Southern blotting technique

8/30/2018 By Hirut G. 1
Southern Blotting technique
By:
Hirut G/Michael(DVM,Lecturer)
April, 2018
Bishoftu, Ethiopia

Outlines of this presentation
Blotting
Types of blotting
Southern blotting
Principle
Apparatus
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Steps involved in Southern blotting
A schematic view of Southern blotting
Application
Advantages and Disadvantages of Southern
blotting
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What is blotting?
• Blots are techniques for transferring DNA,
RNA and proteins onto a solid support (carrier)
generally nylon or nitrocellulose membranes.
so they can be separated, and often follows the
use of a gel electrophoresis.
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• Blotting of nucleic acid is the central technique
for hybridization studies. Nucleic acid labeling
and hybridization on membranes have formed
the basis for a range of experimental
techniques involving understanding of gene
expression, organization, etc.
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• Identifying and measuring specific proteins in
complex biological mixtures, such as blood,
have long been important goals in scientific
and diagnostic practice.
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• More recently the identification of abnormal
genes in genomic DNA has become
increasingly important in clinical research and
genetic counseling. Blotting techniques are
used to identify unique proteins and nucleic
acid sequences.
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• They have been developed to be highly
specific and sensitive and have become
important tools in both molecular biology and
clinical research.
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• A Southern blot is a method used in molecular
biology for detection of a specific DNA
sequence in DNA samples. Southern blotting
combines transfer of electrophoresis -separated
DNA fragments to a filter membrane and
subsequent fragment detection by probe
hybridization.
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The method is named after its inventor, the British
biologist Edwin Mellor Southern.
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• The key to this method is hybridization.
• Hybridization is a process of forming a double-
stranded DNA molecule between a single-
stranded DNA probe and a single-stranded
target patient DNA.
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There are 2 important features of hybridization:
The reactions are specific-the probes will only
bind to targets with a complementary
sequence.
The probe can find one molecule of target in a
mixture of millions of related but non-
complementary molecules.
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1. Extract and purify DNA from cells;
2. DNA is restricted with enzymes;;
3. Denature DNA;
4Separated by electrophoresis;
5. Transfer to nitrocellulose paper;
6. Add labeled probe for hybridization to take place;
7. Wash off unbound probe;
8. Autoradiograph.
Steps for hybridization
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3.The restriction fragments present in the gel are
denatured with alkali and transferred onto
4. 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.
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5. The filter is incubated under hybridization
conditions with a specific radiolabeled DNA
probe.
The probe hybridizes to the complementary
DNA restriction fragment.
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 This procedure preserves the distribution of
the fragments in the gel, creating a replica of
the gel on the filter.
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• 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.
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Southern blots are used in gene discovery , mapping,
To identify specific DNA in a DNA sample.
To Isolate desired DNA for construction of rDNA.
Identify mutations, deletions, and gene
rearrangements.
 Used in prognosis of cancer and in prenatal
diagnosis of genetic diseases.8/30/2018 By Hirut G. 26

 In RFLP (in molecular biology; Southern blot
analysis is used for the detection of DNA or
gene sequence in circular or large DNA. ...
Where as RFLP (Restriction Fragment Length
Ploymorphism) is used for compare the DNA
fragments after reaction with restriction
enzymes.
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 Diagnosis of HIV-1 and infectious disease.
In DNA fingerprinting:
Paternity and Maternity Testing
Criminal Identification and Forensics
Personal Identification
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Effective way to detect a specific DNA
sequence in a large, complex sample of
DNA. Can be used to quantify the amount
of the present DNA.
Cheaper than DNA sequencing
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• Southern blots allow investigators to determine the
molecular weight of a restriction fragment and to
measure relative amounts in different samples.
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More expansive than most other tests.
Complex and labor-intensive.
Time consuming and cumbersome.
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Southern blotting technique

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Southern blotting technique