Table of Contents
What is Southern Blotting?
- Southern blotting is a powerful molecular biology technique used for the detection and quantification of specific DNA sequences within DNA samples. Developed by Edward M. Southern in the 1970s, this method involves the transfer of DNA fragments from an electrophoresis gel onto a solid membrane. The transferred DNA fragments become immobilized on the membrane, allowing for further analysis.
- To perform a Southern blot, the DNA from a biological sample is first purified and then digested with restriction enzymes. These enzymes cleave the DNA at specific recognition sites, generating fragments of varying lengths. Next, the DNA fragments are separated by size using gel electrophoresis, a technique that applies an electric current to move the fragments through a gel matrix. Smaller fragments move more quickly than larger ones, resulting in their separation.
- Once the DNA fragments have been separated, they are transferred from the gel to a solid membrane in a process known as blotting. This transfer is typically achieved by applying pressure or using capillary action. The DNA fragments become permanently attached to the membrane, creating a replica of the original gel pattern.
- The membrane, now containing the immobilized DNA fragments, is then exposed to a labeled DNA probe. The probe is a short, single-stranded DNA molecule that is complementary to the target sequence of interest. The probe is labeled with a radioactive, fluorescent, or chemical tag, which allows for the detection of DNA fragments that have hybridized with the probe.
- During hybridization, the labeled probe binds specifically to DNA fragments containing complementary sequences. By visualizing the labeled probe, researchers can identify and quantify the presence of the target DNA sequence within the original DNA sample. This detection can be achieved through autoradiography (for radioactive probes), fluorescence imaging (for fluorescent probes), or chemiluminescence (for chemically labeled probes).
- Southern blotting has a wide range of applications in molecular biology. It is commonly used for gene identification and cloning, as it allows researchers to analyze DNA fragments and obtain the full-length sequence of genes of interest. It is also valuable in studying somatic rearrangements and transgene identification in immunology research. Additionally, Southern blotting can be used to investigate DNA methylation patterns by analyzing specific methylated sites in genes.
- In summary, Southern blotting is a fundamental technique that enables the detection and analysis of specific DNA sequences within complex DNA samples. It has played a significant role in advancing our understanding of genetics and has paved the way for further advancements in molecular biology research.
Southern Blotting Definition
Southern blotting is a molecular biology technique used to detect specific DNA sequences in a DNA sample by transferring and immobilizing the DNA fragments onto a solid membrane, followed by hybridization with a labeled DNA probe for detection.
- Electrophoretic separation of DNA molecules by agarose gel electrophoresis
- Electrophoretic transfer of DNA from agarose gel to nylon membrane
- Immobilization of DNA on to nylon membrane
- Hybridization and non-isotopic detection of DNA of interest
- Teach the principle of Southern blotting.
- Explain principle of hybridization.
- To find the size of the DNA
History of Southern Blotting
The history of Southern blotting is a fascinating tale of scientific innovation and collaboration. The technique was named after its inventor, Edwin Southern, who combined three key innovations to develop this powerful method for detecting specific DNA sequences.
The first crucial innovation leading to the development of Southern blotting was the discovery of restriction endonucleases. These enzymes, developed at Johns Hopkins University by Tom Kelly and Hamilton Smith, have the ability to cut DNA at specific sequences. This breakthrough allowed researchers to selectively target and manipulate DNA molecules, opening up new possibilities for genetic research.
The second innovation came in the form of gel electrophoresis, which was also developed at Johns Hopkins University by Daniel Nathans and Kathleen Danna in 1971. Gel electrophoresis is a technique that separates mixtures of DNA, RNA, or proteins based on their molecular size. By applying an electric field to a gel matrix, molecules can be separated and visualized, providing researchers with a way to analyze and characterize DNA fragments.
The third innovation crucial to the development of Southern blotting was blotting methods. Frederick Sanger had previously developed a technique for transferring RNA molecules to DEAE paper, and this concept served as the foundation for Southern’s work. By combining the ideas of restriction endonucleases, gel electrophoresis, and blotting, Southern was able to create a groundbreaking technique for specifically detecting DNA sequences.
In 1973, Southern successfully invented the Southern blot technique. However, it wasn’t until 1975 that the method was published, delaying its widespread dissemination. Interestingly, the technique began to spread among scientists before its official publication. Southern shared his technique with Michael Mathews, a scientist at Cold Spring Harbor Laboratory, by drawing the process on a paper. This informal sharing of knowledge contributed to the rapid adoption and further refinement of the Southern blotting technique by the scientific community.
The Southern blotting technique revolutionized molecular biology and became an invaluable tool for DNA analysis. It allowed researchers to detect and identify specific DNA sequences, leading to breakthroughs in genetics, genomics, and medical diagnostics. Southern blotting paved the way for subsequent blotting techniques such as Northern blotting (for RNA analysis) and Western blotting (for protein analysis), expanding the possibilities for studying various biomolecules.
Today, Southern blotting has been largely surpassed by more advanced and high-throughput techniques such as polymerase chain reaction (PCR) and DNA sequencing. Nevertheless, its historical significance and the foundational principles it established continue to shape and inform modern molecular biology techniques. The development of Southern blotting stands as a testament to the power of combining innovative ideas and collaborative efforts in scientific discovery.
Southern Blotting Principle
The principle of Southern blotting revolves around the separation of DNA fragments by gel electrophoresis and the subsequent identification of specific DNA fragments using labeled probe hybridization. The DNA fragments are initially broken down into smaller fragments using restriction endonucleases. These fragments are then separated based on their size and charge through gel electrophoresis.
Following electrophoresis, the DNA fragments are transferred from the gel to a carrier membrane, typically made of nitrocellulose. This transfer is facilitated by capillary action and creates a replica of the original gel pattern on the membrane. The transferred DNA fragments become immobilized on the membrane.
