SOUTHERN BLOTTING

• The technique was developed by E.M. Southern in 1975.
• It is used to find out specific DNA fragments in a DNA sample.

PRINCIPLE

• HYBRIDIZATION - process of forming a dsDNA molecule between a ssDNA probe and single stranded target DNA

PROCEDURE

• STEP 1 -DNA ISOLATION & PURIFICATION

Incubate the cell with detergent to promote cell lysis which will free cell protein and DNA.

Separate protein by proteinase enzyme

Precipitate DNA by alcohol pptn.

Visible DNAs are removed and bufferes.

• STEP 2 - RESTRICTION DIGESTION

Cut the DNA into several sized fragments using restriction endonuclease like Eco R1

• STEP 3 - GEL ELECTROPHORESIS

Fragments are separated by Gel electrophoresis.

Principle is based on size and charges.

Gels are solid agarose with pores and gel is soaked in buffer.

Ethidium Bromide Stain is used for visualisation.

• STEP 4 & 5 - DENATURATION AND BLOTTING

dsDNA is denatured with the help of alkaline solution like NaOH.

DNA is neutralized with NaCl to prevent re-hybridization before adding PROBE.

BLOTTING is the transfer of DNA from gel to nitrocellulose membrane.

The blot is made permanent by drying at 80 c or exposing to UV radiation.

• STEP 6 -  HYBRIDIZATION

The labelled probe is added to the membrane in buffer and incubated for hours to allow the probe to find their targets.

PROBE - small pieces of labelled DNA used to find complimentary DNA fragments

• STEP 7 & 8 - WASHING AND AUTORADIOGRAPHY

Unbound probes are washed out

For radioactive probe, X-Ray Film is placed over the membrane

After development ,there will be dark bands on film wherever the probe bound.

APPLICATION

• To identify specific DNA in a sample
• To isolate desired DNA from rDNA
• Cancer diagnosis and prenatal genetic defect detection
• Used in phylogenetic analysis
• Diagnosis of HIV-1 and other infectious disease
• Other applications of DNA fingerprinting