introduction to southern hybridization michael melzer plant & environmental protection sciences...
TRANSCRIPT
Introduction to Southern Hybridization
Michael MelzerPlant & Environmental Protection
SciencesUniversity of Hawaii at Manoa
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
Example – Looking for Gene X
Arabidopsis thaliana 2 copies of gene X
Capsella rubella ? copies of gene X
extract
DNA
Example – Looking for Gene X
Step 1. Restriction Enzyme Digestion
EcoR I EcoR I EcoR I EcoR I
Step 1. Restriction Enzyme Digestion
Step 2. Gel Electrophoresis
_ +
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
T G A A TC
A C AT T G
Step 3. DNA Denaturation
• 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
Arabidopsis thaliana 2 copies of gene X
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 rubella possess?
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