Brief introduction and uses of Southern Blotting

1. Beijing, 06 October 2018
A Presentation on Southern
Blotting and
Related DNA Detection
TechniquesCourse Name: Advanced Plant
Biology
Course No.: LXG000102
MD ASIQUR RAHMAN
MASTER’S PROGRAM
Student Id: LS20183010014
Major: Crop Cultivation & Farming System
College of Agronomy and Biotechnology
China Agricultural University
Presented By :

2. Background of Southern Blotting
 Southern blotting is one of the central techniques in molecular biology.
 First devised by Sir Edwin Mellor Southern (E. M. Southern) on 1975.
 Southern blotting results in transfer of DNA molecules, usually restriction fragments, from an
electrophoresis gel to a nitrocellulose or nylon sheet, in such way that the DNA banding pattern
present in the gel is reproduced on the membrane.
 During transfer or as a result of subsequent treatment, the DNA becomes immobilized on the
membrane and can be used as a substrate for hybridization analysis with labelled DNA probes that
specifically target individual restriction fragments in the blotted DNA.
 In essence, ‘Southern blotting is therefore a method for detection of a specific restriction fragment
against a background of many other restriction fragments’ ( Brown, 1999)
E. M. Southern

3. The Principle of the Southern Blot
 The process involves the transfer of electrophoresis-separated DNA fragments to a carrier
membrane which is usually nitrocellulose and the subsequent detection of the target DNA fragment
by probe hybridization.
 Hybridization refers to the process of forming a double-stranded DNA molecule between a single-
stranded DNA probe and a single-stranded target DNA.
 Since the probe and target DNA are complementary to each other, the reaction is specific which aids
in the detection of the specific DNA fragment.

4. The Original Methodology for Southern Blotting
 The original methodology for Southern Blotting is illustrated in Figure-1
 An agarose electrophoresis gel, containing the fractionated restriction fragments, is placed
on a filter paper wick that forms a connection between the gel and a reservoir of high-salt
buffer.
 The nitrocellulose membrane is placed on top of the gel and covered with a tower of paper
towels that are held in place with a weight. More recently instead of nitrocellulose
membrane, the Nylon membrane is being used because it is less fragile than nitrocellulose
sheets and can not be damaged by handling.
 Capillary action results in the buffer soaking through the filter paper wick, gel and
membrane and into the paper towels.

5.  Effective transfer of fragments up to 15 kb in length takes approximately 18 h, roughly equivalent to
“overnight”.
 The only technical complication is the possibility that the buffer bypasses the gel by soaking directly
from wick to paper towels, which is unlikely if the setup is assembled carefully.
Figure-1
 As the buffer passes through the gel the DNA fragments are carried with it into the membrane, where
they become bound to the nitrocellulose.

6. Steps Involved in Southern Blotting Process
I. Extraction and purification of DNA from cells
II. Restriction Digestion or DNA Fragmentation
III. Separation by Gel Electrophoresis
IV. Depurination
V. Denaturation
VI. Blotting
VII.Baking
VIII.Hybridization
IX. Washing of unbound probes
X. Autoradiograph

7. I. Extraction and Purification of DNA from Cells
 DNA is first separated from target cells following standard methods of genomic DNA extraction
and then purified.
II. Restriction Digestion or DNA Fragmentation
 The DNA is fragmentized by using suitable restriction enzyme. RE cuts the DNA at specific site
generating fragments.
 The number of fragments of DNA obtained by restriction digest is amplified by PCR

8. III. Separation by Gel Electrophoresis
 Gel electrophoresis separates DNA and RNA molecules according to size, shape and
topological properties.
 The separation may be done by agarose gel electrophoresis in which the negatively charged
DNA fragments move towards the positively charged anode, the distance moved depending
upon its size.
IV. Depurination
 Partial depurination is done by the use of dilute HCl which promotes higher efficiency
transfer of DNA fragments by it breaking down into smaller pieces.

9. V. Denaturation
 DNA is then denatured with a mild alkali such as an alkaline solution of NaOH.
 This causes the double stranded DNA to become single-stranded, making them suitable for
hybridization.
 DNA is then neutralized with NaCl to prevent re-hybridization before addition of the probe.
VI. Blotting
 The denatured fragments are then transferred onto a nylon or nitrocellulose filter membrane
which is done by placing the gel on top of a buffer saturated filter paper, then laying
nitrocellulose filter membrane on the top of gel.
 Finally some dry filter papers are placed on top of the membrane. Fragments are pulled towards
the nitrocellulose filter membrane by capillary action and result in the contact print of the gel.

