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INTRODUCTION

Southern blotting is the first blotting technique developed


which made analysis and recording of DNA easy.
Identification of sequences, correlating of DNA restriction
fragments to RNA and protein, mapping were facilitated
by this technique has become most popular component of
many molecular biology studies.

The name of this technique is derived from the following :

1. The name of its inventor, E.M. southern, and

2. The DNA-DNA hybridization that forms its basis. It is


also called Southern Blotting since the procedure for
transfer of DNA from the gel to the nitrocellulose filter
resembles blotting. This technique has since been
extended to the analysis of RNA (northern blotting) and
protein (western blotting).
PROCEDURE

1) DNA sample is digested with restriction enzymes.

2) Digested sample is electrophoresed on agarose gel.

3) DNA bands in gel are denatured by alkali to get single


stranded DNA.

4) The gel is then neutralized by soaking in Tris buffer.

5) The gel is positioned on top of buffer-soaked filters


and a piece of transfer medium (nitrocellulose or nylon
membrane filter) is placed on top of the gel completing
the gel sandwich.

6) Dry filter papers are then piled on top of the filter and a
weight is put on it.

7) Capillary action pulls the buffer (salt solution)from the


bottom filter through the gel, to the transfer medium and
up through the paper towel stake. A rapid transfer by
applying vacuum has also been developed.
8) The arrangement made for blotting is left undisturbed for
few hours or overnight and then top filters are weight is
removed.

9) SS DNA fragment are trapped on the transfer medium


(nitrocellulose or nylon membrane). Here they are
immobilized in form of bands which is like replica of
original pattern on agarose gel (electrophoresed).

10) DNA fragment can be fixed permanently to


nitrocellulose membrane by baking at 80⁰ C and to nylon
membranes by baking or cross linking the strands with
UV light.

11) DNA fragment on membrane can then be probed for


sequence of interest by hybridization between them.

12) Membrane is washed to remove unbound DNA.

13) X-ray film is then exposed to the (probed DNA)


membrane to get auto radiographs.

DAIGRAM
Fig.- Schematic Diagram of Southern blotting hybridization

DISCUSSION

More recently a versatile nylon membrane material has a


preferred medium for many applications. Nylon is more
amenable to strip washing, in which blot is cleared of the
original probe by exposure to low salt-high temperature
condition for probing. Nylon also does not require baking to
fix DNA on to membrane as does the nitrocellulose. This cuts
2 hours off of the procedure time and avoids the problem of
brittleness associated with baking.

Fig. Schematic of Southern blot hybridization


APPLICATION

1) Southern blotting is extremely sensitive and can be


applied to mapping restriction sites around single-copy
gene sequence in a complex genome such as that of man.

2) When ‘minisatellite’ purpose is used the technique can


be also used for forensic purpose for identification of
minute amounts of DNA.

3) In bacteria, genes consist of contiguous stretch of DNA


and the liner sequence of nucleotides corresponds
directly to a liner sequence of amino acids in the protein.
The discovery that this principal of colinearity did not
exist in the genomes of higher eukaryotes was very
significant finding in molecular biology, and was made
by southern blot analysis of genomic DNA.

4) The use of southern blot technique has also been done for
analyzing the role of DNA methylation in gene
expression.
5) Southern blot analysis is useful in detection of mutated
genes in genetic disorders. Restrictions analysis of genes
studied with southern blot helps in this respect.
REFRENCES

GENE BIO-TECHNOLOGY-
S.N Jogdand

Bio-Technology-
B.D. Singh

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