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:

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

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Step 2. Gel Electrophoresis

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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

(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 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. CSPDDisodium 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?

Capsella rubella

3