Introduction to Southern Hybridization Michael Melzer Plant & Environmental Protection Sciences...
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Transcript of Introduction to Southern Hybridization Michael Melzer Plant & Environmental Protection Sciences...
![Page 1: Introduction to Southern Hybridization Michael Melzer Plant & Environmental Protection Sciences University of Hawaii at Manoa.](https://reader035.fdocuments.us/reader035/viewer/2022062312/551b56db550346d41a8b62e5/html5/thumbnails/1.jpg)
Introduction to Southern Hybridization
Michael MelzerPlant & Environmental Protection
SciencesUniversity of Hawaii at Manoa
![Page 2: Introduction to Southern Hybridization Michael Melzer Plant & Environmental Protection Sciences University of Hawaii at Manoa.](https://reader035.fdocuments.us/reader035/viewer/2022062312/551b56db550346d41a8b62e5/html5/thumbnails/2.jpg)
Outline
• History/Background Info
• Goals of Southern hybridization
• Example
• Other applications
![Page 3: Introduction to Southern Hybridization Michael Melzer Plant & Environmental Protection Sciences University of Hawaii at Manoa.](https://reader035.fdocuments.us/reader035/viewer/2022062312/551b56db550346d41a8b62e5/html5/thumbnails/3.jpg)
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
![Page 4: Introduction to Southern Hybridization Michael Melzer Plant & Environmental Protection Sciences University of Hawaii at Manoa.](https://reader035.fdocuments.us/reader035/viewer/2022062312/551b56db550346d41a8b62e5/html5/thumbnails/4.jpg)
History/Background
• Spawned naming of related techniques:
Southern blot(DNA)
Northern blot(RNA)
Western blot(Protein)
Eastern blot(???)
![Page 5: Introduction to Southern Hybridization Michael Melzer Plant & Environmental Protection Sciences University of Hawaii at Manoa.](https://reader035.fdocuments.us/reader035/viewer/2022062312/551b56db550346d41a8b62e5/html5/thumbnails/5.jpg)
Goals of Southern Hybridization
• Immobilize DNA onto a permanent substrate
• Identify DNA sequence (gene) of interest
![Page 6: Introduction to Southern Hybridization Michael Melzer Plant & Environmental Protection Sciences University of Hawaii at Manoa.](https://reader035.fdocuments.us/reader035/viewer/2022062312/551b56db550346d41a8b62e5/html5/thumbnails/6.jpg)
Example – Looking for Gene X
Arabidopsis thaliana 2 copies of gene X
![Page 7: Introduction to Southern Hybridization Michael Melzer Plant & Environmental Protection Sciences University of Hawaii at Manoa.](https://reader035.fdocuments.us/reader035/viewer/2022062312/551b56db550346d41a8b62e5/html5/thumbnails/7.jpg)
Capsella rubella ? copies of gene X
extract
DNA
Example – Looking for Gene X
![Page 8: Introduction to Southern Hybridization Michael Melzer Plant & Environmental Protection Sciences University of Hawaii at Manoa.](https://reader035.fdocuments.us/reader035/viewer/2022062312/551b56db550346d41a8b62e5/html5/thumbnails/8.jpg)
Step 1. Restriction Enzyme Digestion
EcoR I EcoR I EcoR I EcoR I
![Page 9: Introduction to Southern Hybridization Michael Melzer Plant & Environmental Protection Sciences University of Hawaii at Manoa.](https://reader035.fdocuments.us/reader035/viewer/2022062312/551b56db550346d41a8b62e5/html5/thumbnails/9.jpg)
Step 1. Restriction Enzyme Digestion
![Page 10: Introduction to Southern Hybridization Michael Melzer Plant & Environmental Protection Sciences University of Hawaii at Manoa.](https://reader035.fdocuments.us/reader035/viewer/2022062312/551b56db550346d41a8b62e5/html5/thumbnails/10.jpg)
Step 2. Gel Electrophoresis
_ +
![Page 11: Introduction to Southern Hybridization Michael Melzer Plant & Environmental Protection Sciences University of Hawaii at Manoa.](https://reader035.fdocuments.us/reader035/viewer/2022062312/551b56db550346d41a8b62e5/html5/thumbnails/11.jpg)
Step 2. Gel Electrophoresis
_ +
![Page 12: Introduction to Southern Hybridization Michael Melzer Plant & Environmental Protection Sciences University of Hawaii at Manoa.](https://reader035.fdocuments.us/reader035/viewer/2022062312/551b56db550346d41a8b62e5/html5/thumbnails/12.jpg)
Step 2. Gel Electrophoresis
