Southern Blotting Technique Principle, Procedure, Importance

What is Southern blotting technique?

Southern Blotting is a molecular biology technique used for the identification of a specific DNA sequence in a collected DNA sample. In this method involve the transfer of DNA molecules from an electrophoresis gel to a nitrocellulose or nylon membrane, and is carried out prior to detection of specific molecules by hybridization probing.

• This method was named after its inventor, a  British biologist Edward M. Southern, who stated the method in Edinburgh, Scotland in the 1970s.
• The size and specific activity of the probe is responsible for the amount of DNA needed for this method. Short probes has more specificity. 
• In this method DNA molecules, normally restriction fragments are transferred to a nitrocellulose or nylon sheet from an electrophoresis gel in order to produce the DNA banding pattern on the membrane which is present in the gel.
• After transfer, the DNA immobilized on the membrane and can be utilized as a substrate for hybridization study with labeled DNA or RNA probes that accurately target individual restriction fragments in the blotted DNA.
• Therefore, the Southern Blotting is a method for ‘detection of a specific restriction fragment against a background of many other restriction fragments’.
• The plasmid or bacteriophage clone can be used as a restricted DNA in southern blotting.
• This method used to identify and quantitate specific DNA sequences, in analysis of genome organization and expression, in the study of genetic diseases, in DNA fingerprinting and analysis of PCR products.

Objectives

• 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

Southern Blotting Principle

In this method, a DNA mixture is separated by using agarose gel electrophoresis. It separates the DNA fragments based on molecular size, using an electric charge. After that, the gel is transferred to a nitrocellulose or nylon membrane by capillary or electrophoretic transfer, in order to migrate the DNA into the membrane. The DNA is immobilized onto the membrane by using UV crosslinking for nylon membranes or by baking at 80°C for nitrocellulose membranes.

The membrane is washed, prehybridized and then hybridized with a biotin-labeled probe. A hybridization probe is a short (100-500bp), a single-stranded nucleic acid that will bind to a complementary piece of DNA. Hybridization probes are labeled with a marker so that they can be detected after hybridization. Different types of labels are used such as radioactivity, usually radioactive phosphorous, or fluorescent labels. After hybridization, the unbound probe is removed by washes. 

The membrane is then incubated with the protein block to reduce non-specific interaction. The bound (hybridized) probe is detected by incubating the membrane with streptavidin enzyme conjugate and finally incubated with the substrate solution until sufficient colour (blue) develops.

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

• Prehybridization buffer: Add 1 g of blocking powder to 10 ml of prehybridization buffer
• Hybridization buffer: Add 1 g of blocking powder to 10 ml of hybridization buffer
• Blocking buffer: Add 1 g of blocking powder to 10 ml of blocking buffer
• Wash buffers A, B, C and D: Thaw and mix the buffer provided thoroughly. Dilute 15 ml each of 2X buffers supplied with 15 ml of distilled water to give buffers at 1X concentration, 30 ml each.
• Streptavidin HRP-conjugate buffer: Prepare 9 ml of conjugate dilution buffer by adding 9 μl of Tween-20 to the conjugate dilution buffer. Dilute 3 μl of Streptavidin-HRP conjugate with 9 ml of conjugate dilution buffer for each experiment just before use.
• Substrate solution: Dilute 0.5 ml of substrate solution with 4.5 ml of distilled water to give a final volume of 5 ml substrate solution of 1X concentration.
• Electrotransfer buffer: Dilute 25 ml of 10X electrotransfer buffer with 225 ml of distilled water to give a final volume of 250 ml electrotransfer buffer at 1X concentration.

Southern Blotting Steps

Day 1: Agarose Gel Electrophoresis

1. Prepare a 1.0% agarose gel containing ethidium bromide. (Refer Agarose Gel Electrophoresis)
2. Load 20 μl of ready to use DNA marker supplied onto the gel. Run at 50-100 V until the dye reaches 4.5 cm from the well.
3. Cut the DNA marker lane from the agarose gel as follows: Cut the gel ~ 3 mm above the first band and ~ 2 mm below the last band, ensuring the gel measures about 4 to 4.5 cm.

Note: Mark the appropriate position of the gel to be cut on the gel tank. Take care not to expose yourself to UV light.

4. Cut the filter paper (2 nos.) and nylon membrane exactly to the size of the cut gel. Ensure there is no protrusion of the filter paper and the membrane from the gel.
5. Wet the cut gel, nylon membrane, filter papers and the electrotransfer cassette in 1X electrotransfer buffer.

