PRINCIPLE OF SOUTHERN,NOTHERN,WESTERN BLOTTING AND IMPORTANCE OF DOT BLOT HYBRIDIZATION IN DETECTION OF PLANT PATHOGENS

BLOTTING
Immobilization of nucleic acids, proteins on to a solid support Nylon or nitro-cellulose membrane are generally used as solid support Nucleic acids fixed on the membrane are then used as targets for hybridization with labeled probes

Northern blot(RNA)

Southern Blot (DNA)

Western Blot (Proteins)

SOUTHERN BLOTTING
is a method used to transfer DNA from an agarose gel to a membrane, where the DNA can be subsequently probed for a specific sequence DNA detected can be a single gene, or a larger piece of DNA For the detection of specific DNA sequences, a hybridization probe is used

GOALS OF SOUTHERN HYBRIDIZATION

Immobilize DNA onto a permanent substrate
Membrane paper-like matrix nylon or nitrocellulose usually has a slight positive charge

Identify DNA sequence (gene) of interest

STEPS
1. DNA extraction

Step2:DNA digestion with restriction enzymes

3. Separation by electro- phoresis on an agarose gel

5.DNA transferred to a nylon or nitro cellulose membrane

6.Membrane with DNA fragments is labeled with a probe

PROBE
A probe is a small length of DNA or RNA Complementary to the sequence (gene) of interest Hybridize wit the specific sequence of the sample

Detection of Probes
Radio active labelling with 32P Non radio active labelling Biotin Digoxigenin Horse radish peroxidase system

6. Hybridization of membrane with a labelled probe

7.Detection of DNA fragments on the membrane by auto-radiography

NORTHERN BLOTTING
Similar to Southern Blotting RNA rather than DNA is blotted on the membrane No denaturation process is required

Northern blots are not used very often for diagnostic purposes

WESTERN BLOTTING Also referred to as ImmunoBlotting Identifies proteins specific to antibodies

WESTERN BLOTTING

Proteins transfer to a membrane

Blot for the protein of interest with specific antibodies(Primary antibody)

Probe with an antibody specific to the primary antibody(secondary antibody)

Cont...
Secondary body conjugated with an enzyme
Add enzymes substrate Enzyme substrate reaction produces changes in colour of the substrate

DOT BLOT
DNA or RNA is bound directly to a solid support filter No size separation

Useful when only detection and quantification is required

DOT BLOT:APPLICATION
Rapid detection of specific sequences

Useful to detect and quantify non culturable MLOs infecting plants

Determination of the relative amount of a given species or sequence of DNA or RNA in a complex sample