Southern Blot- Definition, Principle, Steps, Results, Applications

Southern blotting is a molecular biology technique that allows the detection and quantification of specific DNA sequences in complex DNA samples. The technique involves the transfer of DNA fragments that are separated by electrophoresis onto a membrane, where they are immobilized and hybridized with a labeled probe that recognizes the target sequence. The probe can be detected by various methods, such as radioactivity, fluorescence, or chemiluminescence, depending on the type of label used. Southern blotting can be used for various applications, such as gene discovery, mapping, evolution, and diagnosis.

The technique was named after its inventor, Edwin Southern, a British biologist who developed it at Edinburgh University in the 1970s. He first published the method in 1975, after combining three innovations: the use of restriction enzymes to cut DNA at specific sequences, the use of gel electrophoresis to separate DNA fragments by size, and the use of blotting methods to transfer RNA molecules to a membrane. He later modified the technique to transfer DNA molecules instead of RNA. The technique was widely adopted by other researchers and gave rise to other blotting methods that use RNA (northern blotting) or protein (western blotting) as targets.

Southern blotting is based on the principle of transferring DNA fragments that are separated by electrophoresis onto a membrane for immobilization and identification. The process involves the following steps:

• Restriction digestion: The DNA sample to be analyzed is cut into smaller fragments by using specific enzymes called restriction endonucleases. These enzymes recognize and cleave DNA at specific sequences, creating fragments of different sizes and shapes.
• Electrophoresis: The digested DNA fragments are loaded onto a gel matrix, usually made of agarose, and subjected to an electric field. The electric field causes the DNA fragments to migrate through the gel according to their size and charge. Smaller and more negatively charged fragments move faster and farther than larger and less negatively charged ones. This results in the separation of DNA fragments into distinct bands on the gel.
• Denaturation: The DNA fragments on the gel are treated with an alkaline solution, such as sodium hydroxide, to break the hydrogen bonds between the complementary strands of DNA. This converts the double-stranded DNA into single-stranded DNA, which is necessary for the next step of hybridization.
• Blotting: The denatured DNA fragments on the gel are transferred to a solid support, such as a nylon or nitrocellulose membrane, by using a blotting technique. There are different methods of blotting, such as capillary blotting, vacuum blotting, and electroblotting. The most common method is capillary blotting, which uses capillary action to draw the liquid containing the DNA fragments from the gel onto the membrane. The membrane is then placed on a stack of absorbent paper soaked with a salt solution, such as sodium chloride or sodium citrate (SSC), which helps in the transfer of DNA fragments.
• Immobilization: The DNA fragments on the membrane are fixed or immobilized on the surface by using heat or ultraviolet radiation. This prevents the DNA fragments from washing off or moving during the subsequent steps of hybridization and detection.
• Hybridization: The immobilized DNA fragments on the membrane are exposed to a probe, which is a single-stranded DNA or RNA molecule that has a complementary sequence to the target DNA fragment. The probe is labeled with a detectable marker, such as a radioactive isotope, a fluorescent dye, or a chemiluminescent substance. The probe binds to the target DNA fragment by forming hydrogen bonds between the complementary bases, resulting in a hybridization reaction. The hybridization reaction is specific and selective, meaning that only probes that have a perfect or near-perfect match with the target DNA fragment will bind to it.
• Detection: The hybridized regions on the membrane are detected by using a method that depends on the type of label used for the probe. For example, if the probe is labeled with a radioactive isotope, such as 32P or 125I, then the membrane is placed in contact with a photographic film and exposed to X-rays. The radioactive decay of the probe emits radiation that exposes the film, creating an image of the hybridized regions on the membrane. This image is called an autoradiogram. If the probe is labeled with a fluorescent dye, such as fluorescein or rhodamine, then the membrane is scanned with a laser beam that excites the dye molecules and causes them to emit light. The light signals are captured by a detector and converted into an image of the hybridized regions on the membrane. This image is called a fluorogram. If the probe is labeled with a chemiluminescent substance, such as luminol or acridinium ester, then the membrane is incubated with a substrate that reacts with the substance and produces light. The light signals are detected by a camera or a photomultiplier tube and converted into an image of the hybridized regions on the membrane. This image is called a chemilumogram.