To detect specific DNA fragments of interest, a labeled probe is used. The probe is a short, single-stranded DNA molecule that is complementary to the target DNA sequence. The probe is labeled with a marker, such as a radioactive or fluorescent tag, for easy detection. The labeled probe is then incubated with the membrane, allowing it to hybridize specifically with any complementary DNA fragments immobilized on the membrane.
After hybridization, the membrane is washed to remove any unbound probe. The presence of the labeled probe indicates the presence of the target DNA fragments. The detection of the labeled probe can be visualized using autoradiography, fluorescence imaging, or other appropriate methods depending on the labeling method used.
The specificity of hybridization allows for the detection of even a single target molecule among millions of other DNA molecules. This high selectivity and sensitivity make Southern blotting a valuable technique for identifying and quantifying specific DNA sequences within a DNA sample.
In summary, Southern blotting is based on the principles of DNA fragment separation by gel electrophoresis, transfer of the fragments to a membrane, and subsequent detection using labeled probes that hybridize specifically with the target DNA. This technique has been widely used in molecular biology for various applications, including gene identification, DNA analysis, and studying genetic disorders.
Material Required for Southern Blotting
- DNA Marker (Ready to use) – 100 μl store at 20°C
- Biotinylated Probe – 5 x 30 μl store at 20°C
- Prehybridization Buffer – 50 ml store at 4°C
- Hybridization Buffer – 50 ml store at 4°C
- 2X Wash Buffers (A, B, C and D) each – 75 ml store at 4°C
- Blocking Buffer – 50 ml store at 4°C
- Streptavidin HRP Conjugate – 5 μl store at 4°C
- Conjugate Dilution Buffer – 50 ml store at 4°C
- 10X Substrate – 2.5 ml store at 4°C
- 10X Electrotransfer Buffer – 150 ml store at 4°C
- Blocking Powder – 15 g store at 4°C
- Tween-20 – 50 μl
- Agarose – 2.5 g
- 50X TAE 20 ml
- Filter Paper 10 Nos.
- Nylon Membrane 5 Nos.
- Petridish 1 No.
- Equipment: Hot air oven, Incubator shaker (45°C), UV-transilluminator
- Reagents: Distilled water, Ethidium Bromide.
- Other: Crushed ice, Forceps, Gloves, Measuring cylinders, Micropipette, Blade, Scissors, Thermometer, Tips, Transparent polythene sheet.
Preparation of Buffers
When preparing buffers for Southern blotting, it is important to use the correct concentrations of reagents to ensure optimal performance. Here are some commonly used buffers and their preparation methods:
- 10X TBE Buffer:
- Prepare a 1.3 M TRIS solution.
- Dissolve 450 mM boric acid in water.
- Add 25 mM EDTA to the solution.
- Adjust the pH if necessary.
- Mix the solutions thoroughly to obtain the 10X TBE buffer.
- 20X SSPE Buffer:
- Dissolve 2.98 M NaCl in water.
- Add 0.02 M EDTA to the solution.
- Prepare a 0.2 M phosphate buffer with a pH of 7.4.
- Mix the solutions together to obtain the 20X SSPE buffer.
- Denaturing Solution:
- Prepare a 1.5 M NaCl solution.
- Add 0.5 N NaOH to the solution.
- Adjust the pH to approximately 13.
- Neutralizing Solution:
- Dissolve 1.5 M NaCl in water.
- Prepare a 1 M TRIS HCl solution.
- Adjust the pH to 7.5.
- Denhardt’s Solution (50X):
- Dissolve 1% bovine serum albumin, 1% Ficoll, and 1% polyvinylpyrrolidone in water to a final volume of 50 mL.
- Sterilize the solution by filtration.
- 2X Prehybridization Solution:
- Combine 30 mL of 20X SSPE, 10 mL of 100X Denhardt’s solution, 10 mL of 10% SDS, and 50 mL of water to prepare a 1X prehybridization solution.
- Hybridization Solution:
- Mix 30 mL of 20X SSPE, 10 mL of 10% SDS, and 60 mL of water to prepare the hybridization solution.
- 1X Probe Buffer:
- Mix 50 µL of 1 M TRIS (pH 7.6), 5 µL of 2 M MgCl2, 10 µL of 0.5 M DTT, and 35 µL of water to prepare the probe buffer.
- 1X Probe Mix:
- Combine 2.7 µL of probe buffer, 2 µL of oligonucleotide probe (0.2 µg/µL), 1 µL of T4 phosphonucleotide kinase (PNK), 11.3 µL of water, and 10 µL of 32P-ATP to prepare the probe mix.
- 6X Low-Stringency Wash Solution:
- Mix 180 mL of 20X SSPE, 12 mL of 10% SDS, and 408 mL of water to prepare the low-stringency wash solution.
- 1X High-Stringency Wash Solution:
- Combine 30 mL of 20X SSPE, 12 mL of 10% SDS, and 558 mL of water to prepare the high-stringency wash solution.
By following these instructions, you can prepare the necessary buffers for Southern blotting, ensuring accurate and efficient detection of specific DNA sequences in your experiments.
