Introduction to Southern Hybridization

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Introduction to Southern Hybridization

• Michael Melzer
• Plant Environmental Protection Sciences
• University of Hawaii at Manoa

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Outline

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

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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

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History/Background

• Spawned naming of related techniques

Northern blot (RNA)
Western blot (Protein)
Eastern blot (???)
Southern blot (DNA)
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Goals of Southern Hybridization

• Immobilize DNA onto a permanent substrate
• Identify DNA sequence (gene) of interest

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Example Looking for Gene X
Arabidopsis thaliana
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Example Looking for Gene X
Capsella rubella
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Step 1. Restriction Enzyme Digestion
EcoR I
EcoR I
EcoR I
EcoR I
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Step 1. Restriction Enzyme Digestion
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Step 2. Gel Electrophoresis
_

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Step 2. Gel Electrophoresis
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Step 2. Gel Electrophoresis
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Goals of Southern Hybridization

• Immobilize DNA onto a permanent substrate
• Membrane
• paper-like matrix
• nylon or nitrocellulose
• usually has a slight positive charge

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Step 3. DNA Denaturation

• Eliminate hydrogen bonds with sodium hydroxide
  (NaOH)

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Step 4. Transfer DNA to Membrane

• Two methods for transferring DNA to a membrane
• capillary
• electrophoretic

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Step 4. Transfer DNA to Membrane

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Goals of Southern Hybridization

• Immobilize DNA onto a permanent substrate
• Identify DNA sequence (gene) of interest

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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

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Step 5. Making a Probe
Arabidopsis thaliana
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Step 5. Making a Probe
Gene X from Arabidopsis
Partial or full-length probes by PCR
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Step 5. Making a Probe
Gene X from Arabidopsis
Partial probes by random-priming
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Step 5. Making a Probe
Denature template with heat
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Step 5. Making a Probe
Add random primers
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Step 5. Making a Probe
Extend random primers with polymerase
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Step 5. Making a Probe
A probe complementary to the sequence (Gene X) of
interest!
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Step 5. Making a Probe

• How do we detect the probe?
• Radioactivity (32P)

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Step 5. Making a Probe

• How do we detect the probe?
• Digoxigenin (DIG)

U
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Step 4. Transfer DNA to Membrane

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Step 6. Pre-hybridization

Prehybridization buffers contain blocking
reagents that occupy available binding sites on
the membrane
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Step 7. Hybridization

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Step 7. Hybridization
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Step 8. Washes
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Step 9. Anti-DIG
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Step 9. Anti-DIG
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Step 10. Washes
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Step 11. CSPD
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Step 12. Detection

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

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Step 12. Detection

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

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Conclusion

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

3
Capsella rubella
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Other Applications

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

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Other Applications

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

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Other Applications

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

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Other Applications

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

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Other Applications

• Microarray technology