Analysing DNA

Analysis of DNA by Southern blotting

Southern blotting is a widely used technique that allows analysis of specific DNA sequences. DNA is usually first converted into conveniently sized fragments by restriction digestion. The DNA is next run through an agarose gel (6). Southern blotting (named after its inventor, E.M. Southern) refers to the transfer of the DNA to a nylon or nitrocellulose membrane by capillary transfer. The DNA of interest can be identified by hybridization to radioactive or chemiluminescent probes and visualized by autoradiography or staining.

Many variations on the Southern blotting procedure exist. A standard protocol is described here together with recipes for buffers and solutions.

Equipment required:

  • Whatman 3MM filter paper
  • Blotting membrane
  • Paper towels, a stack of approximately 15–20 cm
  • Plastic wrap
  • Two glass or Plexiglas plates
  • Buffer tray (e.g., glass casserole dish) capable of holding 1–2 liters of buffer
  • Support (to be placed in the buffer tray)
  • Flat weight, approximately 1 kg
  • Oven, at 80°C, or UV transilluminator
  • Orbital shaker 
  • Preparation of gels for Southern blotting
Fragmentation of large DNA molecules (optional)
DNA fragments longer than 10 kb do not transfer to blotting membranes efficiently. In order to facilitate their transfer, these fragments are reduced in size, either by acid depurination or by UV irradiation.

Acid depurination — immediately after gel electrophoresis, place the gel in a solution of 0.2 M HCl, so that it is completely covered. Agitate gently for 10 minutes. During this period the color of the bromophenol blue in the samples will change from blue to yellow, indicating that the gel has been completely saturated with the acid. Rinse the gel briefly in distilled water.

Tip: The depurination step should not last too long, since very short fragments attach less firmly to the membrane.

Tip: Depurinated gels may yield “fuzzy” bands on the final autoradiograph, presumably because of increased diffusion of the DNA during transfer. Depurination is therefore recommended only when fragments larger than 10 kb are to be transferred.

UV irradiation — expose the gel to UV light at a wavelength of 254 nm from a source operating at 30 W, for 30–60 seconds.

Denaturation
Double-stranded DNA must be denatured in order to create suitable hybridization targets. Completely cover the gel with denaturation buffer (see table Denaturation buffer) and incubate for 30 minutes with gentle shaking. If acid depurination was used to denature the DNA, the bromophenol blue will return to its original color during this incubation.

Neutralization

Remove the denaturation buffer and completely cover the gel in neutralization buffer (see table Neutralization buffer). Incubate for 30 minutes with gentle shaking

Denaturation buffer
Composition of working solution
Component Amount per liter
1.5 M NaCl NaCl 87.7 g
0.5 M NaOH NaOH NaOH

Neutralization buffer
Composition of working solution
Component Amount per liter
1 M Tris·Cl Tris base 121.1 g
1.5 M NaCl NaCl 87.7 g

20x SSC
Composition of working solution
Component Amount per liter
3 M NaCl NaCl 175.3 g
0.3 M Sodium citrate Sodium citrate-H20 88.2 g

  1. Place a support larger than the gel in a tray containing 10x SSC (see table 20x SSC), and cover the support with a glass or Plexiglas plate (see figure Southern blot setup). 
  2. Cut two lengths of Whatman 3MM paper wider than the gel, long enough to fit under the gel and reach to the bottom of the dish on either side. Wet the sheets briefly in 10x SSC, and place them on the glass plate. Remove any air bubbles between the paper and the support by rolling a pipet several times back and forth over the surface. 
  3. Cut one sheet of blotting membrane and two sheets of Whatman 3MM paper about 1 mm larger than the gel on each side. 

    Tip: Always wear gloves when working with blotting membranes. Handle membranes carefully by the edges or using clean blunt-ended forceps. 

  4. Place the prepared gel upside-down on the platform. Remove any air bubbles trapped between the gel and the platform by rolling a pipet several times back and forth over the gel. 
  5. Surround the gel with plastic wrap. This ensures that the 10x SSC moves only through the gel and not around it. 
  6. Place the precut blotting membrane on top of the gel so that it covers the entire surface. Do not move the blotting membrane once it has been placed on the gel. Remove any air bubbles between the paper and the support as described in step 4. 
  7. Briefly wet the two precut sheets of Whatman 3MM paper in 10x SSC, and place them on top of the nylon membrane. Again, remove any trapped air bubbles as described in step 4. 
  8. Place a 15–20 cm stack of dry paper towels on top of the filter paper. 

    Tip: Make sure that the plastic wrap surrounding the gel prevents contact of the paper towels with the 10x SSC and the wet filter paper under the gel. Ensure that the towels do not droop over since they can cause liquid to flow around the gel instead of through it

  9. Place a second glass or Plexiglas plate on top of the paper towels. Place the weight on top of the plate. Let the transfer proceed for 12–18 h. 

    Tip: Transfer efficiency is improved by removing the wet paper towels and replacing them with dry ones at least once during the transfer.
Agarose gel analysis of plasmid DNA
  1. After the transfer is complete, remove the weight, paper towels, and the two sheets of filter paper. Turn over the gel and the blotting membrane together, and lay them, gel-side up, on a sheet of dry filter paper. Mark the positions of the gel lanes on the membrane using a ballpoint pen or a soft-lead pencil. Peel the gel from the membrane. If desired, keep the gel to assess the efficiency of DNA transfer, otherwise discard. Before removing the gel from the blotting membrane, ensure that the gel lanes are marked so that they can be later identified. In order to assess the efficiency of DNA transfer, stain the gel with ethidium bromide after blotting to see how much DNA remains.
  2. Fix the DNA to the blot, either by baking (see step 3) or by UV-crosslinking 
    (see step 4). 

    Tip: UV-crosslinking generally gives better results and enhanced sensitivity compared with baking. However, effective crosslinking requires optimization of the system.

  3. Use either this step or step 4: To fix the DNA to the membrane by baking, first let the blot air-dry on a sheet of filter paper, then place between two sheets of filter paper, and bake at 80°C for 2 h. Proceed to step 5.
  4. Use either this step or step 3: To fix the DNA by UV-crosslinking, first protect the surface of the membrane by covering the UV source (e.g., a transilluminator) with plastic wrap. Then take the damp blot and expose the side with DNA to the UV source for a predetermined length of time. Proceed to step 5.

    Tip: It is important to optimize the system for UV-crosslinking. To do this, prepare a blot with several control DNA samples. Cut the blot into separate strips for each lane, and irradiate each blot for different times, varying from 0.5 to 5 min. Hybridize all the blots together and determine which time gives the optimal signal intensity. It is important to use the same conditions (UV wavelength, distance from UV source) for each experiment. It is also important to calibrate the system routinely, as the energy emitted from a UV bulb is reduced with use. 

    Tip: UV light can damage the eyes and skin. Always wear suitable eye and face protection.

  5. If the blot will not be used immediately, store it at room temperature covered in plastic wrap.
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