|10X TBE Buffer
|1.3 M TRIS, 450 mM boric acid, 25 mM EDTA
|Mix the reagents in the specified concentrations
|20X SSPE Buffer
|2.98 M NaCl, 0.02 M EDTA, 0.2 M phosphate
|Combine the reagents in the specified amounts
|1.5 M NaCl, 0.5 N NaOH
|Mix the reagents and adjust the pH to ~13
|1.5 M NaCl, 1 M TRIS HCl
|Dissolve the reagents and adjust the pH to 7.5
|Denhardt’s Solution (50X)
|1% bovine serum albumin, 1% Ficoll,
|Dissolve the reagents in water to a final volume
|of 50 mL and sterilize by filtration
|2X Prehybridization Solution
|30 mL 20X SSPE, 10 mL 100X Denhardt’s,
|Mix the reagents in the specified proportions
|10 mL 10% SDS, 50 mL water
|30 mL 20X SSPE, 10 mL 10% SDS, 60 mL water
|Combine the reagents in the specified amounts
|1X Probe Buffer
|50 µL 1 M TRIS, 5 µL 2 M MgCl2,
|Mix the reagents in the specified proportions
|10 µL 0.5 M DTT, 35 µL water
|1X Probe Mix
|2.7 µL probe buffer, 2 µL oligonucleotide
|Combine the reagents in the specified amounts
|probe (0.2 µg/µL), 1 µL T4 phosphonucleotide
|kinase (PNK), 11.3 µL water, 10 µL 32P-ATP
|6X Low-Stringency Wash Solution
|180 mL 20X SSPE, 12 mL 10% SDS, 408 mL water
|Mix the reagents in the specified proportions
|1X High-Stringency Wash Solution
|30 mL 20X SSPE, 12 mL 10% SDS, 558 mL water
|Combine the reagents in the specified amounts
Southern Blotting Steps/Southern Blotting Protocol
1. Sample Preparation
Sample preparation is a crucial step in Southern blotting, as it involves the extraction and purification of DNA from the desired source. The extracted DNA is then used for further analysis and identification of specific DNA fragments. Here is an overview of the sample preparation process for Southern blotting:
- Cell Lysis: a. The DNA of interest is present inside the nucleus of the cells. To extract the DNA, the cells need to be lysed, or broken open. b. Incubate the cell culture with a detergent solution, which lyses the cells and releases their contents, including DNA, proteins, and debris.
- Protein Removal: a. After cell lysis, the lysed sample contains not only DNA but also proteins. To remove the proteins, add a proteinase enzyme to the sample and incubate it. b. The proteinase enzyme digests the proteins, leaving behind purified DNA.
- DNA Purification: a. Once the proteins are removed, the DNA needs to be purified and separated from other cellular materials. b. Alcohol precipitation is a commonly used method for DNA purification. It involves adding alcohol (e.g., ethanol or isopropanol) to the lysed sample, causing the DNA to precipitate out of the solution. c. The precipitated DNA can then be collected by centrifugation and washed with alcohol to remove impurities.
- Removal of Visible DNA Fibers: a. After DNA precipitation, visible DNA fibers may be present in the sample. These fibers can interfere with the subsequent steps of Southern blotting. b. To remove the visible DNA fibers, the DNA is suspended in a suitable buffer solution, which helps in separating the fibers from the purified DNA.
- Quality Control: a. It is important to assess the quality and quantity of the isolated DNA. b. Quantify the DNA concentration using a spectrophotometer or fluorometer, and assess the purity by measuring the A260/A280 ratio.
It is worth mentioning that there are commercially available kits, such as the GenElute™ kits, designed for the isolation of DNA from various sources, including mammalian cells, plants, bacteria, and fungi. These kits ensure high purity of the isolated DNA and provide a convenient and reliable option for sample preparation in Southern blotting.
In Southern blotting, the DNA to be studied is isolated from various tissues, with blood tissue being one of the most suitable sources. However, DNA can be isolated from different tissues such as hair, semen, saliva, and others. The genomic DNA is typically extracted from the target cells using standard methods, and then it undergoes purification to obtain high-quality DNA for subsequent analysis.
Proper sample preparation is essential to ensure the success and accuracy of Southern blotting experiments. By following appropriate techniques and using reliable kits or standard protocols, researchers can obtain purified DNA that is suitable for hybridization and detection in Southern blotting assays.
2. Restriction Digestion/Fragmentation
Restriction digestion is a crucial step in Southern blotting, where the long nucleotide sequences are broken into smaller fragments for purification and identification purposes. Here is an overview of the restriction digestion process in Southern blotting:
- Setup of Digestion Reaction: a. Ensure that all the necessary reagents for the digestion process are kept on ice. b. Set up a restriction digestion reaction by adding the appropriate DNA concentration, suitable restriction enzyme, enzyme buffer, and purified water to multiwell plates or microcentrifuge tubes (PCR tubes). c. Mix the contents gently using a pipette, taking care to avoid the formation of bubbles. d. Add the restriction enzyme at the last step since it should be stored at −20 °C until use. e. It is recommended to add a surplus amount of the enzyme to ensure complete digestion of the DNA, as partial digestion can lead to ambiguous results. f. However, the concentration of the enzyme should not exceed one-tenth of the total volume of the digestion mixture to prevent inhibition of the digestion process by high glycerol content in the enzyme stock. g. Prepare master mixes when analyzing a large number of samples to minimize pipetting errors.
- Incubation: a. Incubate the digestion reaction at 37 °C, preferably in a water bath. b. For DNA samples obtained from cloning, an incubation period of 1-2 hours is usually sufficient. c. In the case of genomic DNA, overnight digestion is often required. To prevent the enzyme from running out during overnight incubation, add half of the enzyme at the beginning of the digestion and add the remaining half in the morning, continuing the incubation for an additional hour.
- Concentration of DNA: a. After digestion, it may be necessary to concentrate the DNA samples to ensure a suitable volume for loading onto the gel. b. A common method for concentrating DNA samples involves precipitation in the presence of ethanol, followed by resuspension in ddH2O. c. Ensure that traces of ethanol are completely removed, as their presence may cause the samples to spill out of the wells during loading onto the gel.
Restriction digestion involves the use of restriction endonucleases, which are enzymes that cut high-molecular-weight DNA strands at specific sites, generating smaller fragments. The choice of restriction enzyme(s) depends on the specific requirements of the experiment, such as the probe used and the complexity of the DNA. The digestion reaction is incubated at an appropriate temperature, and the resulting DNA fragments are further processed for subsequent steps in the Southern blotting protocol.
By following proper procedures and using suitable restriction enzymes, researchers can obtain accurately fragmented DNA for further analysis and identification in Southern blotting experiments.
3. Agarose Gel Electrophoresis
Agarose gel electrophoresis is a vital step in Southern blotting, allowing the separation of DNA fragments based on their size. Here’s an overview of the agarose gel electrophoresis process in Southern blotting:
- Determining Gel Percentage and Size:
- The percentage of the gel and its size need to be determined based on the desired fragment size range.
