1. Hybridization Techniques

2. 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

3. Spawned naming of related techniques:
Northern blot
(RNA)
Western blot
(Protein)
Eastern blot
(???)
Southern blot
(DNA)

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

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

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

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

8. Step 1. Restriction Enzyme Digestion

9. Step 2. Gel Electrophoresis_ +

10. Step 2. Gel Electrophoresis_ +

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

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

13. Step 4. Transfer DNA to Membrane
Two methods for transferring DNA to a membrane
capillary
Electrophoretic (

14. Step 4. Transfer DNA to Membrane

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

16. 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

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

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

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

20. Step 7. Hybridization

21. Step 7. Hybridization

22. Step 8. Washes

23. Step 9. Anti-DIG

24. Step 9. Anti-DIG

25. Step 10. Washes

26. Step 11. CSPD Disodium 3-(4-methoxyspiro {1,2-dioxetane-3,2´-(5'-chloro)tricyclo [ ,7]decan}-4-yl)phenyl phosphate

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

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

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