2. INTRODUCTION
Gel electrophoresis techniques enables us to separate
fragments of DNA in a mixture and by staining we can
visualise and detect them.
Use of specific labelled probes enables in detection and
identifications of fragments of DNA by hybridization
procedure.
To facilitate this hybridization , the bands are often
transferred to a nitrocellulose membrane. This technique
resembles blotting.
For such blotting, DNA has to be single stranded form. This
can be achieved by denaturation of DNA.
4. SOUTHERN BLOTTING
Southern blotting is the first blotting technique which made analysis
and recording easy.
In Southern blotting ,a DNA fragment containing a specific sequence
can be identified by electrophoresis , transferring them into
nitrocellulose & hybridizing with a 32p labelled single stranded DNA
probe complementary to the sequence.
The fragment containing the sequence then visualized by
audioradiography
It was first developed by E.M.Southern in 1975
5. PROCEDURE
For Southern blotting,DNA sample is first digested with a
restriction enzyme and digested sample is electrophoresed.
The DNA bands in the gel are denatured into single strands
with the help of an alkali solution.
Subsequently the gel is laid on top of a buffer saturated filter
paper placed on a solid support(eg.glass plate),with its two
edges immersed in the buffer.
A sheet of nitrocellulose filter membrane is placed on top of the
gel and a stack of many papers on top of this membrane.
6. Capillary action pulls the buffer from the bottom filter through
the gel, to the transfer medium and up through the paper towel
stack.
While passing through the gel, the buffer carries with its single
stranded DNA, which binds on to the nitrocellulose membrane,
when the buffer passes through it to the paper towels.
The nitro cellulose membrane with single stranded DNA bands
blotted on to it, is baked at 80c for 2-3 hours to fix the DNA
permanently on the membrane.
DNA fragments on membrane can then be probed for sequence
of interest by hybridization between them.
Membrane is washed to remove the unbound DNA.
X-ray film is then exposed to the membrane to get
autoradiographs.
8. APPLICATIONS
Southern blotting is extremely sensitive and can be applied to
mapping restriction sites around a single copy gene sequence
in a complex genome such as that of man.
When minisatellite probe is used the technique can be also
used for forensic purpose for identification of minute amount of
DNA.
The use of southern blot technique has also been done for
analyzing the role of DNA methylation in gene expression.
9. Blotting analysis useful in detection of mutated genes in
genetic disorders.
Restriction analysis of genes studied with southern blot helps
in this respect.
RFLP mapping.
DNA finger printing.
10. NORTHERN BLOTTING
The Northern blot procedure essentially identical to that of
southern blotting except that here RNAs are separated by gel
electrophoresis.
Initially southern blotting could not be used directly to blot
transfer RNA from gel to nitrocellulose membrane because
RNA did not bind to cellulose nitrate.
Alwine,Kemp and Stark(1979)developed a procedure for blot
transfer of RNA.
He used a chemically reactive paper(diazotization of amino
benzyl oxymethyl paper which is prepared from Whatsman 540
papers).
11. PROCEDURE
RNA species are separated on the basis of size by
electrophoresis through agarose gel containing
formaldehyde or glyoxal and dimethyl sulfoxide.
The separated RNA bands are then blotted on
chemically reactive filter paper.
RNA species after blotting are hybridized to radiolabelled
DNA probe.
Auto radiography is carried onto locate RNA bands that
are complementary to the probe.
13. APPLICATIONS
Used for detection and quantitative
estimation of hybridized mRNA.
Study RNA degradation
Study RNA half life
Study RNA splicing
It is useful in the studies of gene expression.
14. WESTERN BLOTTING
This technique is used to detect the proteins of a
particular specificity.
When a transferred gene expresses in transformed
cells,
The translated product in the form of proteins can
be identified by this technique.
15. PROCEDURE
First we isolate the protein or extract the protein.
The extracted proteins are subjected to PAGE(Poly
Acrylamide Gel Electrophoresis) and are then
transferred onto nitro cellulose to which they bind.
Then radiolabelled specific antibody is added on such
membrane and it binds only to specific complementary
protein.
The antibody is labelled with 125 I and the signal is
detected again with autoradiography.
If radio active label is not used, bound antibody may be
detected by a second antibody tagged with an enzyme.
16.
17. APPLICATIONS
The confirmatory HIV test employs a western blot to detect anti-
HIV antibody in a human serum sample. Proteins from known
HIV-infected cells are separated and blotted on a membrane as
above. Then, the serum to be tested is applied in the primary
antibody incubation step; free antibody is washed away, and a
secondary anti-human antibody linked to an enzyme signal is
added. The stained bands then indicate the proteins to which
the patient's serum contains antibody.
A western blot is also used as the definitive test for Bovine
spongiform encephalopathy (BSE, commonly referred to as
'mad cow disease').
Western blot can also be used as a confirmatory test for
Hepatitis B infection.
18. DOT BLOT TECHNIQUE
This technique is used to detect the presence of a
given sequence of DNA/RNA in the non-
fractionated(not subjected to electrophoresis) DNA
sample.
DNA from many samples can be tested in a single
test.
A Dot blot (or Slot blot) is a technique used to detect
biomolecules.
It represents a simplification of the northern blot,
Southern blot, or western blot methods. In a dot blot
the biomolecules to be detected are not first separated
19. Instead, a mixture containing the molecule to be
detected is applied directly on a membrane as a
dot.
Then is spotted through circular templates
directly onto the membrane or paper substrate.
.
Then followed by detection by either nucleotide
probes (for a northern blot and Southern blot) or
antibodies (for a western blot).
20. PROCEDURE
Sample DNA/RNA from different individuals are transferred to a
nitrocellulose filter paper in the form of dots. Several samples
are blotted on to a single filter paper.
The DNA is first denatured and then the filter is baked at 80c to
fix DNA firmly on to the filter.
The filter is treated with the appropriate radio active single
stranded probe under conditions favouring hybridization.The
filter is washed to remove the unhybridized probes.
The dots with hybridized probes are detected by
autoradiography intensity of the dots corresponds fairly with the
extent to which DNA/RNA is represented in the sample.
21.
22. The dot blot test can be used to detect the
Chlamydia trachomatis infection and other
sexually transmitted diseases.
Dot blot is used to detect Antidiacyltrehalose
Antibodies in Tuberculosis patients and
typhoid Fever