- Generally, 0.7% to 2% gel concentration is suitable for most applications.
- Longer gels may be required for genomic DNA separation or resolving multiple fragments of similar sizes.
- Low percentage gels are more fragile and may require a higher percentage base gel for support.
- Preparation of Agarose Gel:
- A 1X electrophoresis buffer is prepared by diluting the stock solution in ddH2O.
- Agarose is added to the buffer in a conical flask and stirred to avoid clumping.
- The agarose is melted using a microwave or a stirrer with heating, ensuring regular swirling to maintain consistency.
- Care should be taken to prevent boiling, and a larger conical flask should be used to avoid spillage.
- The molten gel is transferred to a magnetic stirrer and cooled slowly to around 60ºC while stirring gently.
- Ethidium bromide, at a concentration of 5 µg/ml, is added to the gel during cooling.
- Gel Casting and Loading:
- The gel casting tray is prepared with a comb of suitable teeth size based on the sample volume.
- Bubbles should be avoided as they can disrupt the DNA migration pattern.
- After the gel is set, it is transferred to the electrophoresis tank, and 1X running buffer is added to immerse the gel.
- Care should be taken while removing the combs to avoid damaging the wells.
- DNA samples are prepared by mixing them with 1X loading buffer, and the mixture is loaded carefully into the wells.
- A molecular weight marker is loaded in one or two wells to serve as a reference for fragment size.
- The lid is placed on the tank, and the power pack is connected with an appropriate voltage setting.
- The DNA fragments, which carry a negative charge, move toward the positively charged anode during electrophoresis.
- Genomic DNA requires a lower voltage (not exceeding 20 V), while plasmid DNA can be run at around 100 V.
- The correct electrode positions should be ensured for DNA to migrate in the desired direction.
- Small bubbles may be observed as the power supply is switched on, indicating the setup is functioning properly.
Agarose gel electrophoresis allows the separation of DNA fragments based on their size, with larger fragments moving more slowly than smaller ones. The gel is stained with ethidium bromide, enabling the visualization of DNA bands under UV light. By following proper procedures and choosing the appropriate gel percentage and size, researchers can obtain accurate separation and analysis of DNA fragments during the Southern blotting process.
If alkaline transfer methods are used, the double-stranded DNA is denatured by placing the DNA gel into an alkaline solution (typically containing sodium hydroxide). The denaturation in an alkaline environment may improve the binding of the negatively charged thymine residues of DNA to the positively charged amino groups of the membrane, separating it into single DNA strands for subsequent hybridization with the probe (see below) and destroying any residual RNA that may remain in the DNA. However, the selection of alkaline over neutral transfer methods is frequently arbitrary and may yield equivalent results.
4. DNA Transfer (Blotting)
DNA transfer, also known as blotting, is a crucial technique used in molecular biology to transfer fragmented DNA sequences onto a nitrocellulose or nylon membrane. This process, performed through electroblotting or capillary blotting, enables the visualization and analysis of specific DNA fragments. The choice of membrane is an important consideration, with nylon membranes gaining popularity due to their efficient DNA binding capabilities, allowing Southern blotting to be conducted with smaller amounts of target DNA.
Here’s a step-by-step guide to the DNA transfer process, along with some important points to keep in mind:
- Gel Preparation: After DNA fragments are separated using agarose gel electrophoresis, the gel needs to be prepared for transfer. If the gel was run without ethidium bromide, it should be immersed in an electrophoresis buffer mixed with ethidium bromide for 0.5–2 hours. Next, the gel is transferred to a gel documentation system, and a photograph of the gel is captured under ultraviolet light. This step helps in correlating the fluorescence photograph with the final autoradiograph.
- Gel Cutting and Denaturation: If sufficient separation of DNA fragments hasn’t occurred, the gel can be run for a longer duration. Once the desired separation is achieved, the portion of the gel to be transferred is cut using a blade. In the case of large fragments, a dilute acid bath is used for approximately 10 minutes to induce depurination. Subsequently, the gel is completely denatured by submerging it in a solution containing 1.5 M NaCl and 0.5 M NaOH for 15–30 minutes, followed by neutralization with 0.5 M Tris HCl (pH 7) and 3 M NaCl for 15–30 minutes.
- Transfer Set-Up: For strip transfers, a gel transfer set-up is prepared using filter papers and a nitrocellulose membrane. Thick filter paper, dipped in 20X SSC buffer, is laid on a glass or plastic platform. In the case of narrow strips, the filter paper is immersed in 20X SSC, while for wider strips, a tray is filled with 20X SSC, and a glass plate is placed on top. Care should be taken to avoid trapping air between the layers.
- Gel and Membrane Placement: The denatured gel is carefully placed parallel to the glass or plate, leaving a small gap of approximately 2–3 mm. On one side of the gel, a glass or Perspex sheet is laid, and on the opposite side, the nitrocellulose membrane is placed, ensuring there is no air trapped between the membrane and gel. Absorbent paper is placed on top, and caution must be taken not to apply excessive weight that could disorient the gel.
- Transfer Process: The transfer process takes approximately 3 hours, depending on the gel concentration and fragment size. To prevent drying during the transfer, 20X SSC buffer should be replenished to avoid gel shrinkage. It’s important to avoid filling the gap between the gel and glass/sheets with buffer. After the transfer, carefully remove the nitrocellulose membrane, ensuring the gel remains attached.
- DNA Fixation and Storage: Remove the gel from the nitrocellulose membrane, and if necessary, visualize any leftover DNA under UV light. Remove the portion of the membrane that was in contact with the gel using a blade. Immerse the cut portion in 2X SSC for 10–20 minutes, followed by baking at 80 °C for 2 hours in a vacuum oven. Alternatively, UV crosslinking can be used to fix the DNA. Dry membranes can be stored at room temperature for future use.
