Southern Blotting – Principle, Procedure, Results, Applications

Southern blotting was developed by Edwin M. Southern, who published the original method in 1975. The technique was named after his surname “Southern”, not after any geographical direction.

In molecular biology, deoxyribonucleic acid (DNA) can be cut into smaller fragments by using restriction enzymes. These enzymes cut the DNA at specific recognition sites and produce fragments of different lengths. They are known as restriction fragments.

The DNA fragments are separated according to their size by agarose gel electrophoresis. Smaller DNA fragments move faster and travel farther through the agarose gel than the larger fragments.

What is Southern Blotting?

Southern blotting is a molecular biology technique used for detecting specific deoxyribonucleic acid (DNA) sequences by transferring electrophoretically separated DNA fragments onto a membrane and using complementary, labelled nucleic acid probes.

The membrane is usually made up of nylon or nitrocellulose. The separated DNA fragments are immobilized on the membrane, and their relative positions remain the same as obtained after electrophoresis.

This membrane containing the immobilized DNA fragments is referred to as a Southern blot.

Southern blotting is specific for the detection of DNA, not ribonucleic acid (RNA) or proteins. However, the labelled probe used in this technique may be DNA or RNA. For the detection of RNA, northern blotting is used. Proteins are detected by western blotting.

Purpose of Southern Blotting

The main objectives of Southern blotting are as follows-

1. To detect the presence or absence of a specific deoxyribonucleic acid (DNA) sequence in a sample.
2. To determine the size of DNA fragments containing the target sequence.
3. To identify restriction fragment length polymorphisms (RFLPs) by comparing the DNA fragment patterns of different samples.
4. To detect changes in DNA sequences, such as deletions, insertions, and gene rearrangements that alter the restriction fragment patterns.
5. To estimate the number of copies of a particular gene present in the genome.
6. To confirm the integration of foreign DNA into the host genome and examine the number of integration sites.
7. To study DNA methylation patterns using methylation-sensitive restriction enzymes.

Principle of Southern Blotting

The principle of Southern blotting is based on the separation of deoxyribonucleic acid (DNA) fragments according to their size and the binding of a labelled nucleic acid probe to its complementary DNA sequence. This binding is referred to as nucleic acid hybridization.

DNA Fragment Separation and Membrane Transfer

In Southern blotting, restriction-digested DNA fragments are separated by agarose gel electrophoresis. The smaller DNA fragments move faster through the gel than the larger fragments. The fragments of different lengths occupy different positions in the gel.

The double-stranded DNA is denatured into single strands by alkaline treatment, which disrupts the hydrogen bonds between the two complementary strands. These single-stranded DNA fragments are then transferred from the gel onto a nylon or nitrocellulose membrane, usually by capillary action of the buffer. The relative positions of the DNA fragments remain unchanged during transfer.

The DNA fragments are immobilized on the membrane so that they remain attached during hybridization.

Probe Hybridization and Signal Detection

The immobilized DNA fragments are hybridized with a labelled, single-stranded DNA or ribonucleic acid (RNA) probe having a sequence complementary to the target DNA. The probe binds to the complementary region of the DNA through base pairing. Hydrogen bonds are formed between the complementary bases of the probe and target DNA, resulting in the formation of a stable probe-target hybrid.

The specificity of hybridization depends on the stringency conditions, mainly temperature and salt concentration. At higher stringency, such as higher temperature or lower salt concentration, the probes having mismatched sequences bind less strongly and may separate from the target DNA. The probes having sufficient complementary base pairing remain bound under suitable conditions. Unbound and weakly bound probes are removed during washing.

The nucleic acid probes may be labelled with radioactive isotopes or nonradioactive markers. When the labelled probe remains attached to the target DNA, its signal can be detected by autoradiography, chemiluminescence or other suitable detection methods, depending on the label used.

The position of the detected signal on the membrane indicates the DNA fragment containing the complementary target sequence.

Requirements for Southern Blotting

The following equipment, materials, reagents, and buffer solutions are required for Southern blotting.

