Southern blot is a process in which DNA fragments, separated by electrophoresis, are transferred onto a membrane for immobilization and identification.
It is a routine procedure widely adopted for the analysis of DNA samples in various applications.
The technique was discovered by Edwin Southern, and it was named after him.
This discovery led to the development of other related techniques such as Western blotting and Northern blotting, which are based on the same fundamental principle.
In its most basic form, Southern blotting is used to determine the size of a DNA fragment from a complex mixture of genomic DNA.
The technique is also relatively quantitative, allowing researchers to determine the number of copies of a specific DNA segment present in a genome.
Southern blotting can be modified depending on the choice of membrane, transfer buffer, and transfer method.
The nitrocellulose membrane is the most commonly used type because it is robust and can be reprobed multiple times.
The original protocol developed by Edwin Southern used radioactive probes for detection; however, alternative labeling systems have since been introduced that utilize fluorescence and chemiluminescence for safer and more versatile detection.
Over time, Southern blotting has been refined and modified in various ways to enhance its efficiency and adaptability, making it more complex and better suited to different research and diagnostic applications.
Principle of Southern Blot
The principle of Southern blotting is based on the blotting technique, which involves transferring biomolecules from one membrane to another for detection and identification.
In this process, the DNA sample to be analyzed is first digested using restriction enzymes, which cut the DNA into smaller fragments.
These DNA fragments are then separated based on their size through agarose gel electrophoresis.
Following electrophoresis, the DNA strands in the gel are denatured using an alkaline treatment, which separates the double-stranded DNA into single strands.
The single-stranded DNA fragments are then transferred from the gel onto a nylon or nitrocellulose membrane through the blotting process.
Once transferred, the DNA strands are immobilized on the membrane surface by baking or ultraviolet (UV) irradiation to ensure they remain fixed during further processing.
The immobilized DNA sequences on the membrane are then detected using a hybridization process, where labeled probes bind to their complementary target sequences.
Hybridization is a highly specific reaction, as the probe will only bind to DNA fragments with complementary nucleotide sequences.
The probes used in hybridization are labeled with detectable markers, which can vary depending on the method used—such as radioactive isotopes, fluorescent dyes, or chemiluminescent tags—allowing visualization of the hybridized fragments.
Requirements
Water bath
Agarose gel
Power supply
UV radiation source
Hybridization oven
Hybridization bottles
Trays
Film processor
Pipettes
Centrifuge tubes
Glass plate
Whatman 3 mm chromatography paper
Nylon membrane or nitrocellulose membrane
Syringe
Cellulose acetate membrane
Materials
Restriction enzymes
Restriction enzyme buffer
Agarose
TBE buffer
DNA loading buffer
Tris base
Sodium chloride
Sodium hydroxide
Sodium citrate
DNA labeling kit
Nucleic acid detection kit
Sodium dodecyl sulfate (SDS)
Polyvinylpyrrolidone (PVP)
Bovine serum albumin (BSA)
Formamide
Phenol
Solutions and Buffers
Denaturation buffer: Sodium hydroxide (NaOH) and sodium chloride (NaCl) mixed in a 1:6 ratio.
Neutralization buffer: Tris-HCl and sodium chloride (NaCl) mixed in a 5:3 ratio.
SSC (Saline-Sodium Citrate): Prepared by dissolving 175.3 g of NaCl and 88.2 g of sodium citrate in 1 liter of distilled water.
Detection buffer: Tris-HCl and sodium chloride (NaCl) mixed in a 5:1 ratio.
Procedure of Southern Blot
a. Restriction digestion of DNA
About 10 µg of extracted genomic DNA is digested with the appropriate restriction enzyme in a microcentrifuge tube.
The tube is incubated overnight at 37°C to ensure complete digestion of DNA.
In some cases, the tubes are subsequently heated in a water bath at 65°C for 20 minutes to denature the restriction enzymes after digestion.
After incubation, 10 µl of DNA sample buffer is added to the tubes, and the mixture is carefully loaded onto an agarose gel for electrophoresis.
b. Electrophoresis
The percentage and size of the agarose gel are selected based on the expected size of the DNA fragments to be separated.
The electrophoresis buffer is prepared with ethidium bromide and poured into the tank until it is a few millimeters above the gel support.
The gel cast is prepared with a comb to form wells for loading samples. Once the comb is in place, molten gel is poured slowly into the cast and allowed to solidify.
After the gel sets, the comb is removed, and the gel is positioned in the electrophoresis tank.
Running buffer is added to completely cover the gel.
The DNA samples are mixed with loading buffer and carefully pipetted into the wells.
The tank is connected to the power supply and electrophoresis is allowed to run overnight to separate the DNA fragments based on size.
c. Denaturation
After electrophoresis, the gel is transferred to a glass tray containing 500 ml of denaturation buffer (1.5 M NaCl and 0.5 M NaOH) and incubated for 45 minutes at room temperature.