- Preparing for Hybridization: After the transfer, the membrane is incubated in a blocking solution, also known as prehybridization solution, to eliminate non-specific reactions. Denhardt’s solution is commonly used for this purpose, and salmon sperm DNA can be added as a blocking agent. The membrane is incubated in the prehybridization solution for up to 5 hours inside a hybridization chamber.
By following these steps carefully, researchers can perform Southern blotting and analyze specific DNA fragments of interest. The DNA transfer process plays a crucial role in molecular biology research, enabling the detection and characterization of genetic information.
If using a nylon membrane, crosslink the transferred DNA to the membrane using UV irradiation or suitable chemical crosslinkers.
Prehybridization, also known as blocking, is a crucial step in Southern blotting that helps minimize non-specific binding of a probe to the nylon membrane. By using a blocking agent like salmon sperm DNA, researchers can prevent the probe from attaching to the membrane indiscriminately, ensuring that it only interacts with the desired DNA bands that have been transferred onto the membrane. Follow these steps to prepare the prehybridization solution and carry out the prehybridization process effectively:
- Warm the Prehybridization Solution:
- Heat the prehybridization solution to a temperature of 42 °C. This temperature ensures optimal conditions for hybridization and probe binding.
- Prepare the Blocking Agent:
- Take a sample of salmon sperm DNA (D9156) and heat it to 95 °C for 5 minutes.
- Immediately chill the sample on ice to prevent denaturation or degradation of the DNA.
- Add Salmon Sperm DNA to the Prehybridization Solution:
- Once the prehybridization solution has reached the desired temperature, add the heated salmon sperm DNA to the solution.
- Ensure that the final concentration of salmon sperm DNA in the prehybridization solution is 50 µg/mL. This concentration has been found to be effective in blocking non-specific binding of the probe.
- Prepare the Blot for Prehybridization:
- Take the Southern blot out of the UV crosslinker, being careful not to damage the membrane.
- Roll the blot carefully into a hybridization tube. This tube will provide a controlled environment for the prehybridization process.
- Perform Prehybridization:
- Add the prehybridization solution containing salmon sperm DNA into the hybridization tube, ensuring that the blot is fully submerged in the solution.
- Place the hybridization tube in a hybridization oven set to 42 °C.
- Allow the blot to undergo prehybridization for a duration of 5 hours. This time frame allows for adequate blocking and minimizes non-specific interactions.
By following these steps, researchers can effectively carry out the prehybridization process in Southern blotting. This step plays a crucial role in reducing non-specific probe binding, ensuring that the probe specifically interacts with the target DNA bands on the nylon membrane. Prehybridization is an essential component of Southern blotting, enabling accurate detection and analysis of specific DNA fragments.
Hybridization is a crucial step in Southern blotting that involves the binding of a complementary DNA probe to the target sequence on the membrane. Here is a step-by-step guide to carrying out the hybridization process effectively, ensuring accurate detection of the desired DNA fragments:
- Prepare the Probe Mix:
- Prepare a 1X probe mix and incubate it in a water bath at 37 °C for 40 minutes.
- This step allows the probe to reach the appropriate temperature for optimal hybridization.
- Remove Unincorporated Probe:
- To remove any free label or unincorporated probe, pass the probe mix through a G-25 Sephadex column.
- This column effectively separates the labeled probe from any unbound or excess components.
- Determine Probe Specific Activity:
- Assess the specific activity of the probe using liquid scintillation.
- This measurement helps determine the concentration and activity of the labeled probe for accurate detection.
- Prepare the Hybridization Solution:
- Warm 10 mL of hybridization solution to 49 °C.
- Add 10-15 X 106 cpm (counts per minute) of the labeled probe to the hybridization solution and mix well.
- This step ensures that the labeled probe is adequately dispersed in the hybridization solution.
- Perform Hybridization:
- Discard the prehybridization solution from the blot and add the hybridization solution containing the labeled probe.
- Incubate the blot overnight at 49 °C.
- This overnight incubation allows sufficient time for the labeled probe to hybridize with the target DNA sequence on the membrane.
Note: Alternatively, PerfectHyb™ Plus buffer (H7033) can be used for hybridization. It offers a shorter hybridization time with a stronger signal yield. For more detailed protocol information, refer to the PerfectHyb™ Plus Protocol.
- Wash the Blot:
- Warm the 6X wash solution to 49 °C.
- Discard the hybridization solution from the blot and add the 6X wash solution.
- This step helps remove any unbound or non-specifically bound probe from the membrane.
- Perform Low-Stringency Wash:
- Warm the 6X low-stringency wash solution to 49 °C.
- Discard the hybridization solution from the blot and add the 6X low-stringency wash solution.
- The low-stringency wash removes low-homology hybridizations, refining the desired DNA sample.
- Perform High-Stringency Wash:
- Prepare a 1X high-stringency wash solution and warm it to 49 °C.
- Wash the blot for approximately 30 seconds and discard the wash solution.
- This high-stringency wash further refines the desired DNA by removing closely homologous hybridizations.
- Check for Activity:
- Use a Geiger counter to check the blot for activity.
- Desirable readings range between 10-50 counts per second, with clear bands of irradiation peaks.
- If the background radiation is too high, repeat the wash step using 1X wash solution.
- Air Dry the Membrane:
- Once the hybridization and washing steps are completed, remove the membrane from the blotting apparatus.
- Blot the membrane against filter papers to remove excess liquid.
- Air dry the membrane to ensure complete removal of any remaining moisture.
By following these steps, researchers can successfully carry out the hybridization process in Southern blotting. This crucial step enables the specific binding of the labeled probe to the target DNA sequence on the membrane, facilitating accurate detection and analysis of desired DNA fragments.
8. Post-hybridization Washes
a. Prepare suitable wash solutions (e.g., low-stringency and high-stringency wash solutions). b. Perform a series of washes using these solutions to remove unbound probe and reduce background noise.