Electroblotting: Assemble the electrotransfer apparatus as shown below. Start the arrangement by placing filter paper on the cathode cassette cover followed by the cut gel and nylon membrane. Mark and place the soft side of the nylon membrane to the cut gel. Place the wet filter paper on the nylon membrane followed by anode cassette cover. Tighten the electrotransfer cassette tightly with the screws provided.

Note: Ensure no air bubbles are present between any of the layers of filter paper, cut gel and the nylon membrane.

6. Insert the cassette into the apparatus filled with 250 ml of 1X electrotransfer buffer.
7. Connect the cords to power supply according to the convention red: anode, black: cathode and set voltage to 50 V for 3 ½ hours. 
8. Turn off the power supply and remove the cassette from the apparatus. Drain the buffer.

Immobilization of DNA on membrane:

9. Remove the nylon membrane gently from the cassette and place it on a thin transparent polythene sheet and place this on a UV transilluminator (expose the soft side of membrane containing transferred DNA to UV light) with UV lamps switched on for 20 minutes. This helps in fixing the DNA on the membrane.

Note: Do not expose yourself to UV light.

10. Turn off the UV transilluminator. Place the membrane in a plastic petri dish provided and incubate in a hot air oven at 70°C for 30 minutes. This ensures complete immobilisation of DNA onto the membrane.

Hybridization:

11. Bring the petridish containing the membrane to room temperature after incubation. Add 10 ml of prehybridization buffer to the petridish and incubate at 45°C incubator shaker with mild shaking (70-90 rpm) for 45 minutes.
12. After incubation, discard the prehybridization buffer taking care not to discard the membrane.
13. Add 10 ml of hybridization buffer to the petri dish containing the membrane.
14. Keep 1 vial of biotinylated probe for 10 minutes in a boiling water bath and immediately chill by placing it on ice for 5-10 minutes. Add this probe to the hybridization buffer in the petridish. (Rinse the probe vial with 300 μl of hybridization buffer and add it to the petridish.) Incubate the petridish at 45°C incubator shaker with mild shaking 70 rpm for 16 hours.

Day 2: Blocking and Detection

1. Decant the hybridization buffer, add 10 ml of 1X wash buffer A and gently swirl the petridish for 5 minutes at room temperature. Repeat the washes twice (each wash of 5 minutes). Discard the buffer after each wash.
2. Add 10 ml of 1X prewarmed (70°C) wash buffer B. and gently swirl the petridish. Incubate at 70°C for 5 minutes in a hot air oven & gently swirl. Repeat the washes for another two times. Discard the buffer after each wash.
3. Add 10 ml of 1X blocking buffer to the petridish and incubate at room temperature for 1 hour with gentle rocking.
4. Discard the blocking buffer.
5. Add 9 ml of diluted HRP-streptavidin conjugate to the petridish and incubate at room temperature for 20 minutes with gentle rocking. Discard the conjugate buffer.
6. Add 10 ml of 1X wash buffer C to the petridish and incubate at room temperature for 5 minutes with gentle rocking. Repeat the washes two more times. Discard the buffer after each wash.
7. Add 10 ml of 1X wash buffer D to the petridish and incubate at room temperature for 5 minutes with gentle rocking. Repeat the washes two more times. Discard the buffer after each wash.
8. Add 5 ml of 1X substrate solution and gently swirl at room temperature for 15-20 minutes until a blue colour band develops.
9. After the blue colour band is seen, stop the reaction by placing the membrane in distilled water.

Observation

Observe for a single blue band on the nylon membrane.

Why is Southern blotting Important?

• Used for the detection of DNA in a given sample.
• Used for DNA fingerprinting.
• Southern blotting is also used for paternity testing, criminal identification, and victim identification.
• Used for the isolation and identification of desired genes of interest.
• Southern blotting used in restriction fragment length polymorphism.
• The mutation or gene rearrangement in a sequence of DNA can be identified by using the Southern blotting.
• The disease caused by genetic defects can be detected by using this method.
• Helps in identification of infectious agents.
• used to identify a single gene in a pool of DNA fragments.

References

• https://en.wikipedia.org/wiki/Southern_blot
• 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/
• https://www.sigmaaldrich.com/technical-documents/articles/biology/southern-and-northern-blotting.html
• https://www.mybiosource.com/learn/southern-blotting/
• https://laboratoryinfo.com/southern-blot/
• https://geneilabs.com/product/genei-southern-hybridization-teaching-kit-with-ets5-5-expts/