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Southern Blotting
Southern Blotting
Molecular Genetics
Semester: 8th evening
Subject: Adv. in microscopic
technology
Assignment topic: Southern
blotting
Submitted to: Mam Zilursh
Naeem
Submitted by: Group no. 06
Group members:
Hifza Sultan-5013
Sunaina Javed-5027
Aliya Rafique-5031
Rubab Ashraf-5005
Mariyam Abbas-5024
Sheeza Noor-5029
Submission date:17july,2023.
SOUTHERN BLOTTING
DEFINITION
Southern blotting is a molecular biology technique used for DNA detection,
characterization, and quantification.
An example of RFLP(restriction fragment length polymorphism), southern blotting can be
defined as an analytical technique for identifying specific sequences of DNA by
separating fragments on a gel and transferring them to a second medium (carrier
membrane) on which hybridization testing may be carried out.
PRINCIPLE
Southern blotting is a molecular biology technique used to detect specific DNA sequences in
a sample. It is named after its inventor, Edwin Southern, who developed the method in 1975.
The principle of Southern blotting involves several steps and uses DNA hybridization to
identify target sequences.
1. DNA Digestion: The first step is to isolate the DNA from the sample of interest, such
as genomic DNA. The DNA is then cut into smaller fragments using restriction enzymes,
which recognize specific DNA sequences and cleave the DNA at those sites.
2. Gel Electrophoresis: The DNA fragments obtained from the digestion are separated
based on their size using gel electrophoresis. The DNA fragments are loaded into wells of an
agarose gel and subjected to an electric field. Smaller fragments migrate faster through the
gel than larger ones, leading to the separation of DNA fragments by size.
3. DNA Denaturation: After electrophoresis, the DNA fragments are transferred from
the gel to a solid support, usually a nitrocellulose or nylon membrane. This transfer process is
called "blotting." Before blotting, the DNA in the gel needs to be denatured, breaking the
double-stranded DNA into single strands.
4. Blotting: The transfer of DNA from the gel to the membrane is typically achieved
through a capillary or vacuum transfer. As a result, the DNA on the membrane retains the
same pattern of separation as in the gel.
7. Detection: The labeled DNA probes that have successfully hybridized with the
complementary DNA sequences on the membrane can be visualized using autoradiography
(for radioactive probes) or fluorescence imaging (for fluorescent probes).
By following these steps, Southern blotting allows researchers to detect the presence or
absence of specific DNA sequences in a sample and determine their approximate size based
on their position in the gel. This technique has been widely used in molecular biology for
various applications, including gene mapping, genetic testing, and gene expression analysis.
However, it is essential to note that Southern blotting is a time-consuming and labor-intensive
technique, and it has largely been replaced by more advanced methods such as polymerase
chain reaction (PCR) and various DNA sequencing technologies for most applications.
APPLICATIONS
Advantages
Less degraded compared with protein and mRNA as DNA are very stable.
Effective way to detect specific DNA sequence from large complex samples.
Increased sensitivity of fragments detection because of probe label used for amplifying
signals.
Only way to diagnose FSHD (Facioscapulohumerals Muscular Dystrophy).
Disadvantages
Not applicable in routine diagnostic setting.
Time consuming and requires large amount of DNA.
Does not allow morphologic preservation of tissue so historic evaluation features are
not available.
REFERENCES