Hybridization Techniques

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

Spawned naming of related techniques: Northern blot (RNA) Western blot (Protein) Eastern blot (???) Southern blot (DNA)

Goals of Southern Hybridization Immobilize DNA onto a permanent substrate Identify DNA sequence (gene) of interest

Example – Looking for Gene X 2 copies of gene X Arabidopsis thaliana

Example – Looking for Gene X ? copies of gene X extract DNA Capsella rubella

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 _ +

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 Eliminate hydrogen bonds with sodium hydroxide (NaOH) A C T G T G A C

Step 4. Transfer DNA to Membrane Two methods for transferring DNA to a membrane capillary Electrophoretic (www.pnas.org/content/76/9/4350.full.pdf)

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

Step 5. Making a Probe How do we detect the probe? Digoxigenin (DIG) U

Step 6. Pre-hybridization Prehybridization buffers contain ‘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 Disodium 3-(4-methoxyspiro {1,2-dioxetane-3,2´-(5'-chloro)tricyclo [3.3.1.13,7]decan}-4-yl)phenyl phosphate

Step 12. Detection DIG-labeled probes emitting minute amounts of light (chemiluminescence) 32P-labeled probes emitting β-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? 3 Capsella rubella