for MBBS

1. Dr. Vishnu Kumar
Professor, Department of
Biochemistry, SRMSIMS, Bareilly
vkawasthi@hotmail.com
madhwapur1976@gmail.com
Blotting Techniques

2. Learning Objectives
After completion of this lecture learner
should be able to define :
 Blotting Techniques
 Southern Blotting
 Northern Blotting
 Western Blotting

3. Blotting Techniques.
• These are standard techniques for the
identification of a specific DNA, an RNA
or a protein from a vast expanse of
others.
• The technique for specific DNA
identification is termed Southern blot,
whereas Northern blot is for RNA and
Western blot is or protein identification.

5. SOUTHERN BLOTTING
Professor Sir Edwin Southern, Professor of Biochemistry and
Fellow of Trinity, developed this method in 1975.
Southern won the Lasker Award for Clinical Medical Research
prize for the method of finding specific DNA sequences.
He developed this procedure at Edinburgh University more than 30
years ago. The technique is known as DNA transfer or
'Southern blotting‘.
Professor Sir Edwin Southern

6. Cont….
This method Involves Separation, Transfer and
Hybridization.
It is a method routinely used in molecular biology
for detection of a specific DNA sequence in DNA
samples.
The DNA detected can be a single gene, or it can
be part of a larger piece of DNA such as a viral
genome.

7. Cont….
Southern blotting combines Agarose Gel
Electrophoresis for size separation of DNA
with methods to transfer the size separated
DNA to a filter membrane for probe
hybridization.
The key to this method is Hybridization.
Hybridization - Process of forming a double-
stranded DNA molecule between a single-
stranded DNA probe and a single-stranded
target patient DNA.

8. PRINCIPL
E
1. The mixture of molecules is separated.
2. The molecules are immobilized on a matrix.
3. The probe [a hybridization probe is a fragment
of DNA or RNA of variable length (usually 100-
1000 bases long) which is radioactively
labeled. It can then be used in DNA or RNA
samples to detect the presence of nucleotide
sequences (the DNA target) that are
complementary to the sequence in the probe]
is added to the matrix to bind to the
molecules.

9. 4. Any unbound probes are then removed.
5. The place where the probe is
connected corresponds to the location
of the immobilized target molecule.

10. Southern Blot technique.
• DNA of interest is isolated from a cell
line or tissue. It is digested with one or
more restriction enzymes.
• The mixture is pipetted into a well in an
agarose or polyacrylamide gel and
exposed to a direct electric current.
DNA fragments migrate towards the
anode, the smaller fragments move the
most rapidly.

11. Southern Blot technique.
• After a suitable time , the DNA within
the gel is denatured by exposure to
mild alkali and transferred to
nitrocellulose or nylon paper resulting
in an exact replica of the pattern on the
gel, by blotting technique devised by
Southern.
• The DNA is fixed to the paper by
exposure to heat or UV.

12. Southern Blot technique.
• The paper is then exposed to labeled
cDNA probe, which hybridizes to
complimentary fragments of DNA on
the filter.
• After thorough washing, the paper is
exposed to x-ray films, which is
developed to reveal several specific
bands corresponding to the DNA
fragments of interest.

14. APPARATUS

15. Whatman 3MM paper nitrocellulosemembrane

16. Capillary plotting apparatus

17. Steps in southern blotting
1. Digest the DNA with an
appropriate restriction
enzyme.
2. The complex mixture of
fragments is subjected to gel
electrophoresis to separate
the fragments according to
size.

18. Cont….
3. The restriction fragments
present in the gel are
denatured with alkali.
4. It is then transferred onto a
nitrocellulose filter or nylon
membrane by blotting.
This procedure preserves the
distribution of the fragments in
the gel, creating a replica of the
gel on the filter.

19. Cont….
5.The filter is incubated under
hybridization conditions
with a specific radiolabeled
DNA probe.
The probe hybridizes to the
complementary DNA
restriction fragment.

20. Cont….
6.Excess probe is washed away and the
probe bound to the filter is detected by
autoradiography, which reveals the
DNA fragment to which the probe
hybridized.

23. APPLICATIONS
Southern blots are used in gene
discovery , mapping, evolution and
development studies, diagnostics and
forensics (It is used for DNA
fingerprinting, preparation of RFLP
[Restriction Fragment Length
Polymorphism] maps).
Identification of the transferred genes in
transgenic individuals, etc.

24. APPLICATIONS
It allow investigators to determine the
molecular weight of a restriction fragment
and to measure relative amounts in different
samples.
It is used to detect the presence of a
particular bit of DNA in a sample.
Used to analyze the genetic patterns which
appear in a person's DNA.

25. LONG ANSWER QUESTIONS
1. Describe the Southern Blotting technique
and its applications.

26. SHORT ANSWER QUESTIONS
1. Southern Blotting

27. MCQ Blotting Techniques
1. Southern Blotting analysis is used for all except
a. Detecting the presence of a mutant gene
b. Visualizing DNA profile
c. Studying a microarry
d. Detecting Restriction fragment length
polymorphism
Answer: c