Equipment

1. Gel electrophoresis apparatus- A horizontal gel electrophoresis unit with a power supply is used to separate DNA fragments according to their size.
2. Gel casting equipment- Gel casting tray, combs, and other accessories are required for preparing the agarose gel.
3. Micropipettes and laboratory equipment- Micropipettes, sterile tips, microcentrifuge tubes, and a water bath or heating block are used for sample preparation and restriction digestion.
4. Blotting apparatus- A capillary blotting setup containing a buffer reservoir, support platform, and wick arrangement is commonly used for transferring DNA fragments onto the membrane. Vacuum or electrophoretic transfer systems may also be used.
5. Hybridization oven or water bath- A temperature-controlled hybridization oven with rotating tubes or a suitable water bath is used for probe hybridization. A shaking platform may be required for membrane washing.
6. UV crosslinker or vacuum oven- These are used for immobilizing the DNA fragments on the membrane. UV crosslinking is commonly used for nylon membranes, whereas baking may be used for nitrocellulose membranes. Additional fixation is not always required after alkaline transfer onto positively charged nylon membranes.
7. Detection system- An autoradiography cassette with X-ray film or a phosphorimager is used for radioactive probes. Chemiluminescent imaging systems or suitable colour detection equipment are used for nonradioactive probes.

Materials and Reagents

1. DNA sample- The sample contains the deoxyribonucleic acid (DNA) sequences that need to be analysed. Genomic DNA is commonly used for Southern blotting.
2. Restriction enzymes- These enzymes are used to cut DNA at specific recognition sites, producing fragments of different lengths.
3. Agarose- Agarose is used for preparing the gel required for separation of DNA fragments.
4. DNA size markers and loading dye- DNA molecular weight markers are used to estimate fragment sizes, while loading dye is added to the samples before electrophoresis. A suitable nucleic acid stain may be used for visualizing the separated fragments.
5. Blotting membrane- Nylon or nitrocellulose membranes are used for transferring and immobilizing the DNA fragments. Positively charged nylon membranes are commonly preferred for alkaline transfer.
6. Filter papers and absorbent papers- Filter papers are used in the blotting arrangement to maintain the movement of buffer. Paper towels or other absorbent materials draw the buffer through the gel and membrane during capillary transfer.
7. Labelled nucleic acid probes- These are single-stranded DNA or ribonucleic acid (RNA) probes having complementary sequences to the target DNA. The probes may be labelled with radioactive isotopes (e.g., phosphorus-32) or nonradioactive markers such as digoxigenin (DIG) and biotin.
8. Blocking reagents- Denhardt’s reagent, denatured salmon sperm DNA or other suitable blocking agents may be used to reduce nonspecific binding during hybridization. The blocking reagent depends on the hybridization system.
9. Detection reagents- Nonradioactive detection may require specific antibodies or binding reagents and their substrates. For example, DIG-labelled probes can be detected using an anti-DIG antibody conjugated with alkaline phosphatase and a suitable chemiluminescent or chromogenic substrate.

Solutions and Buffers

1. Restriction digestion buffer- It provides the required pH, salts, and other conditions for restriction enzyme activity. The composition depends on the restriction enzyme used.
2. Electrophoresis buffer- Tris-acetate-EDTA (TAE) or Tris-borate-EDTA (TBE) buffer is used during agarose gel electrophoresis. These buffers maintain suitable pH and provide ions for the movement of DNA through the gel.
3. Denaturation solution- An alkaline solution containing sodium hydroxide (NaOH), commonly with sodium chloride (NaCl), is used to separate double-stranded DNA into single strands.
4. Neutralization buffer- A solution containing Tris and NaCl is commonly used to neutralize the alkaline-treated gel before transfer in neutral buffer. It is not required in the same manner for alkaline transfer protocols.
5. Transfer buffer- Saline-sodium citrate (SSC) or saline-sodium phosphate-EDTA (SSPE) buffer is used for transferring DNA from the gel to the membrane under neutral transfer conditions. In alkaline transfer, an NaOH-containing solution is used instead, particularly with positively charged nylon membranes.
6. Prehybridization and hybridization buffer- These solutions provide suitable conditions for binding of the labelled probe to the target DNA. Depending on the protocol, the buffer may contain SSC or SSPE, sodium dodecyl sulfate (SDS), Denhardt’s reagent, denatured carrier DNA, and formamide. The prehybridization solution is used before the labelled probe is added.
7. Washing buffer- SSC or SSPE solutions, often containing SDS, are used to remove unbound and weakly bound probes from the membrane. The salt concentration and washing temperature are adjusted according to the required hybridization stringency.
8. Depurination solution- A dilute hydrochloric acid (HCl) solution may be used before denaturation when large DNA fragments are present. It is an optional treatment used to improve the transfer of high-molecular-weight DNA fragments.