Detection is a crucial step in Southern blotting, allowing researchers to visualize and analyze the target DNA fragments on the membrane. Here are the steps involved in the detection process, ensuring accurate and efficient results:
- Prepare the Blot:
- Place the blot in a film cassette lined with new saran wrap, ensuring there are no air bubbles trapped between the blot and the wrap.
- Carefully wrap the blot, ensuring a tight and secure seal.
- Expose to X-Ray Film:
- Take the cassette to a darkroom and place the X-ray film over the blot.
- Lock the cassette and place it at -80 °C overnight.
- This step allows the labeled DNA or RNA fragments on the membrane to interact with the X-ray film and produce a signal.
- Develop the Film:
- The following day, develop the X-ray film to visualize the signals.
- This step reveals the presence and location of the target DNA fragments on the blot.
- Optional Additional Exposure:
- If necessary, another sheet of X-ray film may be placed over the blot and returned to -80 °C for another exposure.
- This additional exposure can enhance the detection sensitivity, ensuring clear and distinct signals.
The duration of the detection process can vary depending on factors such as the nature of the probe and the desired level of sensitivity. Typically, detection time ranges from approximately 1 to 48 hours.
In the case of radioactively labeled RNA, there are different options for detection. One option is the use of 32P-labeled RNA, where the membrane is wrapped in plastic, ensuring no air bubbles are trapped, and placed on X-ray film. Slight pressure is applied to flatten the membranes for optimal contact with the film.
For the detection of 3H-, 35S-, 125I-, or 14C-labeled RNA, fluorography is employed. The membranes are soaked in a solution of PPO (2,5-diphenyloxazole) in toluene, air dried, and then placed on the X-ray film at -70 °C. This technique allows the radioactive signals to be captured and visualized on the film.
The timing of the various processes involved in Southern blotting, such as restriction digestion, electrophoresis, transfer, blocking, hybridization, and detection, is crucial for obtaining accurate and reliable results. The duration of each step can vary, ranging from minutes to hours or even overnight, depending on factors such as the specific protocol, probe characteristics, and desired sensitivity.
By following these steps and optimizing the detection process, researchers can effectively visualize and analyze the target DNA fragments on the Southern blot membrane, gaining valuable insights into the genetic information they seek.
aCapture images of the Southern blot for further analysis and documentation.
Remember to adjust the protocol according to the specific requirements of your experiment and follow the safety guidelines for handling DNA and radioactive materials
Observe for a single blue band on the nylon membrane.
What is Autoradiography?
Autoradiography is a technique used to detect and visualize radioactive substances in a sample by exposing it to a photographic film or an imaging plate. It is commonly used in molecular biology and biochemistry to study radioactive molecules, such as radioactive isotopes incorporated into DNA, RNA, or proteins.
The process of autoradiography involves the following steps:
- Labeling: The molecule of interest (e.g., DNA, RNA, protein) is labeled with a radioactive isotope. Common isotopes used include 32P, 35S, and 3H. The radioactive isotope emits radiation (such as beta particles or gamma rays) that can be detected.
- Exposure: The labeled sample (e.g., gel, membrane) is placed in close contact with a photographic film or an imaging plate. The film or plate is sensitive to the radiation emitted by the radioactive isotope.
- Exposure Time: The sample is left in contact with the film or plate for a specific period, allowing the radiation emitted by the labeled molecules to expose the film or activate the imaging plate.
- Development: The film or plate is processed using standard photographic development techniques. This involves treating the film with developing chemicals or using specialized equipment to extract and visualize the exposed image on the imaging plate.
- Visualization: After development, the autoradiograph reveals the distribution and intensity of the radioactive signal. Dark areas on the autoradiograph indicate regions where the radioactive molecules were present in higher quantities, while lighter areas represent lower quantities or absence of the radioactive signal.
Autoradiography is commonly used in various applications, such as DNA sequencing, protein labeling, radioactive decay studies, and quantification of radioactive substances. It allows researchers to track and visualize the movement, distribution, and interactions of labeled molecules in biological samples.
Why is Southern blotting Important?
Southern blotting is an important laboratory technique in molecular biology that allows the detection and analysis of specific DNA sequences in a sample. It was named after its inventor, Edwin Southern, who developed the method in 1975.
Southern blotting plays a crucial role in various areas of biological research and diagnostics. Here are some reasons why Southern blotting is important:
- DNA Fragment Analysis: Southern blotting enables the identification and characterization of specific DNA fragments in a complex mixture. By separating DNA fragments based on their size using gel electrophoresis and then transferring them to a solid membrane, researchers can probe the membrane with labeled DNA probes to detect the presence or absence of specific DNA sequences of interest.
- DNA Identification: Southern blotting can be used to determine the presence or absence of a particular DNA sequence in a sample. This is particularly useful in genetic testing and diagnostics. By comparing the pattern of DNA fragments in a sample with a known reference, scientists can identify genetic mutations, gene rearrangements, or genetic variations associated with diseases.
- Gene Mapping: Southern blotting is used in gene mapping to determine the organization and location of specific genes within the genome. By probing a Southern blot with different DNA markers known to be associated with specific genes, researchers can determine the presence or absence of these markers in different individuals or organisms. This information helps in constructing genetic maps and understanding the inheritance patterns of genes.
- DNA Fingerprinting: Southern blotting is a crucial technique in DNA fingerprinting, which is used for forensic analysis and paternity testing. By probing a Southern blot with specific DNA markers that show high variability between individuals, unique DNA patterns can be generated for each individual, allowing for identification or determination of biological relationships.
- Transgene Detection: Southern blotting is employed to detect and confirm the presence of transgenes in genetically modified organisms (GMOs). By using a DNA probe specific to the transgene of interest, Southern blotting can determine if the introduced DNA is integrated into the genome of the organism.
Overall, Southern blotting is a versatile and powerful technique that allows researchers to analyze DNA fragments, identify specific sequences, map genes, and investigate genetic variations. It has made significant contributions to genetic research, diagnostics, and forensic science.
