Its technique is used for Isolation and Identification of DNA and translation process.

1. Blotting Techniques
Dr. Jadhav R.S.
Department of Microbiology
VNBN Mahvidyalaya, Shirala

2. Introduction
• Blotting- Transfer of electrophoretically
separated micro molecules from a gel onto a solid
support.
• Blot- A solid support containing
electrophoretically separated micro molecules.
• Three solids support used-
A) Nitrocellulose paper
B) DBM-Cellulose paper
C) Nylon membrane

3. Mellor Southern in 1975.
• The transfer of electrophoretically separated DNA
molecules from the gel onto the solid support is
called Southern blotting or southern hybridization.
Sr. No. Membrane Blotting
Technique
1 Nitro cellulose and Nylon Southern
2 DBM-Cellulose and Nylon Nothern
3 PVDF and Nylon Western

4. Southern Blotting
• The identification of a specific target DNA
segment on a solid support by using a
radiolabelled probe.
• It is used for the identification of the specific
target DNA segment in genomic DNA.
• The probe used in southern blotting are
radiolabelled ssRNA or ss oligonucleotide
complementary to the target DNA segment which
has to be identified.

5. Method of Southern blotting tech
• Isolation of total genomic DNA from given tissue
or organism.
• Fragmentation of the genomic DNA by using
partial digestion method with one or more
restriction enzymes.
• Electrophoretic separation of the DNA fragment
using Agarose gel electrophoresis (AGE).
• Transfer of the electrophoretically separated DNA
fragment from the Agarose gel onto the solid
support. This transfer can be carried out by either
capillary blotting or electro-blotting.

6. • Denaturation of the transferred DNA by using alkali
(0.5N or 1N NaOH)
• Fixation of the ssDNA fragments on the solid
support by vaccum baking at 80 degree Celsius or
by UV-crosslinking.
• Hybridization with a labelled ssRNA or oligo-
ribonucleotide probe.
• Autoradiography to identify the presence of the
target DNA sequence.

7. Application
• Used for qualitative detection and quantitative
estimation of target DNA in the genomic DNA.
• Used for screening the bacterial colonies for foreign
DNA insert.
• Used for identification of the integrated genome of a
viral pathogen and bacterial, protozoan pathogen.
• Used for DNA profiling or DNA finger printing to
identify the individuals identify through genomic
DNA analysis.
• Used for generation of restriction mapping and
RFLP analysis.

9. Northern Blotting
• The transfer and analysis of electrophoretically
separated m-RNA moleculer form the gel onto the
solid support is called the northern blotting.
• In northern blotting analysis ‘the transcriptome’ is
analyzed rather than the genome in case of Southern
blotting.
• Northern blotting involves the identification of a
specific target m-RNA where as the southern
blotting involves the identification of the target
DNA seq.

10. • A restriction digestion can not be carried out in
northern blotting beq. RNA cant be cleaved by
restriction enzymes.
• A denaturation step is not needed in the case of
northern blotting , m-RNA are single stranded.
• The radiolabelled probes in the case of the northern
blotting are single stranded DNA, ss C-DNA, ss-EST or
oligodeoxyribonucleotides that are complementary to
the target mRNA seq.
• The northern blot analysis involves the expression of a
genome in a given tissue or cell with respect to specific
set of condition where as in southern blot presence or
absence of a specific DNA seq. present in the genomic
DNA but does not give indication regarding expression.

11. • Northern blot analysis can vary from tissue to tissue
and from one developmental stage to another where
as the Southern blotting analysis of an individual
remains the same irrespective of the tissue or
developmental stage.

12. Thanking You