Introduction to Southern Hybridization （英文PPT）.pptVIP

Introduction to Southern Hybridization Michael Melzer Plant Environmental Protection Sciences University 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: Goals of Southern Hybridization Immobilize DNA onto a permanent substrate Identify DNA sequence (gene) of interest Example – Looking for Gene X Example – Looking for Gene X Step 1. Restriction Enzyme Digestion Step 1. Restriction Enzyme Digestion Step 2. Gel Electrophoresis Step 2. Gel Electrophoresis 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 Step 4. Transfer DNA to Membrane 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 Step 5. Making a Probe Step 5. Making a Probe Step 5. Making a Probe Step 5. Making a Probe Step 5. Making a Probe Step 5. Making a Probe 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) Step 4. Transfer DNA to Membrane Step 6. Pre-hybridization 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 ?-particles Step 12. Detecti