- Contamination control: Contamination can lead to false-positive or false-negative results. To minimize contamination, use sterile techniques, work in a clean and dedicated laboratory space, and use disposable gloves and sterile equipment whenever possible. Additionally, separate pre- and post-amplification areas to prevent cross-contamination.
- Proper handling of DNA: DNA is susceptible to degradation by nucleases, so it is crucial to handle DNA samples with care. Use nuclease-free reagents, tubes, and pipette tips. Minimize excessive pipetting or vortexing, which can cause shearing of DNA. Store DNA samples at appropriate temperatures and avoid repeated freeze-thaw cycles.
- Optimization of DNA extraction: The quality and purity of the DNA sample obtained through extraction can affect the success of Southern blotting. Optimize the DNA extraction method to ensure maximum yield and purity. Use appropriate extraction buffers, enzymatic digestion, or column-based purification kits depending on the sample type.
- Proper selection and design of probes: Selecting the appropriate probe and designing it carefully is critical for successful hybridization. Ensure that the probe sequence is specific to the target DNA sequence and has minimal or no cross-reactivity with other sequences. Consider factors such as GC content, secondary structure, and probe length during probe design.
- Optimization of hybridization conditions: The success of Southern blotting depends on achieving optimal hybridization conditions. Factors such as temperature, buffer composition, probe concentration, and hybridization time should be optimized. Test different conditions to determine the best combination that yields specific and strong signals while minimizing background noise.
- Use appropriate controls: Incorporate positive and negative controls in each Southern blotting experiment. Positive controls include known samples containing the target DNA sequence, while negative controls should include samples lacking the target sequence or using non-specific probes. Controls help verify the specificity and sensitivity of the technique.
- Documentation and record-keeping: Maintain accurate records of the experimental details, including sample information, protocols, probe sequences, and hybridization conditions. Proper documentation enables reproducibility, troubleshooting, and validation of results.
- Validation with alternative techniques: Whenever possible, validate the Southern blotting results with alternative techniques, such as PCR or sequencing. This cross-validation can provide additional confidence in the accuracy of the results obtained through Southern blotting.
Application of Southern blotting:
- DNA Fragment Analysis: Southern blotting allows the detection and analysis of specific DNA sequences, such as genes or gene fragments. It is commonly used in genetic research to determine the presence, size, and copy number of a particular DNA sequence in a sample.
- Gene Mapping: Southern blotting can be used to map genes to specific chromosomal locations. By using DNA probes that are specific for certain genetic markers, researchers can determine the presence or absence of these markers in different individuals or populations.
- DNA Methylation Analysis: Southern blotting can also be employed to investigate DNA methylation patterns. Methylation, the addition of a methyl group to DNA, can regulate gene expression. By using specific probes, researchers can identify methylated DNA regions and study their role in gene regulation and epigenetics.
- Genetic Disease Diagnosis: Southern blotting has been used in clinical settings for the diagnosis of certain genetic disorders. By analyzing DNA samples from patients, researchers can detect disease-causing mutations or abnormalities in specific genes.
- Forensic Analysis: Southern blotting has been used in forensic science for DNA profiling and identification. It can be utilized to detect specific DNA sequences, such as short tandem repeats (STRs), which are highly polymorphic and unique to individuals.
- Homology-Based Cloning: Southern blotting transfer can be utilized for homology-based cloning, focusing on the amino acid sequence of the target gene’s protein product. By designing complementary oligonucleotides, which are chemically synthesized and radiolabeled, researchers can screen DNA libraries or cloned DNA fragments. This method allows the identification and acquisition of the full-length sequence of the desired gene.
- Study of Chromosomal and Gene Rearrangements: Southern blotting is instrumental in studying normal chromosomal or gene rearrangements. Researchers can analyze DNA samples using this technique to investigate rearrangements and gain insights into genetic alterations.
- Comparative Genomics: Southern blotting can help identify similar sequences in other species or within a genome by reducing the specificity of hybridization. This approach enables researchers to explore evolutionary relationships and study conserved DNA sequences across different organisms.
- Size Identification of DNA Fragments: In mixtures containing different sizes of digested DNA, Southern blotting can be employed to identify specific restriction fragments. This technique provides a reliable method for determining the size of DNA fragments, aiding in genetic analysis and mapping.
- Detection of Genetic Changes: Southern blotting is a valuable tool for identifying genetic changes, including insertions, rearrangements, deletions, and point mutations that affect restriction sites. By analyzing DNA samples, researchers can pinpoint alterations within specific genes, enabling a better understanding of genetic variations and their implications.
- Restriction Mapping and Single Nucleotide Polymorphism (SNP) Analysis: Southern blotting is crucial in restriction mapping, which involves using different restriction enzymes to identify specific regions of DNA. It can also determine which recognition sites have been altered due to single nucleotide polymorphisms (SNPs) that affect specific restriction enzymes. This capability contributes to the accurate mapping of DNA sequences and the identification of genetic variations.
- Personal Identification and Disease Diagnosis: Southern blotting has practical applications in personal identification through DNA fingerprinting. It can also be utilized in disease diagnosis by detecting genetic mutations or alterations associated with various disorders. This technique provides a reliable and accurate method for genetic profiling and disease detection.
Limitations of Southern Blot
- Time-consuming and labor-intensive: Southern blotting is a time-consuming technique that requires multiple steps, including DNA extraction, digestion, electrophoresis, transfer, hybridization, and detection. Each step requires careful optimization and can take several days to complete. Moreover, the technique involves manual handling and manipulation of DNA, making it labor-intensive.
- Low sensitivity: Southern blotting may have limited sensitivity compared to other DNA detection methods. The sensitivity of the technique depends on the abundance of the target DNA sequence and the efficiency of probe hybridization. If the target DNA sequence is present in low amounts, it may be challenging to detect using Southern blotting, especially when working with complex samples.
- Limited dynamic range: Southern blotting is not well-suited for quantifying the abundance of DNA sequences accurately. The intensity of the hybridization signal on the blot does not always correspond linearly to the amount of DNA present in the sample. Consequently, it may be challenging to accurately quantify DNA fragments using Southern blotting.
