What Is Southern Blotting?
Southern blot analysis is a laboratory technique for determining the identity, size, and quantity of DNA. It is the process of transferring DNA (deoxyribonucleic acid) fragments separated by electrophoresis onto a membrane for identification and immobilization. The technique is named for its inventor, British biologist Edwin Southern. The most basic form of the method is used to measure the size of a DNA fragment from a complicated mixture of genomic DNA. Southern blotting can be altered by changing the membrane, transfer buffer, and methodology. The nitrocellulose membrane is the most often used because it is stable and can be re-probed multiple times. Similar to how the original southern blotting method uses radioactive probes, other labeling technologies use fluorescence and chemiluminescence. It has been changed in various ways to serve the application better, and it has become more complicated and efficient.
What Is the Principle of Southern Blotting?
The principle of Southern blotting is based on the separation of DNA fragments by gel electrophoresis, followed by the identification of labeled probe hybridization. The DNA fragments are divided according to their size and charge during electrophoresis. The alkaline treatment denatures the DNA strands, which are then transferred to a nylon or nitrocellulose membrane via blotting. The strands on the membrane are fixed on the surface by baking or UV irradiation and can be identified using the hybridization procedure. Hybridization processes are specific because the probes bind to target fragments of complementary sequences. Depending on the type of probes used, the probes are labeled with different components that can be viewed using various methods.
What Are the Requirements for Southern Blot?
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Agarose gel
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Ethidium bromide for staining the DNA
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Cellulose nitrate or nitrocellulose membrane filter with uniform porosity.
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Enzymes such as restriction nucleases, RNase A
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DNA loading buffer
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TBE Buffer for electrophoresis
What Steps Are Involved in the Southern Blotting Procedure?
Extraction and Purification of DNA From Cells
DNA is extracted from target cells using standard genomic DNA extraction procedures and subsequently purified.
Restriction Digestion or DNA Fragmentation
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An appropriate restriction enzyme is used to fragment the DNA. Restriction endonucleases are enzymes that cut high molecular weight DNA at certain places, resulting in fragments.
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By using PCR (polymerase chain reaction), the number of DNA fragments acquired after restriction digestion is amplified.
Gel Electrophoresis
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The separation can be accomplished via agarose gel electrophoresis, in which negatively charged DNA fragments migrate toward the positively charged anode.
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In this step, the distance traveled depends on the size of the fragment.
Depurination
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Diluted HCl is used for partial depurination, which increases the effectiveness of DNA fragment transfer by breaking them up into smaller pieces.
Denaturation
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The next step is to denature the DNA using a moderate alkali, such as an alkaline solution of NaOH. It permits the double-stranded DNA to become single-stranded, making it ideal for hybridization.
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The DNA is then neutralized with NaCl to stop re-hybridization before the probe is added.
Blotting
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The denatured fragments are transferred onto a nylon or nitrocellulose filter membrane by first laying the gel on top of a filter paper that has been saturated with buffer, followed by the nitrocellulose filter membrane.
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On top of the membrane, some dry filter papers are placed.
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Capillary motion causes fragments to be drawn toward the direction of the nitrocellulose filter membrane, creating the contact print of the gel.
Baking
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After the target DNA has been attached to the membrane, it is baked for two to three hours at 80 °C in an autoclave to bind to the membrane.
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The membrane is subsequently treated with casein or bovine serum albumin (BSA), which saturates all of the membrane's binding sites.
Hybridization
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The membrane is then subjected to a hybridization probe, a single DNA fragment with a specified sequence whose presence in the target DNA is to be detected.
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The probe DNA is labeled to allow detection by adding radioactivity or tagging the molecule with a fluorescent or chromogenic dye.
Autoradiogram
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It is possible to see the membrane-bound DNA labeled with a probe under an autoradiogram, which displays a pattern of bands.
What Are the Applications of Southern Blotting?
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Southern blotting has a wide range of applications in gene identification, mapping, evolution, and diagnostic studies.
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The method can be applied to DNA analysis to find point mutations and other structural rearrangements in the DNA sequences.
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Used for forensic investigations, personal identification, criminal identification, and recombinant DNA technology.
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Used in RFLP (Restriction Fragment Length Polymorphism) maps.
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Finding and identifying the transgene in a transgenic individual.
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To identify and isolate the desired genes.
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Used for the detection of infectious diseases.
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Used in cancer prognosis and genetic disease prenatal diagnosis.
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Determining the molecular weight of a restriction fragment and determining relative levels in different samples.
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To determine if chromosomal integration has occurred.
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It is used in immunology to examine clonal rearrangements of immunoglobulins and T-cell receptor genes.
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To investigate the structure of a gene or to map restriction enzymes.
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This technique makes it possible to identify a specific DNA segment, making it useful for fingerprint-based personal identification.
What Are the Limitations of the Southern Blot?
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The southern blot method is expensive because it requires more expensive equipment and reagents than other tests.
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It is a multi-step process that is complicated. The procedure is also labor intensive and requires the use of trained staff.
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It is a time-consuming procedure that can be substituted by speedier processes such as Polymerase Chain Reaction.
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The procedure is semi-quantitative and provides an estimation of the size of the DNA fragments.
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It is not a suitable method for detecting base-pair mutations.
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This method needs a significant amount of samples and better DNA isolation techniques to produce DNA of higher quality.
Conclusion
Overall, Southern blotting is a helpful technique for diagnosing and studying disorders such as fragile X syndrome and sickle cell anemia and analyzing DNA for forensic and paternity testing. However, southern blotting is very expensive, time-consuming, and technically challenging, and it needs many DNA samples. As a result, new techniques are gradually replacing southern blotting, like real-time PCR.
