To know and understand the types of blotting techniques and their applications in various fields

2. HISTORY OF BLOTTING
• BLOTTING - The immobilization of Nucleic acids
and Proteins onto a solid support generally nylon
or nitrocellulose membranes.
• The blotting method was originally developed by
British Biologist Edwin Mellor Southern in 1975.
• He demonstrated that Restricted DNA fragments
had been electrophoretically fractioned on
agarose gels could be transferred to a solid
support and detected as discrete bands following
hybridization to a complementary DNA Probe.

3. TYPES OF BLOTTING
TECHNIQUES

4. Genomic DNA is digested with one or more
restriction enzymes.
The resulting fragments are separated according
to size by Agarose
Gel Electrophoresis.
Agarose gel is mounted on filter paper wick.
A piece of nylon membrane is sandwiched
between the gel and the
filter paper towels.
The electrophoresed DNA is exposed to a short
depurination treatment
followed by alkali treatment.
It shortens the DNA fragments at depurinated sites
and denatures the

5. DNA can be fixed to the membranes by the UV Cross linking
method between a small fraction of thymine residues in the DNA
and positively charged amino groups on the nylon membrane.
The membrane can then be used for the hybridization labeling
reaction.
Probes are oligodeoxynucleotides or an RNA that is a
complementary sequence to the blotted DNA.
Labeling of probe can be done by radioactive or nonradioactive
methods.
After hybridization, the membrane is washed to remove unbound
probe and regions of hybridization are detected as per the
protocol for a particular probe is used.
If a radioactive probe sequence has been employed, the regions
of hybridization are detected by the autoradiographic method.
The localization and recording of a radiolabel within a solid
specimen is known as autoradiography and involves the
production of an image in a photographic emulsion.

7. APPLICATIONS OF SOUTHERN BLOT
Detecting major genome rearrangements and deletions found in a variety of
human diseases.
To identify structurally related genes in the same species and homologous
genes in other species.
Zoo blots, reveal the degree of evolutionary conservation of a gene.
For eg. Identified genes in yeast related to the RAS oncogene in human
tumors, a remarkable example of evolutionary conservation.
In DNA fingerprinting for:
-Paternity & Maternity tests
-Forensics
-Personal/Bio identification
Phylogenetic analysis
Recently used in conjunction with electrophoretic separation of very large DNA
molecules to prepare restriction maps over distances of hundreds of Kilo
bases.
Important for understanding of a variety of biological processes such as RNA
Splicing and Genomic rearrangements to form antibodies and T Cell receptors
and in the detection of rearranged oncogenes.

9. Northern blotting is a technique for detection of specific RNA
sequences.
Developed by James Alwine & George stark at Stanford University in
1979.
RNA molecules have defined length and much more shorter than
genomic DNA.
It is not necessary to cleave RNA before electrophoresis.
RNA is more susceptible to degradation than DNA.
RNA sample is separated based on size by gel electrophoresis.
The procedure of northern blot is similar to southern blot .
Initially northern blotting was carried out on chemically reactive
paper, because this was not able to bind on nitrocellulose.
The RNA was blotted or immobilized on diazotized cellulose paper
(diazobenzyloxymethylcellulose - DBM).
Thus, presently nitrocellulose membranes are used under appropriate
conditions because of the convenience of using these membranes.

11. APPLICATIONS OF NORTHERN BLOT
Useful as an adjunct to cDNA cloning because the size of cloned cDNA s,
revealing whether the cloned cDNA is in full length.
A Standard for direct study of the gene expression at the level of mRNA.
Study of RNA Splicing can detect alternatively spliced transcripts.
Detection of mRNA transcript size.
Study RNA Half life.
Indicate which tissues or cell types express a particular gene or the
factors that regulate its expression.
To study gene expression by detecting RNA in a sample during
differentiation, morphogenesis as well as abnormal or diseased condition.
Can determine whether the gene is transcribed or not.

12. In 1979, Towbin et Al., developed the western
blotting technique to find out the newly encoded
protein by a transformed cell.
The extracted proteins are subjected to
polyacrylamide gel electrophoresis and are then
transferred onto nitrocellulose to which they bind.
Nitrocellulose membrane is then used for probing
with a specific labeled antibody
The antibody may labeled with 125 I and the signal
is detected again with autoradiography.
WESTERN BLOT ANALYSIS

15. APPLICATIONS OF WESTERN BLOT
Diagnosis of HIV by ELISA involves the western blotting technique.
Used to detect some form of lyme diseases.
Confirmatory test for Hepatitis – B
Analysis of Biomarkers such as hormones, growth factors and cytokines.
Employed in gene expression studies.
Definitive test for Bovine Spongiform Encephalopathy..
Detect auto - antibodies that causes auto immune diseases.
Double confirm the presence of abnormal cellular proteins.
Western blot analysis can analyze any protein sample whether from cells or
tissues, but also can analyze recombinant proteins synthesized in vitro.
Western blot is dependent on the quality of antibody you use to probe for your
protein of interest, and how specific it is for this protein.

16. COMPARISON OF SOUTHERN, NORTHERN AND
WESTERN BLOTS