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In this article we will discuss about the technique of southern blotting for transfer of DNA from gels to nitrocellulose.
This technique was first described by E.M. Southern in 1975. It is useful for analysis of Restriction Fragment Length Polymorphisms (RFLPs). Using gel electrophoresis in combination with restriction endonuclease analysis, a detailed and accurate physical map of a DNA molecule can be constructed.
However, in order to construct a genetic map, indicating the size and location of the genetic coding regions on this molecule, more information is required. With agarose gel, further experiments on DNA fragments are difficult since gel is very delicate to handle. This has been made possible by transferring the DNA fragments by elution or electrophoretically to nitrocellulose. This blotting technique described by Southern is known as ‘Southern Blotting.”
Principle:
From agarose gel, DNA molecules are transferred to nitrocellulose membrane either by capillary action (capillary blotting) or by electrophoresis (electroblotting). Smaller fragments are more readily transferred, compared to larger ones which also are transferred with more time. The DNA molecules are immobilized to nitrocellulose when heated at 80°C under vacuum after transfer or cross linked by using a U.V. cross linker.
Requirements:
Reagents:
1. 0.25N HCl
2. Denaturation solution:
NaCl 1.5M; NaOH: 0.5M.
3. Neutralisation solution:
NaCl : 1.5 M
Tris HC1 pH 7.5: 0.5 M.
4. 20x SSC:
NaCl: 3.0 M
Sodium citrate: 0.2 M.
5. Nylon/nitrocellulose membrane.
6. Whatman 3 mm filter paper.
7. Tray.
8. Vortex mixer.
9. U.V. transilluminator.
U.V. light box for nylon membrane cross linking. Nylon membrane is preferable to nitrocellulose since it does not dissolve in NaOH solution. It is sufficiently strong to allow multiple stripping and reprobing of the membrane.
Procedure:
Capillary Blotting:
This is the most commonly used method. Here agarose gel is mounted on a filter paper wick which dips into a reservoir containing transfer buffer which is a high salt buffer, like 20xSSC. The hybridisation membrane (nylon) is sand-witched between the gel and a stack of filter papers which serves to draw transfer buffer through the gel by capillary action. Due to the flow of buffer, DNA molecules are carried out of the gel and immobilized on the membrane.
For efficient southern blotting, pretreatment of the gel viz. depurination, denaturation and neutralisation is important.
i. Depurination:
1. Destain agarose gel in sufficient amount of distilled water with gentle shaking at room temperature. Destaining improves clarity of gel by reducing the colour of the background.
2. Decant water and soak the gel in 0.25(N) HCl for 15 minutes with gentle shaking, at room temperature. Acid treatment allows depurination of DNA and facilitates transfer of large molecules of DNA. Completion of acid treatment makes bromophenol blue tracking dye change from blue to yellow.
ii. Denaturation:
Decant acid solution and soak the DNA on the gel in several volumes of denaturation solution for 30 minutes at room temperature with constant shaking. This alkali treatment denatures DNA fragments prior to transfer, ensuring that they are in single stranded state, accessible for probing. When denaturation step is complete the bromophenol blue will once again be blue.
iii. Neutralisation:
Decant denaturation solution and neutralise the gel in several volumes of neutralisation solution for 30 minutes at room temperature with constant shaking.
Setting up of capillary blots:
1. Wrap a piece of Whatman 3 mm filter paper on a glass plate and place it on a platform in a large plastic tray with ends of 3 mm filter paper dipping into the solution in the tray.
2. Cut a piece of nylon membrane (use gloved hands and forceps since oil present on hands can get on to the membrane and cause transfer artifacts) exactly of the same size as that of the gel.
3. Float the membrane on 20xSSC until it wets completely.
4. Place the nylon piece on top of the gel. Remove all air bubbles that are trapped between the gel and the membrane.
5. Wet two pieces of Whatman 3 mm filter paper cut to exactly the same size as the gel and place them on top of the membrane. Remove all air bubbles.
6. Cut a stack of coarse filter paper slightly smaller than the gel and place it on the pieces of Whatman 3 mm filter paper.
7. Place a glass plate on top of this and keep a weight of approximately 500 g.
8. Allow the transfer of DNA to proceed for about 12-24 hours. Transfer takes place more quickly from thinner gels than thicker ones. Similarly smaller DNA fragments are transferred more rapidly than larger ones which requires more time. The extend of transfer can be monitored after it is completed by noting the extent of transfer of the loading dye.
9. Remove the stack of coarse filter papers and the 3 mm Whatman filter paper from above the gel.
10. Turn over the dehydrated gel and membrane and lay them, with gel side up, on a dry sheet of Whatman 3 mm paper and mark the position of wells on the membrane with a pencil or ball point pen. Peal off the gel. Transfer it and confirm that gel is retained. If transfer is proper, little or no DNA will be retained on the gel.
11. Soak the membrane in 20xSSC at room temperature for few minutes.
12. Allow excess fluid to strain off from the membrane and set it to dry at room temperature on a sheet of 3 mm paper.
13. Place the dried membrane between two sheets of 3 mm paper. Bake for 2 hours at 80°C under vacuum. Alternately a U.V. cross linker also can be used which immobilises DNA on nitrocellulose membrane. The blot can be stored at room temperature till further use.