Procedure of Southern Blotting

Southern blotting involves the separation of DNA fragments by agarose gel electrophoresis, transfer of DNA onto a membrane and detection of specific DNA sequences using labelled nucleic acid probes. The procedure is carried out in the following steps-

Step 1 – DNA Isolation and Restriction Digestion

1. At first, the deoxyribonucleic acid (DNA) is isolated from the biological sample using a suitable DNA extraction method. The isolated DNA must be free from proteins and other contaminants that may interfere with restriction digestion.
2. Take a suitable amount of the isolated DNA and add the selected restriction endonucleases along with their appropriate reaction buffer. These enzymes cut the DNA at specific recognition sites and produce fragments of different lengths. The restriction enzyme and digestion conditions are selected depending on the target DNA sequence.

Step 2 – Agarose Gel Electrophoresis

1. Prepare an agarose gel of suitable concentration using an electrophoresis buffer. The restriction-digested DNA is mixed with loading dye and loaded into the wells of the gel. A DNA size marker is also loaded in a separate well to estimate the sizes of the DNA fragments.
2. The gel is subjected to electrophoresis by applying an electric field. DNA fragments move towards the positive electrode due to the presence of negatively charged phosphate groups. Smaller DNA fragments move faster and travel farther through the agarose gel than the larger fragments.
3. After electrophoresis, the separated DNA fragments may be visualized using a suitable nucleic acid stain. The band pattern is recorded before transfer.

Step 3 – Gel Treatment and DNA Denaturation

1. In the case of very large DNA fragments (approximately 15–20 kilobases or more), the gel may be treated briefly with dilute hydrochloric acid for depurination. This treatment makes the large DNA fragments more susceptible to strand cleavage during subsequent alkaline treatment, which facilitates their transfer. This step is optional.
2. The agarose gel is then treated with an alkaline denaturation solution containing sodium hydroxide (NaOH). The alkali breaks the hydrogen bonds between the two complementary strands of DNA, converting the double-stranded DNA into single strands. These single-stranded DNA fragments are required for hybridization with the complementary probe.
3. After denaturation, the gel is neutralized using a suitable neutralization buffer if a neutral transfer method is followed. Neutralization is generally not required before alkaline transfer onto positively charged nylon membranes.

Step 4 – Transfer of DNA onto a Membrane

1. Take a suitable nylon or nitrocellulose membrane and prepare it according to the selected transfer method. For conventional capillary blotting, a filter paper wick is arranged over a support, with its two ends dipped into the transfer buffer.
2. The treated agarose gel is placed over the wet filter paper wick. Now place the prepared membrane directly on the upper surface of the gel, followed by wet filter papers and a stack of absorbent paper towels. Air bubbles between the gel and membrane must be removed, as they may interfere with DNA transfer.
3. The absorbent papers draw the buffer upward through the gel and membrane by capillary action. During this process, the single-stranded DNA fragments move from the gel onto the membrane and become attached to its surface. The relative positions of the DNA fragments remain unchanged. Transfer may require several hours or overnight depending on the fragment sizes and transfer method.
4. DNA transfer can also be carried out by vacuum blotting or electroblotting using suitable equipment. For neutral transfer, saline-sodium citrate (SSC) or saline-sodium phosphate-EDTA (SSPE) buffer is commonly used, whereas alkaline transfer is performed using an alkaline solution.

Step 5 – DNA Immobilization and Probe Hybridization

1. After completion of transfer, carefully remove the membrane from the gel and mark the positions of the sample lanes. The DNA fragments are immobilized on the membrane using a suitable fixation method, such as ultraviolet (UV) crosslinking or baking. Additional fixation is generally not required when alkaline transfer is performed onto positively charged nylon membranes.
2. The membrane is incubated in a prehybridization solution containing suitable blocking reagents, when required. It reduces the nonspecific binding of the labelled probe to the membrane.
3. Now add the labelled, single-stranded DNA or ribonucleic acid (RNA) probe to the hybridization solution and incubate the membrane under suitable conditions. The probe binds to the complementary sequence of the immobilized DNA through base pairing. This forms a stable probe-target hybrid. The incubation temperature and salt concentration depend on the sequence and length of the probe and the detection system.
4. The nucleic acid probe may be labelled with radioactive isotopes (e.g., phosphorus-32) or nonradioactive markers such as digoxigenin (DIG), biotin, or fluorescent dyes.

