Southern blotting is a technique used to detect specific DNA sequences. It involves extracting DNA, restricting it with enzymes, separating fragments via electrophoresis, transferring DNA to a membrane, then using a probe with a complementary sequence and radioactive tag to hybridize and identify the target DNA fragment through autoradiography. It is useful for detecting gene copies, identifying mutations, and diagnosing genetic diseases.

2. SOUTHERN BLOTTING
REGISTRATION : 074452
COURSE TITLE : MOLECULAR GENETICS
COURSE CODE : ZOL-508
DEPARTMENT OF ZOOLOGY
GOVERNMENT UNIVERSITY FAISALABAD

3. CONTENT
 BLOTTING
 SOUTHERN BLOTTING
 Principle
 STEPS IN SOUTHERN BLOTTING
 PROCEDURE
 ADVANTAGES
 DISADVANTAGES
 REFERNCES

4. Blotting
 Technique
 Detect and analyze biological macro-molecules
 Types
o Southern blotting
o Northern blotting
o Western blotting

5. TYPES OF BLOTTING
SOUTHERN
BLOTTING
BLOTTING
WESTERN
BLOTTING
NORTHERN
BLOTTING

6. SOUTHERN BLOTTING
 Method for detection of specific DNA sequence
 Transfer of electrophoresis – separated DNA to
membrane
 Subsequent fragment detect by probe hybridization
 Southern hybridization

7. Historical background
Southern blotting was invented in 1973 but it was not
published until 1975
IN 1975 , EDMIN SOUTHERN published it .

8. Principle
 Transfer of electrophoresis – separated DNA
fragments to a membrane
 Hybridization refers to the process of forming double
– standard DNA
 Probe and target DNA are complementary to each
other

9. Southern blotting set up

10. STEPS IN SOUTHERN
BLOTTING
i. EXTRACTION OF DNA
ii. PURIFICATION
iii. RESTRICTION RNDONUCLEASES
iv. ELECTROPHORESIS
v. DEPURINATION
vi. DENATURATION
vii. BLOTTING

11. STEPS IN SOUTHERN
BLOTTING
viii. BAKING
ix. HYBRIDIZATION
x. WASHING
xi. AUTORADIOGRAPHY

12. STEPS INVOLVED IN SOUTHERN BLOTTING

13. 1. Extraction of DNA
o DNA is separated from cell

14. 2. Purification of DNA
 purification is done by adding phenol

15. 3. RESTRICTION
ENDONUCLEASES
 Fragmentation of DNA by enzyme(endo-nucleases)

16. 4. Separation by Electrophoresis
 Agraose gel electrophoresis
 Negatively charged DNA move toward positively
charged anode

17. 5. Depurination
 It is done by dilute HCl
 N – Glycosidic bond cleaved to release adenine or
guanine

18. 6. DENATURATION
 It is denatured by mild alkali (alkaline solution of
NaoH)
 double – standard DNA become single – standard
 It is denatured by heat

19. 7. Blotting
 Transfer of fragments of DNA to nitrocellulose
membrane
 It is done by CAPILLARY BLOTTING

20. 8. BAKING
 Membrane is removed and regular oven at 80°C for
2-3 hours
 Transferred DNA permanently onto membrane

21. 9. HYBRIDIZATION
 Membrane with DNA fragment exposed to a
hybridization probe
 Probe is single standard complementary sequence of
DNA nucleotides
 Probe is tagged with fluorescent or chromogenic dye

23. 10. Washing of unbound probes
 After hybridization is thoroughly washed with buffer to
remove unbound probe

24. 11. AUTORADIOGRAH
 Hybridized region are detected autoradiographically
with a photographic film

25. Advantages
 Effective way to detect specific DNA sequence in a
large , complex sample of DNA
 Only way to diagnose FSHD (Facioscapulohumeral
muscular dystrophy)
 Detect exact location of disorder
 Cheaper than DNA sequencing

26. Disadvantages
 It requires expensive equipment and reagents
 Time – consuming takes 7 – 14 days
 Radioactive
 Difficult to troubleshoot
 Complex

27. Application of southern blotting
 It is used to determine number of sequence (e . g . ,
gene copies)
 Find out specific DNA sequence present in different
animal
 FOR EXAMPLE , presence of insulin gene in animal
 DNA fingerprinting
 Used to identify mutation

28. Application
 Used in diagnose of disease caused by genetic
defect
 To isolate desire gene

29. REFERENCES
 https://mybiosource.com
 https://www.news-medical.net/life-sciences
 https://www.nature.com