The images obtained from these detection methods show the position and size of the target DNA fragments on the membrane as bands or spots. These images can be used to analyze various aspects of the DNA sample, such as its molecular weight, copy number, structure, and function.

Southern blotting is a technique that requires various equipment, materials and solutions to perform the steps of DNA transfer and detection. The following is a list of some of the common requirements for southern blotting:

Equipment

• Water bath: A water bath is used to incubate the DNA samples with restriction enzymes and to heat the samples after digestion to inactivate the enzymes.
• Agarose gel: An agarose gel is used to separate the DNA fragments by size by electrophoresis. The gel is prepared by dissolving agarose powder in a buffer solution and pouring it into a mold with a comb to form wells for loading the samples.
• Power supply: A power supply is used to provide an electric current to the electrophoresis tank. The current causes the negatively charged DNA molecules to migrate towards the positive electrode through the gel matrix.
• UV radiation: A UV radiation source is used to visualize the DNA bands on the gel after electrophoresis. The DNA molecules are stained with ethidium bromide, which fluoresces under UV light.
• Hybridization oven: A hybridization oven is used to incubate the membrane with the labeled probe under controlled temperature and humidity conditions. The oven ensures uniform hybridization of the probe to the target DNA sequences on the membrane.
• Hybridization bottles: Hybridization bottles are used to hold the membrane and the hybridization solution during incubation in the oven. The bottles are designed to minimize the volume of the solution and prevent evaporation.
• Trays: Trays are used to hold the gel and the membrane during denaturation, neutralization and blotting steps. The trays are filled with appropriate buffers and solutions for each step.
• Film processor: A film processor is used to develop the photographic film that is exposed to the membrane after hybridization. The film processor reveals the bands corresponding to the hybridized regions on the membrane.
• Pipettes: Pipettes are used to transfer liquids and solutions during various steps of southern blotting. Pipettes can be manual or automatic, depending on the volume and accuracy required.
• Centrifuge tubes: Centrifuge tubes are used to store and mix the DNA samples, restriction enzymes, buffers and other reagents. Centrifuge tubes can be spun at high speed to separate different phases or components of a mixture.
• Glass plate: A glass plate is used to support the blotting structure during DNA transfer. The glass plate is placed on top of a stack of filter papers, gel, membrane and more filter papers. The glass plate provides weight and pressure to facilitate DNA transfer from the gel to the membrane.
• Whatman 3 mm chromatography paper: Whatman 3 mm chromatography paper is used as a filter paper during blotting. The paper absorbs excess buffer and solution from the gel and transfers it to a sponge soaked in SSC (saline-sodium citrate) buffer. The paper also helps in maintaining contact between the gel and the membrane.
• Nylon membrane/ Nitrocellulose membrane: A nylon membrane or a nitrocellulose membrane is used as a solid support for immobilizing the DNA fragments transferred from the gel. The membrane has a high affinity for nucleic acids and binds them covalently or non-covalently depending on the type of membrane.
• Syringe: A syringe is used to inject liquid or air into a system or container. A syringe can be used to apply pressure or vacuum to a system or container, such as a hybridization bottle or a cellulose acetate membrane.
• Cellulose acetate membrane: A cellulose acetate membrane is used as a filter for removing air bubbles from hybridization solutions. The membrane is attached to a syringe and inserted into a hybridization bottle. The syringe is then pulled back to create a vacuum that draws out air bubbles from the solution.

