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TRANSCRIPT
Introduction to Southern Hybridization
Outline
• History/Background Info
• Goals of Southern hybridization
• Example
• Other applications
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
History/Background
• Spawned naming of related techniques:
Southern blot(DNA)
Northern blot(RNA)
Western blot(Protein)
Eastern blot(???)
Goals of Southern Hybridization
• Immobilize DNA onto a permanent substrate
• Identify DNA sequence (gene) of interest
Step 1. Restriction Enzyme Digestion
EcoR I EcoR I EcoR I EcoR I
Step 1. Restriction Enzyme Digestion
Step 2. Gel Electrophoresis
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Step 2. Gel Electrophoresis
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Step 2. Gel Electrophoresis
Goals of Southern Hybridization
Immobilize DNA onto a permanent substrate
• ‘Membrane’– paper‐like matrix
– nylon or nitrocellulose
– usually has a slight positive charge
Step 3. DNA Denaturation
T G A A TC
A C AT T G
• Eliminate hydrogen bonds with sodium hydroxide (NaOH)
Step 4. Transfer DNA to Membrane
• Two methods for transferring DNA to a membrane– capillary– electrophoretic
Step 4. Transfer DNA to Membrane
Goals of Southern Hybridization
• Immobilize DNA onto a permanent substrate
• Identify DNA sequence (gene) of interest
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
Step 5. Making a Probe
Gene Xfrom Arabidopsis
Partial or full-lengthprobes by PCR
Step 5. Making a Probe
Gene Xfrom Arabidopsis
Partial probes by random-priming
Step 5. Making a Probe
Denature template with heat
Step 5. Making a Probe
Add random primers
Step 5. Making a Probe
Extend random primers with polymerase
Step 5. Making a Probe
A probe complementary to the sequence (Gene X) of interest!
Step 5. Making a Probe
• How do we detect the probe?– Radioactivity (32P)
Step 5. Making a Probe
• How do we detect the probe?– Digoxigenin (DIG)
U
Step 4. Transfer DNA to Membrane
Step 6. Pre‐hybridization
Prehybridization bufferscontain ‘blocking reagents’that occupy available binding sites on the membrane
Step 7. Hybridization
Step 7. Hybridization
Step 8. Washes
Step 9. Anti‐DIG
Step 9. Anti‐DIG
Step 10. Washes
Step 11. CSPD
Step 12. Detection
• DIG‐labeled probes emitting minute amounts of light (chemiluminescence)
• 32P‐labeled probes emitting ß‐particles
Step 12. Detection
• DIG‐labeled probes emitting minute amounts of light (chemiluminescence)
• 32P‐labeled probes emitting ß‐particles
• Autoradiography film can detect this radiation
Conclusion
• How many copies of ‘Gene X’ does Capsella rubellapossess?
Capsella rubella
3
Other Applications
• DNA fingerprinting– RFLP of VNTRs
• Dot or slot blot
• Colony or plaque lifts
• Microarray analysis
Other Applications
• DNA fingerprinting– RFLP of VNTRs
• Dot or slot blot
• Colony or plaque lifts
• Microarray analysis
Other Applications
• DNA fingerprinting– RFLP of VNTRs
• Dot or slot blot
• Colony or plaque lifts
• Microarray analysis
Other Applications
• DNA fingerprinting– RFLP of VNTRs
• Dot or slot blot
• Colony or plaque lifts
• Gene Expression
Other Applications
• Microarray technology