Biology Today — December 2017

Southern Blotting

Southern blotting is a technique that involves transfer of DNA fragments from an electrophoresis gel onto a

membrane support, in such a way that DNA banding pattern present in the gel is reproduced on the membrane.

During transfer, DNA becomes immobilised on the membrane and can be used as a substrate for hybridisation

analysis with labelled DNA or RNA probes that specifically target individual restriction fragments in blotted

DNA. This technique is named after its inventor Edwin Southern (1975). It involves the following steps:

BIO-GRAM

Power supply

Electrophoresis

tank

40-60 volts

Direction of

movement

Buffer solution

(conducts electric current)

Electrode

Electrode

DNA Sample

(Loaded into the

wells with the help

of micropipette.)

Larger molecules

Shorter

molecules

Separation of Fragments by Gel Electrophoresis

Fragmented DNA is typically electrophoresed on an agarose

gel to separate them on the basis of their molecular weights.

DNA size is determined by running the DNA ladders (or

molecular weight markers) in the lane adjacent to the sample.

By matching the distance covered by fragment and marker, the

molecular weight and size of DNA fragment can be estimated.

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Blotting

After electrophoresis, the DNA fragments in the gel are denatured for which it is

soaked in about 0.5 M NaOH which separates dsDNA into ssDNA and then transferred

onto positively charged nylon or nitrocellulose membrane. The traditional transfer of

DNA is done overnight using an upward capillary transfer of DNA from an agarose

gel onto a membrane and its subsequent immobilisation by UV irradiation (for

nylon) or baking (for nitrocellulose).

3

DNA Digestion

DNA containing gene

of interest is extracted

from cells with the

help of enzymes and

subjected to restriction

digestion so as to obtain

DNA fragments.

1

Detection

The labelled probe is detected

using the method required

for the type of label used. For

example, radiolabelled probes

may be detected using X-ray

film or a phosphorimaging

instrument.

6 Expose X-ray
Probe hybridised film to filter
to complementary
sequence

Gel

DNA transferred

to filter

Southern blot

tray lid

The buffer contains a strong

alkali that denatures DNA.

Denaturation facilitates the

transfer and prepares the DNA

for hybridisation by probe.

A minimum of 500 gm weight is placed

on top of the entire set up.

Southern blot tray

Sponge

Agarose gel

Filter paper

At least six dry sheets of

filter paper are placed on

top of the nylon membrane.

Filter paper bridge

Briefly soaked in buffer

and placed across the

support so that it hangs

down into tray.

Nylon

membrane

Absorbent paper-Stack of

paper towels are placed

on top of filter paper.

Buffer

Obtained from gel

electrophoresis after

separation of fragments

and is carefully placed

onto filter paper bridge.

Restriction

enzyme cuts

DNA into

fragments

Gene of interest

Hybridisation

Hybridisation is promoted when the labelled probe

is incubated with the DNA fragments immobilised

on the blot in a tube that is constantly rotated or in a

sealed plastic bag that is placed on a shaker. Random

collisions bring small regions of complementary

sequences together to start the renaturation. Base

pairing proceeds in a zipper-like fashion as flanking

sequences are complementary; probe sticks even

after washing. The unhybridised probe is removed

by washing several times in buffer. Only the fully

hybridised labelled probe molecules with sequences

complementary to gene of interest remain bound.

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C C

CCC C GCC

T

GAG GATTTATTAAA TGGTTGG

Base pairing with

incorrect fragment

CCC C CC C

AT A AAACA

GCATTAATAT TACGGGCTCGGGT

Base pairing with

correct fragment

The nylon membrane is carefully removed from the setup

and is incubated in a buffer for 15 minutes containing

radioactively labelled probe. Labelled probe is a nucleic

acid probe with sequence homologous to the target

sequence under study, labelled with radioactivity,

fluorescent dye or an enzyme that can generate a

chemiluminescent signal when incubated with the

appropriate substrate.

4 Prehybridisation of Southern blot

Probe falls away

1000 bp900 bp

800 bp700 bp

600 bp500 bp

400 bp

300 bp

200 bp

100 bp

1,200 bp