Materials

• Restriction enzymes: Restriction enzymes are enzymes that cut DNA molecules at specific sequences called restriction sites. Restriction enzymes are used to digest genomic DNA into smaller fragments that can be separated by electrophoresis and analyzed by southern blotting.
• Restriction enzyme buffer: Restriction enzyme buffer is a buffer solution that provides optimal conditions for restriction enzyme activity. The buffer contains salt, pH stabilizers, cofactors and inhibitors that affect enzyme activity and specificity.
• Agarose: Agarose is a polysaccharide derived from seaweed that forms a gel when dissolved in hot water and cooled. Agarose gel is used as a medium for electrophoresis of DNA fragments. Agarose gel has different pore sizes depending on the concentration of agarose, which affects the resolution of DNA fragments.
• TBE buffer: TBE buffer is a buffer solution that contains Tris, boric acid and EDTA. TBE buffer is used as a running buffer for electrophoresis of DNA fragments. TBE buffer maintains a constant pH and electric conductivity during electrophoresis and prevents DNA degradation by chelating metal ions.
• DNA loading buffer: DNA loading buffer is a solution that contains dye, glycerol and EDTA. DNA loading buffer is added to the DNA samples before loading them on the gel. The dye helps in visualizing the samples during loading and electrophoresis. The glycerol increases the density of the samples and helps them sink into the wells. The EDTA prevents DNA degradation by chelating metal ions.
• Tris base: Tris base is a chemical compound that acts as a pH buffer. Tris base is used to prepare various buffers and solutions for southern blotting, such as neutralization buffer and detection buffer.
• Sodium chloride: Sodium chloride is a common salt that dissolves in water to form sodium and chloride ions. Sodium chloride is used to prepare various buffers and solutions for southern blotting, such as denaturation buffer, SSC and detection buffer.
• Sodium hydroxide: Sodium hydroxide is a strong base that dissolves in water to form sodium and hydroxide ions. Sodium hydroxide is used to prepare denaturation buffer for southern blotting. Denaturation buffer contains sodium hydroxide and sodium chloride and is used to break the hydrogen bonds between the two strands of DNA on the gel.
• Sodium citrate: Sodium citrate is a salt of citric acid that dissolves in water to form sodium and citrate ions. Sodium citrate is used to prepare SSC for southern blotting. SSC contains sodium chloride and sodium citrate and is used as a transfer buffer and a washing buffer for southern blotting. SSC helps in maintaining the stability and specificity of hybridization reactions.
• DNA labeling kit: A DNA labeling kit is a kit that contains reagents and instructions for labeling DNA probes with radioactive or non-radioactive components. DNA probes are short segments of DNA that are complementary to the target DNA sequences on the membrane. DNA probes are labeled so that they can be detected by autoradiography or other methods after hybridization.
• Nucleic acid detection kit: A nucleic acid detection kit is a kit that contains reagents and instructions for detecting nucleic acids on a membrane after hybridization. The kit may use different methods of detection, such as colorimetric, chemiluminescent or fluorescent methods. The kit may also include blocking agents, washing buffers, substrates and enhancers to improve the sensitivity and specificity of detection.
• Sodium dodecyl sulfate (SDS): SDS is an anionic detergent that dissolves in water to form micelles. SDS is used to prepare hybridization solution for southern blotting. Hybridization solution contains SDS, formamide, labeled probe and other components that facilitate hybridization reactions. SDS helps in reducing the secondary structure of DNA molecules and increasing the accessibility of target sequences on the membrane.
• Polyvinylpyrrolidone: Polyvinylpyrrolidone is a synthetic polymer that binds nonspecifically to proteins and nucleic acids. Polyvinylpyrrolidone is used as a blocking agent for southern blotting. Blocking agents are added to the membrane before hybridization to prevent nonspecific binding of probes or detection reagents to the membrane.
• Bovine serum albumin: Bovine serum albumin is a protein derived from cow blood that binds nonspecifically to proteins and nucleic acids. Bovine serum albumin is used as a blocking agent for southern blotting. Blocking agents are added to the membrane before hybridization to prevent nonspecific binding of probes or detection reagents to the membrane.
• Formamide: Formamide is a solvent that dissolves in water to form formamidium and hydroxide ions. Formamide is used to prepare hybridization solution for southern blotting. Hybridization solution contains formamide, SDS, labeled probe and other components that facilitate hybridization reactions. Formamide helps in lowering the melting temperature of DNA molecules and increasing the rate of hybridization.
• Phenol: Phenol is an organic compound that acts as an antiseptic and a preservative. Phenol is used to store DNA probes after labeling for southern blotting. Phenol prevents bacterial growth and degradation of labeled probes.