![Page 13: Introduction to Southern Hybridization Michael Melzer Plant & Environmental Protection Sciences University of Hawaii at Manoa.](https://reader035.fdocuments.us/reader035/viewer/2022062312/551b56db550346d41a8b62e5/html5/thumbnails/13.jpg)
Goals of Southern Hybridization
Immobilize DNA onto a permanent substrate
• ‘Membrane’– paper-like matrix– nylon or nitrocellulose– usually has a slight positive charge
![Page 14: Introduction to Southern Hybridization Michael Melzer Plant & Environmental Protection Sciences University of Hawaii at Manoa.](https://reader035.fdocuments.us/reader035/viewer/2022062312/551b56db550346d41a8b62e5/html5/thumbnails/14.jpg)
T G A A TC
A C AT T G
Step 3. DNA Denaturation
• Eliminate hydrogen bonds with sodium hydroxide (NaOH)
![Page 15: Introduction to Southern Hybridization Michael Melzer Plant & Environmental Protection Sciences University of Hawaii at Manoa.](https://reader035.fdocuments.us/reader035/viewer/2022062312/551b56db550346d41a8b62e5/html5/thumbnails/15.jpg)
Step 4. Transfer DNA to Membrane
• Two methods for transferring DNA to a membrane– capillary– electrophoretic
![Page 16: Introduction to Southern Hybridization Michael Melzer Plant & Environmental Protection Sciences University of Hawaii at Manoa.](https://reader035.fdocuments.us/reader035/viewer/2022062312/551b56db550346d41a8b62e5/html5/thumbnails/16.jpg)
Step 4. Transfer DNA to Membrane
![Page 17: Introduction to Southern Hybridization Michael Melzer Plant & Environmental Protection Sciences University of Hawaii at Manoa.](https://reader035.fdocuments.us/reader035/viewer/2022062312/551b56db550346d41a8b62e5/html5/thumbnails/17.jpg)
Goals of Southern Hybridization
• Immobilize DNA onto a permanent substrate
• Identify DNA sequence (gene) of interest
![Page 18: Introduction to Southern Hybridization Michael Melzer Plant & Environmental Protection Sciences University of Hawaii at Manoa.](https://reader035.fdocuments.us/reader035/viewer/2022062312/551b56db550346d41a8b62e5/html5/thumbnails/18.jpg)
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
![Page 19: Introduction to Southern Hybridization Michael Melzer Plant & Environmental Protection Sciences University of Hawaii at Manoa.](https://reader035.fdocuments.us/reader035/viewer/2022062312/551b56db550346d41a8b62e5/html5/thumbnails/19.jpg)
Step 5. Making a Probe
Arabidopsis thaliana 2 copies of gene X
![Page 20: Introduction to Southern Hybridization Michael Melzer Plant & Environmental Protection Sciences University of Hawaii at Manoa.](https://reader035.fdocuments.us/reader035/viewer/2022062312/551b56db550346d41a8b62e5/html5/thumbnails/20.jpg)
Step 5. Making a Probe
Gene Xfrom Arabidopsis
Partial or full-lengthprobes by PCR
![Page 21: Introduction to Southern Hybridization Michael Melzer Plant & Environmental Protection Sciences University of Hawaii at Manoa.](https://reader035.fdocuments.us/reader035/viewer/2022062312/551b56db550346d41a8b62e5/html5/thumbnails/21.jpg)
Step 5. Making a Probe
Gene Xfrom Arabidopsis
Partial probes by random-priming
![Page 22: Introduction to Southern Hybridization Michael Melzer Plant & Environmental Protection Sciences University of Hawaii at Manoa.](https://reader035.fdocuments.us/reader035/viewer/2022062312/551b56db550346d41a8b62e5/html5/thumbnails/22.jpg)
Step 5. Making a Probe
Denature template with heat
![Page 23: Introduction to Southern Hybridization Michael Melzer Plant & Environmental Protection Sciences University of Hawaii at Manoa.](https://reader035.fdocuments.us/reader035/viewer/2022062312/551b56db550346d41a8b62e5/html5/thumbnails/23.jpg)
Step 5. Making a Probe
Add random primers
![Page 24: Introduction to Southern Hybridization Michael Melzer Plant & Environmental Protection Sciences University of Hawaii at Manoa.](https://reader035.fdocuments.us/reader035/viewer/2022062312/551b56db550346d41a8b62e5/html5/thumbnails/24.jpg)
Step 5. Making a Probe
Extend random primers with polymerase
![Page 25: Introduction to Southern Hybridization Michael Melzer Plant & Environmental Protection Sciences University of Hawaii at Manoa.](https://reader035.fdocuments.us/reader035/viewer/2022062312/551b56db550346d41a8b62e5/html5/thumbnails/25.jpg)
Step 5. Making a Probe
A probe complementary to the sequence (Gene X) of interest!
