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: Northern blot
(RNA)
Western blot
(Protein)
Eastern blot
(???)
Southern blot
(DNA)

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

6. Example – Looking for Gene X
2 copies of gene X
Arabidopsis thaliana

7. Example – Looking for Gene X
? copies of gene X
extract
DNA
Capsella rubella

8. Step 1. Restriction Enzyme Digestion
EcoR I
EcoR I
EcoR I
EcoR I

9. Step 1. Restriction Enzyme Digestion

10. Step 2. Gel Electrophoresis_ +

11. Step 2. Gel Electrophoresis_ +

12. Step 2. Gel Electrophoresis

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

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

15. Step 4. Transfer DNA to Membrane
Two methods for transferring DNA to a membrane
capillary
electrophoretic

16. Step 4. Transfer DNA to Membrane

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

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

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

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

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

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

23. Step 5. Making a Probe
Add random primers

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

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

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

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

28. Step 4. Transfer DNA to Membrane

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

30. Step 7. Hybridization

31. Step 7. Hybridization

32. Step 8. Washes

33. Step 9. Anti-DIG

34. Step 9. Anti-DIG

35. Step 10. Washes

36. Step 11. CSPD

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

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

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

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

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

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

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

45. Other Applications
Microarray technology