EAST WEST UNIVERSITY

Department of pharmacy

Assignment on
Southern blotting
Course:Pharmaceutical Biotechnology
Course code: PHRM 407
Submitted to:
Dr.ABM Md.Khademul Islam
Associate professor
Dept of Genetic Engineering and Biotechnology
University of Dhaka
Submitted by:
Name: Md: Shukur ali
ID No:-2014-1-70-038
Submission Date: 6th October,2016

Introduction:- A Southern blotting is a method of routinely used in molecular biologically to

checking the presence of a DNA sequence in a DNA sample. Southern blotting which was named
after, the British Biologist Edwin Southern. Edward M. Southern who developed this procedure
at Edinburgh University in the 1970s(technique et al., 2016).Southern blotting technique is
used to separate and characterize the DNA. It is an effective way to identify a specific DNA
pattern. Southern blotting is the process used to identify the presence of a
specific DNA sequence within a mixture via agarose gel electrophoresis. Southern blotting
combines with agarose gel electrophoresis for size separation of DNA with methods to transfer
the size-separated DNA to a filter membrane for probe hybridization.Southern Blotting could be
used to locate a particular gene within an entire genome .The Southern blotting is an example of
a basic nucleic acid hybridization technology. Hybridizations are useful for understanding gene
organization and copy number, cloning genes from libraries, detecting polymorphisms (such as
RFLPs used in forensics) and detecting expressed transcripts.
Principle:It is a technique for transferring DNA molecules from agarose gel to a solid materials support
such as nitrocellulose filter or nylon membrane. In Southern blotting, DNA fragments are first
separated using gel electrophoresis and then the separated molecules are transferred to a
membrane surface . Southern blotting is a method that combines gel electrophoresis and nucleic
acid hybridization. It allows detection of specific nucleotide sequences in a DNA sample, that
the DNA used does not have to be pure to be identified.Gel embedded DNA is depurinated in the
presence of HCl followed by denaturation in alkali. Denaturation prior to blotting is essential for
dissociating the two polynucleotide chain which are then hybridized with the probe. Denatured
molecules move upward by capillary action of buffer and on coming in contact with the nylon
membrane they get bound to it.In the next step, hybridization analysis is carried out on the
membrane using labeled probes complimentary to the target sequence to be identified, thus
detecting the presence of DNA fragment of interest. Bulky target molecules need to be broken
down before electrophoretic separation so as to facilitate molecule movement through the gel. In
Southern blotting, enzymes called restriction endonucleases are used to cut large DNA sequences
in to smaller pieces.Restriction fragments are cut up and run through electrophoresis. Restriction
fragments of DNA to be separated on the basis of their size prompt to development of techniques
for the transfer of separated fragments en masse from gel to nitrocellulose support. The
procedure described by Southern (1975), involving capillary transfer of DNA from the gel to a
nitrocellulose sheet placed on top of it, was simple and eective. DNA gets transferred to a
nitrocellulose membrane, which makes the blot and denatures the DNA. The single strands of
DNA are positioned in bands corresponding to those in the gel. The blot is then exposed to a
solution with probes which are usually radioactive single-stranded DNA molecules
complementary to the genes.This combination of electrophoresis and nucleic acid allows for
identification of impure DNA.

Figure: Southern blotting.

Materials and reagent:-

Agarose gel containig DNA fragments Nylon membrane Whatman 3 mm paper Glass
tray Rough Filter papers 0.25 M HCl Denaturing Solution Neutralizing Solution 10x
SSC

Method of blotting:Gel electrophoresis process:By the 1970 the DNA fragments was isolated by using gravity. After that time, the scientists
found other way to separate DNA fragment. That is Gel Electrophoresis. Electrophoresis
uses electricity to separate different sized molecules.

1.The DNA sample, if necessary, is digested with an appropriate restriction enzymes, or

restriction endonuclease.The digest is then separated by gel electrophoresis, usually on an
agarose gel. If a large amount of restriction fragments are present in the sample, the sample may
likely appear as faint smears rather than discrete bands.
2. The digest is then denatured to allow for transfer onto a membrane. Since the sample DNA is
double-stranded and only single-stranded DNA can be transferred, the sample must be denatured
by making in an appropriate alkaline solution (e.g. 0.5M NaOH). If the sample is still too large to
be transferred (more than 15kb), the sample may be treated with an appropriate acid to
depurinate (e.g. remove the purines) the sample and break it down into smaller fragments. Then
the sample is then neutralized.
3. The sample is transferred into a membrane which is a sheet of special blotting paper. The
transfer to another membrane is performed in order to preserve the position of the DNA
fragments. A Nitrocellulose membrane is generally used, though some may prefer the use of
nylon because nylon is better binding capacity. The membrane used is laid on top of the gel and a
usually paper towels are placed on top of the membrane to ensure an even distribution by
applying pressure. The transfer is done usually be capillary action which may take several hours.
Alternatively, a vacuum apparatus can be used; this is very similar to capillary action, though
transferring via vacuum apparatus may be faster. The transfer can also occur by moving
the DNA out of the gel and onto the membrane by electrophoresis, a process called
electrotransfer.
4. The sample is then treated with UV light to irreversibly cross-link the sample to the membrane
covalently. The temperature around 80C for several hours.
5. The membrane is then probed with labeled, single-stranded DNA. This process is also known
as hybridization. The labeled DNA binds to the membrane DNA via the binding of
complementary strands. The label is generally a 32P probe label, though a bioluminescent probe
or biotin/streptavidin may also be used. The reason that hybridization is important is that it
allows you to physically. It should be noted that prior to hybridization, a prehybridization process
should be run to restrict the labeled DNA binding to specific sites, because otherwise the

labeled DNA may bind to unwanted or unimportant sites within the sample. To do so, salmon
sperm DNA treated with varying concentrations of SSC, SDS, and formamide is used.
32

P labeled:-

Treat the dsDNA fragment that you are using as a probe with a limiting amount of Dnase, which
causes double-stranded nicks in DNA. Add sup>32P, dATP, and other dNTPs toDNA
polymeraseI which has 5' to 3' polymerase activity and 5' to 3' exonuclease activity.
6. The results are analyzed. If a 32P labeled probe was used, the sample will be analyzed with an
autoradiograph or the use of an X-ray film. The target sequence is needed, the X-ray film will
show blackened bands caused by beta emissions from the 32P radio labeled probe. Sequences in
the sample that are very similar to the target sequence will also be shown. If a bio-luminescent
probe was used, the luminescence will be a method of visualization. If biotin/streptavidin was
used,the
sample
is
analyzed
by
colorimetric
methods.
FIGUREOGELELECTROPHtechnique, S., Porterfield, A., Nanodrop, S. and Erk, J. (2016).
Southern (blot) exposure remains a useful technique - Bitesize Bio. [online] Bitesize Bio.
Available at: http://bitesizebio.com/25429/southern-blot-exposure-remains-a-useful-technique/
[Accessed 5 Oct. 2016].pA
Reference:S., Porterfield, A., Nanodrop, S. and Erk, J. (2016). Southern (blot) exposure remains a useful
technique
Bitesize
Bio.
[online]
Bitesize
Bio.
Available
at:
http://bitesizebio.com/25429/southern-blot-exposure-remains-a-useful-technique/ [Accessed 5
Oct. 2016].