![Page 26: Introduction to Southern Hybridization Michael Melzer Plant & Environmental Protection Sciences University of Hawaii at Manoa.](https://reader035.fdocuments.us/reader035/viewer/2022062312/551b56db550346d41a8b62e5/html5/thumbnails/26.jpg)
Step 5. Making a Probe
• How do we detect the probe?– Radioactivity (32P)
![Page 27: Introduction to Southern Hybridization Michael Melzer Plant & Environmental Protection Sciences University of Hawaii at Manoa.](https://reader035.fdocuments.us/reader035/viewer/2022062312/551b56db550346d41a8b62e5/html5/thumbnails/27.jpg)
Step 5. Making a Probe
• How do we detect the probe?– Digoxigenin (DIG)
U
![Page 28: Introduction to Southern Hybridization Michael Melzer Plant & Environmental Protection Sciences University of Hawaii at Manoa.](https://reader035.fdocuments.us/reader035/viewer/2022062312/551b56db550346d41a8b62e5/html5/thumbnails/28.jpg)
Step 4. Transfer DNA to Membrane
![Page 29: Introduction to Southern Hybridization Michael Melzer Plant & Environmental Protection Sciences University of Hawaii at Manoa.](https://reader035.fdocuments.us/reader035/viewer/2022062312/551b56db550346d41a8b62e5/html5/thumbnails/29.jpg)
Step 6. Pre-hybridization
Prehybridization bufferscontain ‘blocking reagents’that occupy available binding sites on the membrane
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Step 7. Hybridization
![Page 31: Introduction to Southern Hybridization Michael Melzer Plant & Environmental Protection Sciences University of Hawaii at Manoa.](https://reader035.fdocuments.us/reader035/viewer/2022062312/551b56db550346d41a8b62e5/html5/thumbnails/31.jpg)
Step 7. Hybridization
![Page 32: Introduction to Southern Hybridization Michael Melzer Plant & Environmental Protection Sciences University of Hawaii at Manoa.](https://reader035.fdocuments.us/reader035/viewer/2022062312/551b56db550346d41a8b62e5/html5/thumbnails/32.jpg)
Step 8. Washes
![Page 33: Introduction to Southern Hybridization Michael Melzer Plant & Environmental Protection Sciences University of Hawaii at Manoa.](https://reader035.fdocuments.us/reader035/viewer/2022062312/551b56db550346d41a8b62e5/html5/thumbnails/33.jpg)
Step 9. Anti-DIG
![Page 34: Introduction to Southern Hybridization Michael Melzer Plant & Environmental Protection Sciences University of Hawaii at Manoa.](https://reader035.fdocuments.us/reader035/viewer/2022062312/551b56db550346d41a8b62e5/html5/thumbnails/34.jpg)
Step 9. Anti-DIG
![Page 35: Introduction to Southern Hybridization Michael Melzer Plant & Environmental Protection Sciences University of Hawaii at Manoa.](https://reader035.fdocuments.us/reader035/viewer/2022062312/551b56db550346d41a8b62e5/html5/thumbnails/35.jpg)
Step 10. Washes
![Page 36: Introduction to Southern Hybridization Michael Melzer Plant & Environmental Protection Sciences University of Hawaii at Manoa.](https://reader035.fdocuments.us/reader035/viewer/2022062312/551b56db550346d41a8b62e5/html5/thumbnails/36.jpg)
Step 11. CSPD