- Requirement for specific probes: Southern blotting relies on the use of specific DNA probes complementary to the target sequence of interest. Designing and generating suitable probes can be a challenging task. Additionally, the availability of probes for every DNA sequence of interest may be limited, especially for novel or rare sequences.
- Potential for DNA degradation: During the various steps involved in Southern blotting, there is a risk of DNA degradation, especially if not handled carefully. DNA can be prone to degradation by nucleases or physical shearing, leading to reduced sensitivity or loss of the target DNA signal.
- Inability to detect novel or unknown DNA sequences: Southern blotting requires prior knowledge of the DNA sequence of interest. It is not suitable for the detection of novel or unknown DNA sequences. Other techniques like PCR-based methods or next-generation sequencing are better suited for such applications.
Troubleshooting of Southern blotting
Troubleshooting is an important aspect of Southern blotting to identify and resolve issues that may arise during the process. Here are some common troubleshooting steps to consider:
- Leaking or Floating Samples:
- Samples may leak out of the wells if there is damage or puncture. Be cautious when removing the comb and loading the gel.
- Floating of samples can occur due to residual ethanol or hasty gel loading. Ensure ethanol is completely removed and load the gel slowly.
- Frowned Appearance of Bands: If the bands on the gel have a frowned appearance, it may be due to running the gel at a high voltage. Choose a lower voltage to resolve this issue.
- Invisibility of DNA and Molecular Weight Markers: If DNA, including the molecular weight markers, is not visible, it could be due to using a low amount of ethidium bromide. Adjust the concentration of ethidium bromide appropriately.
- Non-separation of DNA and Molecular Weight Markers: If the DNA and molecular weight markers are not separated, it may be because the gel is made up of water instead of 1X running buffer. Ensure the gel is prepared using the correct running buffer.
- DNA Sticking to Wells: When the DNA concentration in the sample is too high, it can cause the DNA to stick to the wells. Reduce the DNA concentration in the sample to prevent this issue.
- Background Spots: Appearance of background spots could be due to powder from gloves. Use powder-free gloves to eliminate this problem. Increasing the number of washes can also help remove the background.
- Low Signal Intensity:
- If the signal intensity is low, it may be due to a low sample content. Increase the DNA concentration in the sample to improve the signal.
- Additionally, check the transfer time and hybridization timing. A shorter transfer time or inadequate hybridization can affect the signal intensity. Optimize the transfer time and hybridization conditions accordingly.
By following these troubleshooting steps, researchers can identify and address common issues that may occur during Southern blotting, ensuring accurate and reliable results.
Southern Blotting Concept map
What is Southern blotting?
Southern blotting is a laboratory technique used to detect specific DNA sequences in a sample. It involves the separation of DNA fragments by gel electrophoresis, transfer of the fragments to a membrane, and hybridization with a labeled probe to visualize the target sequence.
What is the purpose of Southern blotting?
Southern blotting is primarily used to detect and analyze specific DNA sequences in a sample. It can be employed for various applications, such as gene mapping, identification of genetic mutations, DNA fingerprinting, and studying gene expression patterns.
How does Southern blotting work?
Southern blotting involves several steps. First, DNA is extracted from the sample and digested with restriction enzymes to produce DNA fragments. These fragments are then separated by size using gel electrophoresis. After that, the DNA fragments are transferred to a membrane, where they are immobilized. Finally, the membrane is exposed to a labeled DNA probe that hybridizes specifically to the target DNA sequence, allowing detection and visualization of the sequence of interest.
What are the key components required for Southern blotting?
The key components for Southern blotting include DNA samples, restriction enzymes, agarose gel, electrophoresis apparatus, a membrane (such as nitrocellulose or nylon), transfer buffer, labeled DNA probe, hybridization buffer, and detection methods (such as autoradiography or chemiluminescence).
What is the role of restriction enzymes in Southern blotting?
Restriction enzymes are used in Southern blotting to cleave DNA at specific recognition sites. By digesting the DNA sample with appropriate restriction enzymes, it is possible to generate fragments of different sizes, allowing the identification and analysis of specific DNA sequences.
How is the DNA transferred from the gel to the membrane during Southern blotting?
The DNA fragments are transferred from the gel to the membrane through a process called capillary or electroblotting. The gel is placed on top of the membrane, and a buffer solution is allowed to flow through the gel by capillary action or through the application of an electric current. This transfers the DNA fragments onto the membrane, where they become immobilized.
What is the purpose of the DNA probe in Southern blotting?
The DNA probe is a labeled single-stranded DNA molecule that is complementary to the target DNA sequence of interest. It is used to specifically hybridize with the target sequence on the membrane after the transfer step. The labeled probe allows the detection and visualization of the target DNA sequence.
How is the DNA probe labeled in Southern blotting?
The DNA probe can be labeled with a variety of markers, such as radioactive isotopes (e.g., 32P), fluorescent dyes, or enzymes. These markers allow the visualization and detection of the probe after hybridization with the target DNA sequence.
What are the detection methods used in Southern blotting?
The two common methods of detecting the DNA probe in Southern blotting are autoradiography and chemiluminescence. Autoradiography involves exposing the membrane to X-ray film, while chemiluminescence relies on the emission of light from an enzymatic reaction between the probe and a substrate.
What are the advantages of Southern blotting?
Southern blotting is a versatile technique that allows the specific detection of DNA sequences. It provides valuable information about DNA structure, gene organization, and genetic variations. It can also be used for diagnostic purposes in medical research and forensic science. However, it is a time-consuming and labor-intensive technique and has been largely replaced by more advanced methods like PCR and DNA sequencing for routine DNA analysis.
- Gaurab Karki, Southern Blotting: principle, procedure and application, Published on December 3, 2017, Online Biology Notes. https://www.onlinebiologynotes.com/southern-blotting-principle-procedure-application/