The denaturation buffer is then replaced with neutralization buffer, and the gel is soaked for 1 hour while being gently rotated on a platform rotator.
d. Blotting
An oblong sponge, slightly larger than the gel, is placed in a glass dish filled with SSC buffer, ensuring the sponge is about half-submerged.
Three pieces of Whatman 3 mm filter paper are cut to the same size as the sponge, placed on top, and wetted with SSC.
The gel is placed on the filter paper, and any air bubbles are removed by gently rolling a glass pipette over the surface.
A nylon or nitrocellulose membrane, just large enough to cover the gel, is placed on top and wetted with SSC.
Several additional filter papers are placed over the membrane, followed by a glass plate to keep the setup stable.
The transfer is left overnight to allow DNA fragments to move from the gel onto the membrane by capillary action.
e. Baking/ Immobilization
The nylon membrane is removed from the blotting setup and baked in a vacuum or conventional oven at 80°C for 2–3 hours to fix the DNA onto the membrane.
Alternatively, immobilization can be achieved by exposing the membrane to ultraviolet (UV) radiation to crosslink the DNA strands to the membrane surface.
f. Hybridization
The membrane is then exposed to a labeled hybridization probe, which may be a DNA or RNA fragment designed to specifically detect the target DNA sequence.
The probe is labeled using radioactive isotopes, fluorescent dyes, or chromogenic molecules to enable visualization after hybridization.
The hybridization conditions (temperature, buffer, and salt concentration) are optimized to promote binding between the probe and the complementary target sequence.
After hybridization, the membrane is washed with buffer to remove any unbound or nonspecifically bound probes, leaving only those that have hybridized to the correct target DNA.
g. Detection
Detection of hybridized regions is carried out using autoradiography by placing the membrane in contact with photographic film.
The developed film reveals bands corresponding to hybridized DNA fragments, which can be compared to a DNA marker to determine fragment length.
The autoradiogram also provides information about the number and size of hybridizing fragments.
If fluorescent or chromogenic probes are used instead of radioactive ones, visualization can be achieved either on X-ray film or directly on the membrane through color development.
Result Interpretation of Southern Blot
The results of a Southern blot are visualized as distinct bands on the membrane, representing the DNA fragments that have successfully hybridized with the labeled probe.
Each band corresponds to a DNA fragment containing the target sequence complementary to the probe used during hybridization.
The size of the DNA fragments is determined by comparing the migration distance of the bands with those of a DNA marker or ladder containing fragments of known lengths.
The intensity of the bands can also provide semi-quantitative information about the copy number or abundance of the specific DNA sequence in the sample.
Therefore, the Southern blot result not only identifies the presence of a specific DNA sequence but also helps estimate the fragment size and relative quantity of the target DNA in the analyzed sample.
Applications of Southern Blot
Southern blotting has numerous applications in gene discovery, genetic mapping, evolutionary studies, and diagnostic research.
It is widely used for DNA analysis to detect point mutations, insertions, deletions, and other structural rearrangements within DNA sequences.
The technique allows for the determination of molecular weights of restriction fragments, aiding in the analysis and characterization of these DNA fragments.
Because Southern blotting can specifically detect a particular DNA segment, it is employed in personal identification and DNA fingerprinting for forensic and paternity testing purposes.
It is a valuable tool in medical diagnostics, where it is used to identify genetic diseases, monitor gene expression patterns, and assist in prenatal diagnosis of inherited disorders.
Limitations of Southern Blot
Southern blotting is a costly technique because it requires expensive equipment, reagents, and labeled probes compared to other molecular tests.
The procedure is complex and involves multiple steps, including digestion, electrophoresis, blotting, hybridization, and detection, which makes it technically demanding.
It is a labor-intensive process that requires skilled and trained personnel to perform each step accurately.
The technique is time-consuming, often taking several days to complete, and has largely been replaced in many applications by faster methods such as Polymerase Chain Reaction (PCR).
Southern blotting is semi-quantitative, meaning it provides only an approximate estimation of the DNA fragment size and quantity rather than precise measurements.
It is not suitable for detecting mutations at the single base-pair level, as it lacks the resolution needed for such detailed analysis.
The method requires a large amount of high-quality DNA, which must be isolated using superior extraction techniques to ensure successful results.
References
Brown, T. (2001). Southern blotting. Current Protocols in Immunology, Chapter 10, Unit 10.6A. https://doi.org/10.1002/0471142735.im1006as06. PMID: 18432697.
Glenn, G., & Andreou, L. V. (2013). Analysis of DNA by Southern blotting. Methods in Enzymology, 529, 47–63. https://doi.org/10.1016/B978-0-12-418687-3.00005-7. PMID: 24011036.
