Southern hybridization is a technique developed by Edwin Southern in 1975 to detect specific DNA sequences. It involves immobilizing DNA fragments on a membrane, making a labeled probe complementary to the target sequence, and detecting binding between the probe and target. The goals are to immobilize DNA on a substrate and identify a specific DNA sequence or gene of interest. It has applications like DNA fingerprinting, detecting gene expression, and microarray analysis.

1. Introduction to
Southern Hybridization
Michael Melzer
Plant & Environmental Protection Sciences
University of Hawaii at Manoa

2. Outline
• History/Background Info
• Goals of Southern hybridization
• Example
• Other applications

3. History/Background
• ‘Southern’ hybridization
named after Sir Edwin
Southern
• Developed in 1975
• One of the most highly
cited scientific publications
• Earned Sir Southern a
Lasker Award in 2005

4. History/Background
• Spawned naming of related techniques:
Southern blot
(DNA)
Northern blot
(RNA)
Western blot
(Protein)
Eastern blot
(???)

5. Goals of Southern Hybridization
• Immobilize DNA onto a permanent
substrate
• Identify DNA sequence (gene) of interest

6. Southern Blot
Restriction Digest of DNA
Electrophoresis
Denaturation/Depurination
Blotting Step (overnight)
Detection of DNA (next lab)

7. Capsella rubella
The key to this method is
hybridization.
Hybridization-process of
forming a double-
stranded DNA molecule
between a single-stranded
DNA probe and a single-
stranded target patient
DNA.

8. 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

9. Steps for hybridization
1. The mixture of molecules
is separated.
2. The molecules are
immobilized on a matrix.
3. The probe is added to
the matrix to bind to the
molecules.
4. Any unbound probes are
then removed.
5. The place where the
probe is connected
corresponds to the location
of the immobilized target
molecule.

10. Blotting
Blotting
Blotting
Transfer the DNA from the
gel to a solid support.
The blot is usually done on a
sheet of nitrocellulose paper
or nylon.
DNA is then neutralized with NaCl to
prevent re-hybridization before adding the
probe.
Transferred by either electrophoresis or
capillary blotting.

11. Step 1. Restriction Enzyme Digestion

12. Step 2. Gel Electrophoresis
_ +

13. Step 2. Gel Electrophoresis
_ +

14. Step 2. Gel Electrophoresis

15. Goals of Southern Hybridization
Immobilize DNA onto a permanent substrate
• ‘Membrane’
– paper-like matrix
– nylon or nitrocellulose
– usually has a slight positive charge

16. T G A A T
C
A C A
T T G
Step 3. DNA Denaturation
• Eliminate hydrogen bonds with sodium
hydroxide (NaOH)

17. Step 4. Transfer DNA to Membrane
• Two methods for transferring DNA to a
membrane
– capillary
– electrophoretic

18. Step 4. Transfer DNA to Membrane

19. Goals of Southern Hybridization
• Immobilize DNA onto a permanent
substrate
• Identify DNA sequence (gene) of interest

20. Step 5. Making a Probe
• A probe is a small (25-2000 bp) length of
DNA or RNA
– Complementary to the sequence (gene) of
interest
– Labeled for subsequent detection procedures

21. Step 5. Making a Probe
Arabidopsis thaliana 2 copies of gene X

22. Step 5. Making a Probe
Gene X
from Arabidopsis
Partial or full-length
probes by PCR

23. Step 5. Making a Probe
Gene X
from Arabidopsis
Partial probes by
random-priming

24. Step 5. Making a Probe
Denature template with heat

25. Step 5. Making a Probe
Add random primers

26. Step 5. Making a Probe
Extend random primers with polymerase

27. Step 5. Making a Probe
A probe complementary to the sequence (Gene X) of interest!

28. Step 5. Making a Probe
• How do we detect the probe?
– Radioactivity (32P)

29. Step 5. Making a Probe
• How do we detect the probe?
– Digoxigenin (DIG)
U

30. Step 4. Transfer DNA to Membrane

31. Step 6. Pre-hybridization
Prehybridization buffers
contain ‘blocking reagents’
that occupy available binding
sites on the membrane

32. Step 7.
Hybridization

33. Step 7. Hybridization

34. Step 8. Washes

35. Step 9. Anti-DIG

36. Step 9. Anti-DIG

37. Step 10. Washes

38. Step 11. CSPD

39. Step 12. Detection
• DIG-labeled probes
emitting minute
amounts of light
(chemiluminescence)
• 32P-labeled probes
emitting ß-particles

40. Step 12. Detection
• DIG-labeled probes
emitting minute
amounts of light
(chemiluminescence)
• 32P-labeled probes
emitting ß-particles
• Autoradiography
film can detect this
radiation

42. Conclusion
• How many copies of
‘Gene X’ does Capsella
rubella possess?
Capsella rubella
3

43. Other Applications
• DNA fingerprinting
– RFLP of VNTRs
• Dot or slot blot
• Colony or plaque lifts
• Microarray analysis

44. Other Applications
• DNA fingerprinting
– RFLP of VNTRs
• Dot or slot blot
• Colony or plaque lifts
• Microarray analysis

45. Other Applications
• DNA fingerprinting
– RFLP of VNTRs
• Dot or slot blot
• Colony or plaque lifts
• Microarray analysis

46. Other Applications
• DNA fingerprinting
– RFLP of VNTRs
• Dot or slot blot
• Colony or plaque lifts
• Gene Expression

48. Other Applications
• Microarray
technology