Solutions and Buffers

• Denaturation buffer: Denaturation buffer is a solution that contains sodium hydroxide and sodium chloride in the ratio 1:6. Denaturation buffer is used to denature the DNA molecules on the gel after electrophoresis. Denaturation buffer breaks the hydrogen bonds between the two strands

Southern blotting is a technique that involves transferring DNA fragments from an agarose gel to a membrane, where they can be detected by hybridization with a specific probe. The procedure consists of the following steps:

• Restriction digestion of DNA: The DNA sample is cut into smaller fragments by using restriction enzymes that recognize specific sequences and cleave the DNA at those sites. The restriction digestion is performed in a microcentrifuge tube and incubated overnight at 37°C. The digested DNA is then mixed with a loading buffer and loaded onto an agarose gel for electrophoresis .
• Electrophoresis: The agarose gel is prepared by dissolving agarose powder in a buffer solution and pouring it into a gel cast with a comb that creates wells for the samples. The gel is allowed to solidify and then placed in an electrophoresis tank filled with running buffer. The DNA samples are pipetted into the wells and a voltage is applied across the gel. The DNA fragments migrate through the gel according to their size, with smaller fragments moving faster than larger ones. The gel is stained with ethidium bromide, which binds to DNA and fluoresces under UV light, allowing the visualization of the DNA bands .
• Denaturation: The DNA fragments in the gel are denatured by soaking the gel in an alkaline solution of sodium hydroxide and sodium chloride. This breaks the hydrogen bonds between the complementary strands of DNA and converts them into single-stranded molecules. The denaturation step is necessary for the transfer of DNA to the membrane and for the hybridization with the probe .
• Blotting: The denatured DNA fragments are transferred from the gel to a membrane, such as nitrocellulose or nylon, by capillary action or vacuum blotting. A blotting apparatus is set up by placing a sponge soaked in transfer buffer at the bottom of a glass dish, followed by layers of filter paper, the gel, the membrane, more filter paper, and a glass plate with a weight on top. The transfer buffer (usually SSC) moves up through the layers by capillary action and carries the DNA fragments along with it. The DNA fragments bind to the membrane according to their position on the gel. Alternatively, a vacuum blot apparatus can be used to suck the transfer buffer through the gel and membrane, which speeds up the process .
• Baking/Immobilization: The membrane with the transferred DNA fragments is removed from the blotting apparatus and baked in an oven at 80°C for 2-3 hours or exposed to UV light. This cross-links the DNA molecules to the membrane and prevents them from washing off during hybridization .
• Hybridization: The membrane is exposed to a labeled probe that is complementary to the target sequence of interest. The probe can be labeled with radioactivity, fluorescence, or chemiluminescence. The hybridization step involves incubating the membrane with the probe in a hybridization buffer that contains ingredients such as formamide, SDS, BSA, and polyvinylpyrrolidone to enhance specificity and reduce background noise. The hybridization conditions depend on the type and length of the probe and can vary from several hours to overnight .
• Detection: After hybridization, the membrane is washed with a detection buffer that removes any unbound or nonspecifically bound probe molecules. The hybridized regions on the membrane are then detected by autoradiography (for radioactive probes), fluorescence imaging (for fluorescent probes), or chemiluminescence assay (for chemiluminescent probes). The detection method produces an image of bands on the membrane that correspond to the position and size of the target DNA fragments .

The results of a Southern blot are observed in the form of bands on the membrane. The size of the DNA fragments can be determined by comparing their relative size with the DNA bands of known lengths. The presence or absence of a band indicates the presence or absence of a specific DNA sequence in the sample. The intensity of the band reflects the amount of DNA that hybridized with the probe.

Proper interpretation of Southern blots necessitates the use of controls throughout the procedure. In order to compare the gene copy number between one sample and another, it is imperative that the same quantity of DNA be loaded onto the gel for all samples being compared. A loading control, such as a housekeeping gene, can be used to verify that equal amounts of DNA were loaded. A positive control, such as a known DNA sample containing the target sequence, can be used to confirm that the probe hybridized correctly. A negative control, such as a DNA sample without the target sequence, can be used to rule out non-specific binding or cross-hybridization.

