Blotting Techniques

BY
SHEELA
09HA013
It is a tool that is widely used for identification
of desired DNA or RNA fragments from
thousands of molecules.

Blotting refers to the process of sample nucleic
acids on solid support(nitrocellulose & nylon
membrane).
SOUTHERN BLOTTING (for DNA)

NORTHERN BLOTTING (for RNA)

WESTERN BLOTTING (PROTEINS)

DOT BLOTTING (DNA/RNA)
IMMOBILIZATION OF NUCLEIC ACID
SOUTHERN BLOT(DNA)
NORTHERN BLOT(RNA)
WESTERN BLOT(PROTEINS)
PREHYBRIDIZATION
HYBRIDIZATION
STRINGENCY WASHES
DETECTION
It was named after scientist Southern who
developed it in 1975.

Southern blotting is mainly meant for DNA.

Genomic DNA
DNA fragments
The genomic DNA is isolated from
cells/tissues

Digestion of the DNA using one or more
restriction enzymes.

The fragments are loaded into the well of
agarose or polyacrylamide gel it is subjected to
electrophoresis.

DNA being negatively charged it migrate
towards anode & we know that smaller
fragments move faster.
The separated DNA molecules are denatured
by exposure to a mild alkali , then it is
transferred to nitrocelluose or nylon
membrane which creates exact replica of DNA
fragments on the gel

The DNA is annealed to nitrocellulose by
exposing to heat of (80 C)

The nitrocellulose is exposed to labeled cDNA
probes.

These probes hybridize with complementary
DNA .
After thorough washing the paper is exposed
to X ray film to develop autoradiograph.

This shows the specific bands corresponding to
the DNA fragments recognized by cDNA
probe.
It is similar to southern blotting expect that
RNA is blotted through this method this was
named as jargons (since it was named after
scientist Southern).

Instead of nitrocellulose membrane chemically
reactive paper prepared by diazotization of
aminobenzyloxymethyl to create
diazobenzyloxymethyl (DBM) paper. The RNA
covalently bind to DBM paper.

But they modified nitrocellulose membrane, at
present it is been used for northern blotting.

The same procedure follows where the blot is
transferred RNA molecules hybridize with
DNA probe which can be detected.

Northern blotting is a technique for
determining the number of genes present in
given DNA, each DNA gives rise to two or
more RNA transcripts, but the drawback is the
presence of exons an introns.

Modification of southern and northern blotting
where the nucleic acids(DNA or RNA) are
directly spotted onto filters and not subjected
to electrophoresis the hybridization technique
is similar.

This technique is particularly useful in
obtaining quantitative data for the evaluation
of gene expression.

This involves identification of proteins, useful
to understand the nucleic acid functions. This is
also called as immunoblottings

This involves the transfer of electrophoresed
protein bands from polyacrylamide gel to
nylon or nitrocellulose membrane. These
proteins can be detected by specific protein
ligand interactions. Antibodies or lectins are
commonly used for this purpose.
Applications of Blotting
Techniques -

It is method of gene analysis.

Important for the confirmation of DNA cloning results

Forensically applied to detect minute quantities of DNA
(parenthood, thieves etc.)

DNA pieces from one species (eg; human) can be used to
detect DNA molecules from related species ( e.g:
chimpanze) this is referred to as Zoo- blotting.

Southern blotting technique is widely used to find
specific nucleic acid sequence present in different
animals including man. For example if we want to
know whether there is a gene like insulin in sea
anemone, then DNA of sea anemone is mobilized on
membrane and blotted by using insulin probes against
it.

Northern blotting technique is widely used to find
gene expression and regulation of specific genes. For
example if we find human insulin like gene in oyster,
then by isolating and immobilizing RNA and blotting
it with insulin probe we call tell whether the gene is
expressing or not.

By using blotting technique we can identify infectious
agents present in the sample.

We can identify inherited disease.

It can be applied to mapping restriction sites in
single copy gene.

Highly useful for the determination of
restriction fragment length polymorphism
(RFLP) associated with pathological conditions.

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