What is Southern Blotting?
Southern blotting is a technique that allows researchers to detect and identify specific DNA sequences from a complex mixture of DNA samples. It is based on the principle of transferring DNA fragments that have been separated by gel electrophoresis to a carrier membrane, such as nitrocellulose or nylon, and then hybridizing them with a complementary probe that is labeled for detection. The probe can be radioactive, fluorescent, or chromogenic, depending on the method of visualization. Southern blotting can be used for various purposes, such as gene mapping, gene expression analysis, DNA fingerprinting, diagnosis of genetic diseases, and forensic investigations.
The technique was named after its inventor, Edwin M. Southern, a molecular biologist who developed it in 1975 while working at the University of Edinburgh. He published his first paper on the method in the Journal of Molecular Biology in 1975, titled "Detection of specific sequences among DNA fragments separated by gel electrophoresis". The technique was soon adopted by other researchers and became a widely used tool in molecular biology. In recognition of his contribution, Southern was awarded the Lasker Award in 2005 and the Royal Medal in 2012. The term "Southern blotting" has also inspired the names of other similar techniques that use RNA or protein as the target molecule, such as Northern blotting and Western blotting.
Southern blotting is based on the principle of transfer of electrophoresis-separated DNA fragments to a carrier membrane and detection by probe hybridization. The DNA fragments are separated by size on an agarose gel and then transferred to a nitrocellulose or nylon membrane by capillary action. The membrane is then exposed to a labeled probe that is complementary to the target DNA sequence of interest. The probe can be detected by various methods, such as radioactivity, fluorescence or colorimetry, depending on the type of label used. The probe hybridizes only to the DNA fragments that contain the target sequence, allowing the identification and characterization of the specific DNA fragment. Southern blotting is a powerful technique for analyzing the structure and organization of genes and genomes.
Southern blotting involves several steps that are performed in a sequential manner to transfer and detect the DNA fragments of interest. The following are the main steps involved in Southern blotting:
- Extraction and purification of DNA from cells: The first step is to obtain the DNA sample from the cells of interest. This can be done by using standard methods of genomic DNA extraction, such as phenol-chloroform extraction or column-based purification kits. The extracted DNA should be free of contaminants and impurities that may interfere with the subsequent steps.
- Restriction Digestion or DNA Fragmentation: The next step is to cut the DNA sample into smaller fragments using restriction enzymes. Restriction enzymes are molecular scissors that recognize and cleave specific sequences of DNA. Depending on the research question, one or more restriction enzymes can be used to generate a specific pattern of DNA fragments. The size and number of the fragments will depend on the restriction sites present in the DNA sample.
- Separation by Electrophoresis: The DNA fragments are then separated by size using agarose gel electrophoresis. Agarose gel is a porous matrix that allows the movement of charged molecules under an electric field. Since DNA is negatively charged, it will migrate towards the positive electrode (anode) when an electric current is applied. The smaller fragments will move faster and farther than the larger ones, creating a band pattern on the gel.
- Depurination: After electrophoresis, the gel is treated with dilute hydrochloric acid (HCl) to partially depurinate the DNA fragments. Depurination is the removal of purine bases (adenine and guanine) from the DNA backbone, resulting in breaks in the DNA strands. This step facilitates the transfer of DNA fragments from the gel to the membrane by making them smaller and more flexible.
- Denaturation: The gel is then soaked in an alkaline solution, such as sodium hydroxide (NaOH), to denature the DNA fragments. Denaturation is the process of converting double-stranded DNA into single-stranded DNA by disrupting the hydrogen bonds between the complementary bases. This step makes the DNA fragments available for hybridization with the probe.
- Blotting: The denatured DNA fragments are then transferred from the gel to a carrier membrane, such as nitrocellulose or nylon, by blotting. Blotting is done by placing the gel on top of a buffer-saturated filter paper, then laying the membrane on top of the gel, and finally placing some dry filter papers on top of the membrane. The buffer solution moves up through the layers by capillary action, carrying along the DNA fragments that bind to the membrane.
- Baking: The membrane is then removed from the blotting stack and baked in an oven or exposed to ultraviolet radiation to permanently fix the DNA fragments onto the membrane. This step prevents the loss or displacement of the DNA fragments during subsequent steps.
- Hybridization: The membrane is then incubated with a hybridization probe, which is a single-stranded DNA fragment that has a specific sequence complementary to the target DNA fragment. The probe is labeled with a detectable marker, such as a radioactive isotope, a fluorescent dye, or a chromogenic enzyme. The probe binds to its complementary target DNA fragment on the membrane by forming hydrogen bonds, resulting in a hybridization complex.
- Washing of unbound probes: After hybridization, the membrane is washed with a buffer solution to remove any excess or nonspecifically bound probes from the membrane. This step reduces background noise and increases signal specificity.
- Autoradiograph: The hybridized regions on the membrane are detected by autoradiography, which is a technique that uses photographic film to capture radiation emitted by radioactive probes or fluorescence emitted by fluorescent probes. Alternatively, colorimetric detection methods can be used to visualize chromogenic probes that produce a color change when exposed to a substrate. The resulting image shows the location and intensity of hybridization signals on the membrane, corresponding to the presence and abundance of target DNA fragments in the sample.
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