Some examples of Southern blot applications and their result interpretations are:

• DNA fingerprinting: Southern blotting can be used to identify individuals based on their unique DNA patterns. The DNA samples are digested with restriction enzymes that cut at specific sites and generate different-sized fragments for each individual. The fragments are separated by electrophoresis and transferred to a membrane. The membrane is probed with a radioactive or fluorescent DNA probe that binds to a highly variable region of DNA, such as a microsatellite or a minisatellite. The resulting bands on the membrane represent the alleles of the individual at that locus. By comparing the band patterns of different individuals, one can determine their genetic relatedness or identity.
• Gene mapping: Southern blotting can be used to locate a gene on a chromosome by using DNA probes that are linked to the gene of interest. The DNA samples are digested with restriction enzymes that cut at different sites along the chromosome and generate different-sized fragments containing the gene. The fragments are separated by electrophoresis and transferred to a membrane. The membrane is probed with a labeled DNA probe that binds to the gene of interest. The resulting band on the membrane indicates the size and position of the fragment containing the gene. By comparing the band patterns of different individuals or populations, one can determine the genetic variation or linkage of the gene.
• Disease diagnosis: Southern blotting can be used to detect mutations or alterations in genes that are associated with diseases. The DNA samples are digested with restriction enzymes that cut at specific sites near or within the gene of interest. The fragments are separated by electrophoresis and transferred to a membrane. The membrane is probed with a labeled DNA probe that binds to the normal or mutated version of the gene. The resulting bands on the membrane indicate the presence or absence of the mutation or alteration in the gene. For example, Southern blotting can be used to diagnose sickle cell anemia by detecting a single base pair substitution in the beta-globin gene.

Southern blotting is a powerful technique that has many applications in the field of molecular biology, genetics and biotechnology. Some of the major applications are:

• Gene discovery and mapping: Southern blotting can be used to identify and locate specific genes or DNA sequences in a genome. For example, the technique can be used to map restriction fragment length polymorphisms (RFLPs), which are variations in the length of DNA fragments generated by restriction enzymes. RFLPs can serve as genetic markers that can help in constructing genetic maps and identifying genes associated with certain traits or diseases.
• Evolutionary studies: Southern blotting can be used to compare the DNA sequences of different species or populations and study their evolutionary relationships. For example, the technique can be used to compare the DNA sequences of mitochondrial DNA (mtDNA) or ribosomal RNA (rRNA) genes, which are highly conserved and can provide information about the phylogenetic history and divergence of organisms.
• Diagnostic studies: Southern blotting can be used to diagnose various genetic diseases and disorders by detecting mutations or abnormalities in the DNA sequences. For example, the technique can be used to detect deletions, insertions, duplications, inversions or translocations in the DNA that can cause diseases such as sickle cell anemia, cystic fibrosis, hemophilia or cancer. The technique can also be used to detect viral or bacterial infections by hybridizing the DNA samples with specific probes that target the pathogen`s DNA.
• Personal identification: Southern blotting can be used for personal identification by analyzing the DNA fingerprints of individuals. DNA fingerprints are unique patterns of DNA bands that are generated by restriction digestion and hybridization with probes that target variable regions of the DNA. The technique can be used for forensic purposes, such as identifying suspects or victims of crimes, as well as for paternity testing or kinship analysis.

Southern blotting is a powerful technique for DNA analysis, but it also has some limitations that should be considered before using it. Some of the limitations are:

• Costly and complex process: Southern blotting requires expensive equipment and reagents, such as restriction enzymes, labeled probes, membranes, and hybridization ovens. It also involves multiple steps that require careful handling and optimization. The process is labor-intensive and requires trained personnel to perform it.
• Time-consuming process: Southern blotting can take several days to complete, depending on the size and complexity of the DNA sample. It can be replaced by other faster processes, such as polymerase chain reaction (PCR), which can amplify and detect specific DNA sequences in a few hours.
• Semi-quantitative process: Southern blotting only provides estimated sizing of the DNA fragments by comparing them with DNA markers of known lengths. It does not provide accurate quantification of the DNA amount or copy number. It also does not detect mutations at the base-pair level, which may affect the gene function or expression.
• Large and high-quality DNA sample required: Southern blotting requires a large amount of DNA sample (about 10 µg) to obtain clear and reliable results. The DNA sample also needs to be of high quality and purity, which may require superior isolation methods. The DNA sample may degrade or contaminate during the process, affecting the results.

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