![Page 37: Introduction to Southern Hybridization Michael Melzer Plant & Environmental Protection Sciences University of Hawaii at Manoa.](https://reader035.fdocuments.us/reader035/viewer/2022062312/551b56db550346d41a8b62e5/html5/thumbnails/37.jpg)
Step 12. Detection
• DIG-labeled probes emitting minute amounts of light (chemiluminescence)
• 32P-labeled probes emitting ß-particles
![Page 38: Introduction to Southern Hybridization Michael Melzer Plant & Environmental Protection Sciences University of Hawaii at Manoa.](https://reader035.fdocuments.us/reader035/viewer/2022062312/551b56db550346d41a8b62e5/html5/thumbnails/38.jpg)
Step 12. Detection
• DIG-labeled probes emitting minute amounts of light (chemiluminescence)
• 32P-labeled probes emitting ß-particles
• Autoradiography film can detect this radiation
![Page 39: Introduction to Southern Hybridization Michael Melzer Plant & Environmental Protection Sciences University of Hawaii at Manoa.](https://reader035.fdocuments.us/reader035/viewer/2022062312/551b56db550346d41a8b62e5/html5/thumbnails/39.jpg)
![Page 40: Introduction to Southern Hybridization Michael Melzer Plant & Environmental Protection Sciences University of Hawaii at Manoa.](https://reader035.fdocuments.us/reader035/viewer/2022062312/551b56db550346d41a8b62e5/html5/thumbnails/40.jpg)
Conclusion
• How many copies of ‘Gene X’ does Capsella rubella possess?
Capsella rubella
3
![Page 41: Introduction to Southern Hybridization Michael Melzer Plant & Environmental Protection Sciences University of Hawaii at Manoa.](https://reader035.fdocuments.us/reader035/viewer/2022062312/551b56db550346d41a8b62e5/html5/thumbnails/41.jpg)
Other Applications
• DNA fingerprinting– RFLP of VNTRs
• Dot or slot blot
• Colony or plaque lifts
• Microarray analysis
![Page 42: Introduction to Southern Hybridization Michael Melzer Plant & Environmental Protection Sciences University of Hawaii at Manoa.](https://reader035.fdocuments.us/reader035/viewer/2022062312/551b56db550346d41a8b62e5/html5/thumbnails/42.jpg)
Other Applications
• DNA fingerprinting– RFLP of VNTRs
• Dot or slot blot
• Colony or plaque lifts
• Microarray analysis
![Page 43: Introduction to Southern Hybridization Michael Melzer Plant & Environmental Protection Sciences University of Hawaii at Manoa.](https://reader035.fdocuments.us/reader035/viewer/2022062312/551b56db550346d41a8b62e5/html5/thumbnails/43.jpg)
Other Applications
• DNA fingerprinting– RFLP of VNTRs
• Dot or slot blot
• Colony or plaque lifts
• Microarray analysis
![Page 44: Introduction to Southern Hybridization Michael Melzer Plant & Environmental Protection Sciences University of Hawaii at Manoa.](https://reader035.fdocuments.us/reader035/viewer/2022062312/551b56db550346d41a8b62e5/html5/thumbnails/44.jpg)
Other Applications
• DNA fingerprinting– RFLP of VNTRs
• Dot or slot blot
• Colony or plaque lifts
• Gene Expression
![Page 45: Introduction to Southern Hybridization Michael Melzer Plant & Environmental Protection Sciences University of Hawaii at Manoa.](https://reader035.fdocuments.us/reader035/viewer/2022062312/551b56db550346d41a8b62e5/html5/thumbnails/45.jpg)
Other Applications
• Microarray technology