Introduction to Southern Hybridization

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
Arabidopsis thaliana
7
Example Looking for Gene X
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)

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 (25-2000 bp) length of DNA or
  RNA
• Complementary to the sequence (gene) of interest
• Labeled for subsequent detection procedures

19
Step 5. Making a Probe
Arabidopsis thaliana
20
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 (a32P)

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 7. Hybridization
33
Step 8. Washes
34
Step 9. Anti-DIG
35
Step 9. Anti-DIG
36
Step 10. Washes
37
Step 11. CSPD
38
Step 12. Detection

• DIG-labeled probes emitting minute amounts of
  light (chemiluminescence)
• 32P-labeled probes emitting ß-particles

39
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
(No Transcript)
41
Conclusion

• How many copies of Gene X does Capsella rubella
  possess?

3
Capsella rubella
42
Other Applications

• DNA fingerprinting
• RFLP or VNTRs
• Dot or slot blot
• Colony or plaque lifts
• Microarray analysis

43
Other Applications

• DNA fingerprinting
• RFLP or VNTRs
• Dot or slot blot
• Colony or plaque lifts
• Microarray analysis

44
Other Applications

• DNA fingerprinting
• RFLP or VNTRs
• Dot or slot blot
• Colony or plaque lifts
• Gene expression

45
Other Applications

• DNA fingerprinting
• RFLP or VNTRs
• Dot or slot blot
• Colony or plaque lifts
• Gene expression

46
Other Applications

• Microarray technology