Step 6 – Washing and Detection

1. After hybridization, wash the membrane with suitable washing buffers to remove the unbound and weakly bound probes. The washing temperature and salt concentration are adjusted according to the required stringency. Probes having sufficient complementarity with the target DNA remain attached to the membrane.
2. For radioactive probes, the membrane is exposed to X-ray film or a phosphorimaging screen for detecting the radioactive signal. Nonradioactive probes are detected using suitable detection methods, such as chemiluminescence or fluorescence. Some nonradioactive detection systems require additional binding reagents and substrates before detecting the signal.
3. The detected signal appears as a band at the position of the DNA fragment containing the complementary target sequence. The approximate size of this fragment can be estimated by comparing its position with the DNA size marker.

Results and Interpretation of Southern Blotting

The result of Southern blotting is observed from the bands produced after the labelled probe binds with complementary deoxyribonucleic acid (DNA) fragments on the membrane. With a radioactive probe, the bands are detected on an autoradiograph. Chemiluminescent, fluorescent or coloured signals are obtained with the respective nonradioactive detection systems.

The band position, number and intensity are then compared with the DNA ladder and control samples.

Interpretation of DNA Band Patterns

1. Positive Result- A distinct band is considered as a positive result. It indicates that a DNA fragment containing sequence complementary to the probe is detected in that sample.
2. Negative Result- No detectable band is observed. The target sequence may be absent. It may also be present but not detected due to an experimental problem, so the controls are checked before reporting a true negative result.
3. Fragment Size- The position of the band is compared with the DNA size marker (ladder). Smaller DNA fragments move farther in the gel and larger fragments remain nearer to the upper region. From this position, the approximate fragment length is estimated in base pairs (bp) or kilobases (kb).
4. Single or Multiple Bands- Sometimes only one probe-reactive band is present. More than one band may also be obtained when complementary sequences occur in DNA fragments of different sizes, due to related sequences, different restriction fragments or variation in the DNA. The number of bands alone cannot be taken as the exact gene copy number.
5. Altered Band Pattern- A sample may show a band at a different position or may contain an additional or missing band compared to the expected pattern. Such changes can occur with restriction-site variation, DNA insertion, deletion or rearrangement. The restriction enzyme, probe sequence and expected fragment pattern must be considered before assigning the cause.
6. Signal Intensity- A strong band generally contains a greater hybridization signal and a faint band gives a weaker signal. Under properly controlled conditions, the intensity can be used for relative comparison of DNA abundance or copy number. Signal intensity alone is not an exact measurement of gene copy number.

Example of Southern Blot Band Pattern

Suppose all samples are digested with the same restriction enzyme and hybridized with the same probe.

Lane M is used for estimating the fragment sizes.

Lane A gives a band at approximately 5 kb, similar to the positive control. In lane B, two probe-reactive bands are observed at about 5 kb and 7 kb. It contains two detectable fragment-size classes complementary to the probe. The reason for the extra 7 kb band cannot be established from this pattern alone.

No band is present in lane C. It may represent a negative result, but only when the sample and the experimental controls are satisfactory.

Controls and Common Interpretation Errors

1. Positive Control- The positive control contains the target sequence and should give its expected band. If this band is absent, failure may have occurred during restriction digestion, DNA transfer, hybridization or detection.
2. Negative Control- No target-specific band should occur in the negative control. A visible signal may be due to nonspecific probe binding, contamination or another experimental error.
3. Incomplete Restriction Digestion- If all required restriction sites are not cut, larger DNA fragments remain and unexpected bands can appear. These bands may be mistaken for an actual change in DNA.
4. Poor DNA Transfer- Weak or missing bands can occur when DNA is not transferred efficiently from the gel to the membrane. Large fragments may transfer less efficiently. Their signals can become faint or disappear.
5. Nonspecific Probe Binding- Extra bands or high background may occur when the probe binds with partially complementary DNA sequences. Unsuitable hybridization conditions or insufficient washing can produce such signals.
6. Weak Signal- A faint band may result from a low amount of target DNA, degraded DNA, poor probe labelling or inefficient hybridization. No visible band can also occur for the same reasons. Such result cannot be interpreted as true absence of the target sequence without checking the controls.
7. Band Intensity Error- DNA loading, transfer efficiency, probe binding and signal exposure can change band intensity. Two or more fragments having the same size may also appear as a single band. Relative DNA comparison is made only under suitable controls and within the linear range of detection.

