Southern blotting is a molecular technique to find target DNA sequences in a sample. It is a multi-step process, that begins with the electrophoresis of DNA, transfer of DNA fragments into nitrocellulose strip, and exposing those fragments to a DNA probe labeled with a radioactive or chemical tag.
The Southern blotting technique was named after Edwin Southern who introduced the technique in 1975.
Table of Contents
Principle
The target DNA is broken into small fragments using restriction endonucleases and is separated by electrophoresis. Following separation, the double-stranded pieces of DNA are denatured into single strands within the gel and transferred from the gel onto a blotting membrane. The membrane is then treated with a small piece of DNA or RNA called a probe, which has a complementary sequence to the target DNA.
The probe also has a radioactive atom or a fluorescent dye label, that following hybridization, permits the DNA fragment of interest to be detected from different DNA fragments present on the membrane.
Requirements
- Agarose gel
- Cellulose nitrate or nitrocellulose membrane filter with uniform porosity.
- Ethidium bromide for staining the DNA
- Enzymes: restriction nucleases, RNase A
- DNA loading buffer, TBE Buffer for electrophoresis
Steps involved in Southern blotting
(Image source: National Human Genome Research Institute)
Extraction, purification, fragmentation, and separation of target DNA
DNA is extracted from the target source and is broken into small fragments using restriction endonuclease enzyme.
Fragmented DNA is then electrophoresed on an agarose gel to separate the fragments according to their molecular weights. Acrylamide gels can alternatively be used for good resolution of smaller DNA fragments (<800 bp). DNA thus obtained are double-stranded, and is therefore denatured to single-stranded DNA by dipping the gel in an alkaline solution.
Blotting refers to the transfer of the fragmented DNA sequence to the nitrocellulose membrane or nylon membrane. Although this step determines the name of this technique “Southern blotting,” the term is typically used to describe the entire procedure.
Blotting
The process is done by either electroblotting or capillary blotting. A sheet of nitrocellulose membrane is placed on top of (or below, depending on the direction of the transfer) the gel and gentle pressure is applied evenly to the gel (either using suction or by placing a stack of paper towels and a weight on top of the membrane and gel), to ensure good and even contact between gel and membrane. Fragments are pulled towards the nitrocellulose filter membrane by capillary action and result in the formation of an imprint or blot of the gel. The portion of the nitrocellulose membrane, touching the gel should be gently removed using a blade.
Baking
The membrane is then baked in a vacuum or regular oven at 80 °C for 2 hours or can be fixed via UV crosslinking mediated by exposure to short-wavelength UV light to permanently attach the transferred DNA to the membrane.
Pre hybridization and blocking
Prehybridization and blocking are done to eliminate non-specific reactions. There are various kinds of blocking agents, commonly, salmon or herring sperm DNA is used for blocking the membrane surface and target DNA.
In this step, the membrane is incubated in Denhardt’s solution for 1 hour or more, depending on the type of reaction. After incubating the prehybridization solution at 42°C, the heat snap chilled salmon sperm DNA is added to it at a concentration of 50 µg/mL.
Hybridization with the probe
Hybridization is usually carried out in a sealed bag which will contain the Southern blot, and hybridization fluid containing the labeled probe. The time required for hybridization usually 1–16 hours depending on factors like the complexity of the probe and concentration.
Visualization of the result
After hybridization, the excess probe is washed from the membrane using a buffer, and the pattern of hybridization is visualized on X-ray film by autoradiography in the case of a radioactive or fluorescent probe, or by the development of color on the membrane if a chromogenic detection method is used.
Result Interpretation
Hybridization of the probe to a specific DNA fragment on the filter membrane indicates that this fragment contains DNA sequence that is complementary to the probe. A probe that has hybridized with a single fragment of DNA not being digested by restriction enzymes will result in only one band in the final blot. In case the probe binds to many of the similar sequences it will result in multiple bands.
Modification of the hybridization conditions (for example, increasing the hybridization temperature or decreasing salt concentration) may be used to increase specificity and decrease hybridization of the probe to sequences that are less similar.
Applications
Southern blotting is used to analyze the genome of organisms of interest in order to identify a specific sequence. Southern blot analysis can be used to:
- To study mutation and gene rearrangement, this property is used to diagnose neonatal disease and genetic disease.
- In phylogenetic studies, recombinant DNA technology, paternity & maternity analysis, forensic studies, and personal identification.
- To demonstrate the presence or absence of a specific target sequence in DNA,
- To determine whether chromosomal integration has occurred,
- In immunology, the clonal rearrangements of the immunoglobulins, as well as the T Cell receptor genes, can be analyzed by Southern blotting.
- To study the structure of a gene or to elucidate restriction enzyme maps.
- To determine the copy number of transgenes etc.,
References and Further Readings
- ‘Southern Blot’, National Human Genome Research Institute.
- ‘Southern Blotting‘, MyBioResource.com
- ‘Southern Blot‘, World Library of Science
