the use of an electric current to facilitate transfer

10. Southern blot hybridization
-transfer of DNA from a gel to a membrane (e.g., nitrocellulose, nylon)
-developed by Edwin Southern

12. Southern blotting of genomic DNA
Steps
1. Extraction of genomic DNA
2. Restriction digestion
Cut the DNA into different sized fragments
USE RESTRICTION ENDONUCLEASE ( RE)
3- Electrophoresis of genomic DNA (or more commonly restriction enzyme-digested genomic DNA)

13. Southern blotting of genomic DNA
Step 4 and 5 Denaturation and blotting
DNA is then denatured with an alkaline solution such as NAOH.
This causes the double stranded to become single-stranded.

14. SOUTHERN BLOTTING
DNA is then neutralized with NaCl to prevent re-hybridization before adding the probe.
Transferred by either electrophoresis or capillary blotting.

15. SOUTHERN BLOTTING
1) Electrophoresis- takes advantage of the molecules negative charge.

17. B) Wick method

18. 5. After the transfer, cross-link the DNA to the nitrocellulose membrane
A. UV- cross linking
B. Bake at 120°C for 30 min
C. Bake at 80°C for 2 hrs

19. 6. 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.

20. Radioactive probes enable autoradiographic detection.
7. 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
8- Detection
Radioactive probes enable autoradiographic detection.
Expose to X-ray …… Pos give black band

22. 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. Hyberidization
8. Wash off unbound probe
9. Autoradiograph

23. USES Identify mutations, deletions,
Used in prognosis of cancer and in prenatal diagnosis of genetic diseases
Leukemias
Diagnosis of HIV-1 and infectious disease

24. USES Applications of DNA fingerprinting include:
Paternity and Maternity Testing
Criminal Identification and Forensics
Personal Identification

25. THANK YOU