1. SOUTHERN BLOT
Developed by Edward M. Southern in 1975.
Routinely used in molecular biology for
detection of a specific DNA.
Also used for ‘detection of a specific
restriction fragment against a background of
many other restriction fragments’
(Brown, 1999).
2. PROTOCOL
1. Digest the DNA with an appropriate restriction enzyme.
2. The complex mixture of fragments is subjected to gel
electrophoresis to separate the fragments according to size.
3. The restriction fragments present in the gel are denatured with
alkaline and transferred onto,
4. A nitrocellulose filter or nylon membrane by blotting.
5. The filter is incubated under hybridization conditions with a
specific radiolabeled DNA probe.
6. Excess probe is washed away and the probe bound to the filter is
detected by autoradiography, which reveals the DNA fragment to
which the probe hybridized.
3.
4. FUNCTIONS
To determine the molecular weight of a restriction
fragment.
To measure relative amounts in different samples and
to locate a particular sequence of DNA within a
complex mixture.
To confirm the identity of a cloned fragment or to
identify an interesting sub fragment from within the
cloned DNA.
5. INTERPRETATION OF RESULTS
Single band:
A probe that hybridizes only to a single DNA segment.
Multiple bands:
Will likely be observed when the probe hybridizes to
several highly similar sequences (sequence duplication).
6. The presence of a band - a DNA sequence homologous to the probe DNA
is present in clones ## 3, 4, & 8, with the same size as the probe, and
absent in clones ##1, 2, 5, & 9.
The probe sticks to itself in #10, as expected.
In lane #6, a homologous sequence is present, but with an additional
restriction site that cuts the fragment into two smaller fragments of 300bp
and 100bp. This analysis indicates that the gene of interest has been
successfully cloned in a particular set of plasmids, and that there is genetic
variation in clone #6.
7. APPLICATIONS
Gene discovery , mapping, evolution and development studies,
diagnostics and forensics. (DNA fingerprinting)
Identification of the transferred genes in transgenic individuals, etc.
Allow investigators to determine the molecular weight of a restriction
fragment and to measure relative amounts in different samples.
To detect the presence of a particular bit of DNA in a sample.
Analyze the genetic patterns which appear in a person's DNA.
8. ADVANTAGES
Detecting multiple homologous genes in a genome.
Detecting orthologous or paralogous genes in similar
or distant species where you might not know anything
about the sequence divergence and hence primer sites
(you can increase or decrease specificity depending on
hybridization conditions).
Easier to multiplex / detect multiple products.
Easier and time efficient.
9. DISADVANTAGES
More expensive than most other tests
Complex and labor-intensive
Time consuming and cumbersome
Requires a large amount of the targeted DNA.