10. VII. Baking
 The nitrocellulose membrane is removed from the blotting stack, and the membrane with
single stranded DNA bands attached on to it is baked in a vacuum or regular oven at 80 °C
for 2-3 hours or exposed to ultraviolet radiation to permanently attach the transferred DNA
onto the membrane.
VIII. Hybridization
 The membrane is then exposed to a hybridization probe which is a single DNA fragment
with a specific sequence whose presence in the target DNA is to be determined.
 The probe DNA is labeled so that it can be detected, usually by incorporating radioactivity or
tagging the molecule with a fluorescent or chromogenic dye. Here generally, 32P is used as a
radioactive label.

11. Types of Hybridization Probes
 Any DNA molecule that is complementary to the restriction fragment being sought can
be used as the probe.
 Depending on the application, the probe might itself be a restriction fragment, or it might
be a DNA molecule obtained by the polymerase chain reaction (PCR), or a cDNA
prepared by complementary DNA synthesis from an mRNA template.
 Synthetic oligonucleotides are also frequently used as hybridization probes.
 The another popular type of hybridization probe is one made of RNA rather than DNA.

12. IX. Washing of Unbound Probes
 After hybridization, the membrane is thoroughly washed with a buffer to remove the probe that is bound
nonspecifically or any unbound probes present.
X. Autoradiograph
 The hybridized regions are detected autoradiographically by placing the nitrocellulose membrane in
contact with a photographic film which shows the hybridized DNA molecules.
 The pattern of hybridization is visualized on X-ray film by autoradiography in case of a radioactive or
fluorescent probe is used or by the development of color on the membrane if a chromogenic detection
method is used.

13. Figure-2: Steps of Southern Blotting Process

14. Applications of Southern Blotting
 Identifying specific DNA in a DNA sample.
 Preparation of RFLP (Restriction Fragment Length Polymorphism) maps
 Detection of mutations, deletions or gene rearrangements in DNA
 For criminal identification and DNA fingerprinting (VNTR)
 Detection and identification of trans gene in transgenic individual
 Mapping of restriction sites
 For diagnosis of infectious diseases
 Prognosis of cancer and prenatal diagnosis of genetic diseases
 Determination of the molecular weight of a restriction fragment and to measure
relative amounts in different samples.

15. Identifying specific
DNA in a DNA
sample.

16.  Preparation of
RFLP (Restriction
Fragment Length
Polymorphism)
maps

17. Detection of mutations,
deletions or gene
rearrangements in DNA

18. For Criminal
Identification and
DNA Fingerprinting
(VNTR)

19. Detection and identification of
trans gene in transgenic
individual

20. Mapping of
restriction sites

21. For diagnosis of
infectious diseases

22. Prognosis of cancer and
prenatal diagnosis of
genetic diseases

23. Summary
 Southern blotting is a technique that enables a specific restriction fragment to be detected against a
background of many other restriction fragments.
 It involves transfer of DNA fragments from an electrophoresis gel to a nitrocellulose or nylon
membrane in such a way that the DNA banding pattern present in the gel is reproduced on the
membrane.
 Hybridization probing is then used to detect the restriction fragment that is being sought.
 The basic methodology for Southern blotting has not changed since original technique was
described in 1975, but modifications have been introduced with the aim of speeding up the process
and achieving a more efficient transfer.

24. Reference
s
 Amasino RM (1986) Acceleration of nucleic acid hybridization rate by polyethylene glycol. Analytical
Biochemistry 152: 304-307
 Bittner M, Kupferer P and Morris CF (1980) Electrophoretic transfer of proteins and nucleic acids
from slab gels to diazobenzyloxymethyl cellulose or nitrocellulose sheets. Analytical Biochemistry
102: 459-471
 Brown TA (1999) Genomes. Oxford: BIOS Scientific Publishers.
 Southern EM (1975) Detection of specific sequences among DNA fragments separated by gel
electrophoresis. Journal of Molecular Biology 98: 503-517

25. Any
Question?