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).

SOUTHERN BLOT

PROTOCOL1. 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.

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.

FUNCTIONS

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).

INTERPRETATION OF RESULTS

 

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.

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.

APPLICATIONS

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.

ADVANTAGES

More expensive than most other tests Complex and labor-intensive Time consuming and cumbersome Requires a large amount of the targeted DNA.

DISADVANTAGES

By

Khaisa Jerry