5. Goals of Southern Hybridization
• Immobilize DNA onto a permanent
substrate
• Identify DNA sequence (gene) of interest
6. Southern Blot
Restriction Digest of DNA
Electrophoresis
Denaturation/Depurination
Blotting Step (overnight)
Detection of DNA (next lab)
7. Capsella rubella
The key to this method is
hybridization.
Hybridization-process of
forming a double-
stranded DNA molecule
between a single-stranded
DNA probe and a single-
stranded target patient
DNA.
8. The technique was
developed by E.M.
Southern in 1975.
The Southern blot is used
to detect the presence of
a particular piece of DNA
in a sample.
The DNA detected can be
a single gene, or it can be
part of a larger piece of DNA
such as a viral genome
9. Steps for hybridization
1. The mixture of molecules
is separated.
2. The molecules are
immobilized on a matrix.
3. The probe is added to
the matrix to bind to the
molecules.
4. Any unbound probes are
then removed.
5. The place where the
probe is connected
corresponds to the location
of the immobilized target
molecule.
10. Blotting
Blotting
Blotting
Transfer the DNA from the
gel to a solid support.
The blot is usually done on a
sheet of nitrocellulose paper
or nylon.
DNA is then neutralized with NaCl to
prevent re-hybridization before adding the
probe.
Transferred by either electrophoresis or
capillary blotting.
15. Goals of Southern Hybridization
Immobilize DNA onto a permanent substrate
• ‘Membrane’
– paper-like matrix
– nylon or nitrocellulose
– usually has a slight positive charge
16. T G A A T
C
A C A
T T G
Step 3. DNA Denaturation
• Eliminate hydrogen bonds with sodium
hydroxide (NaOH)
17. Step 4. Transfer DNA to Membrane
• Two methods for transferring DNA to a
membrane
– capillary
– electrophoretic
19. Goals of Southern Hybridization
• Immobilize DNA onto a permanent
substrate
• Identify DNA sequence (gene) of interest
20. 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
21. Step 5. Making a Probe
Arabidopsis thaliana 2 copies of gene X
22. Step 5. Making a Probe
Gene X
from Arabidopsis
Partial or full-length
probes by PCR
23. Step 5. Making a Probe
Gene X
from Arabidopsis
Partial probes by
random-priming