Applications of Southern Blotting

• Used to detect specific deoxyribonucleic acid (DNA) sequences and determine the size of the restriction fragments containing them.
• Used for restriction mapping and studying the organization of genes within the genome.
• Used in restriction fragment length polymorphism (RFLP) analysis for genotyping, genetic mapping and gene identification.
• Used for DNA fingerprinting and paternity analysis based on polymorphic DNA fragments.
• Used to detect changes in DNA such as deletions, insertions, rearrangements and restriction-site variations.
• Used for detecting immunoglobulin gene rearrangements and studying clonality in lymphoproliferative disorders.
• Used to confirm transgene integration and study its copy number, integrity and integration pattern in genetically modified organisms or cells.
• Used for studying DNA methylation with methylation-sensitive restriction enzymes.

Advantages of Southern Blotting

• It has high sequence specificity because only deoxyribonucleic acid (DNA) fragments complementary to the labelled probe are detected.
• It detects the target DNA and also gives the approximate size of the restriction fragment containing it.
• Changes such as insertions, deletions, rearrangements and restriction-site variations can be identified from altered band patterns.
• It is useful for restriction mapping and studying the organization of genes within a genome.
• It can estimate transgene copy number and provide information about insert integrity and independent integration sites.
• The transferred DNA remains immobilized on the membrane and can be hybridized again with another probe after removing the previous probe.

Limitations of Southern Blotting

• It is a time-consuming and labor-intensive technique involving several experimental steps.
• A relatively large amount of good-quality deoxyribonucleic acid (DNA) is generally required.
• Its sensitivity is lower compared to amplification-based methods such as PCR, especially for low-copy target sequences.
• Prior information about the target sequence is required for selecting a suitable probe and restriction enzyme.
• Most single-base changes cannot be detected unless they alter a restriction site or produce a detectable change in the fragment pattern.
• It has low throughput and is less suitable for analyzing a large number of samples.
• Radioactive probe systems require special handling and disposal and also have radiation hazards. Nonradioactive probes can be used instead.
• Incomplete restriction digestion or inefficient DNA transfer can produce additional, weak or missing bands and affect the interpretation.

Southern Blotting vs Related Techniques

Southern blotting is related to several molecular techniques, but they do not examine the same type of molecule or give the same information.

Southern vs Northern vs Western Blotting

Southern and Northern blotting both depend on nucleic acid hybridization. The main difference is the sample. Southern blotting analyzes DNA, whereas Northern blotting analyzes RNA.

Western blotting follows the same general idea of electrophoretic separation followed by transfer to a membrane, but the target is protein. Antibodies are used for the specific detection instead of a complementary nucleic acid probe.

Southern Blotting vs Polymerase Chain Reaction (PCR)

PCR can detect very small amounts of DNA because the target sequence is amplified before detection. Southern blotting does not amplify the target. It detects the DNA already present in the sample.

Southern blotting has another type of information. The target remains associated with a particular restriction fragment, so changes in fragment size or band pattern can be detected. Large insertions, deletions or rearrangements may change this pattern. Conventional PCR generally examines only the region defined by its two primers.

Southern Blotting vs Gel Electrophoresis

Gel electrophoresis separates DNA fragments mainly according to their size. Smaller fragments move farther through the agarose gel than the larger fragments. It does not itself identify which fragment contains a particular DNA sequence.

Southern blotting uses this electrophoretic separation first. The separated fragments are then transferred onto a membrane and a complementary probe is used to detect the required DNA fragment. Gel electrophoresis is therefore one part of the conventional Southern blotting process, not an equivalent sequence-specific detection method.

Southern Blotting and Restriction Fragment Length Polymorphism (RFLP) Analysis

A restriction fragment length polymorphism (RFLP) is a variation in the length of DNA fragments produced after restriction enzyme digestion. It is a DNA variation or molecular marker, not another type of blotting technique.

Southern blotting can be used to detect these different restriction fragments. DNA from different samples is digested, separated, transferred and hybridized with a probe for the required locus. Different fragment lengths then appear as different band positions.

RFLPs detected by Southern blotting were widely used in genetic linkage mapping and DNA fingerprinting. RFLP can also be analyzed by other methods, including PCR-based approaches, depending on the polymorphism being studied.

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