Analysing DNA

DNA analysis using analytical gels

Gels allow separation and identification of nucleic acids based on charge migration. Migration of nucleic acid molecules in an electric field is determined by size and conformation, allowing nucleic fragments of different sizes to be separated. However, the relationship between the fragment size and rate of migration is non-linear, since larger fragments have greater frictional drag and are less efficient at migrating through the polymer.

Agarose gel analysis is the most commonly used method for analyzing DNA fragments between 0.1 and 25 kb, while pulse-field gel electrophoresis enables analysis of DNA fragments up to 10,000 kb. This section provides useful hints for effective gel analysis of DNA.

Agarose concentration

The concentration of agarose used for the gel depends primarily on the size of the DNA fragments to be analyzed. Low agarose concentrations are used to separate large DNA fragments, while high agarose concentrations allow resolution of small DNA fragments (see table Concentration of agarose used for separating fragments of different sizes). Most gels are run using standard agarose, although some special types of agarose are available for particular applications. For example, low-melt agarose allows in situ enzymatic reactions and can therefore be used for preparative gels. Genomic DNA can be isolated directly from cells immobilized in low-melt agarose gels (see reference 6 for more information).


Tip:
 Use ultrapure-quality agarose since impurities such as polysaccharides, salts, and proteins can affect the migration of DNA. Agarose quality is particularly important when running high-percentage agarose gels.

Concentration of agarose used for separating fragments of different sizes
Agarose concentration (% w/v) DNA fragment range (kb)
0.3* 5–60
0.5 1–30
0.7 0.8–12
1.0 0.5–10
1.2 0.4–7
1.5 0.2–3
2.0* 0.1–2

Electrophoresis buffers

The most commonly used buffers for agarose gel electrophoresis are TBE (Tris·borate–EDTA) and TAE (Tris·acetate–EDTA) (see tables TAE, TBE, and Gel loading buffer). Although more frequently used, TAE has a lower buffering capacity than TBE and is more easily exhausted during extended electrophoresis. TBE gives better resolution and sharper bands, and is particularly recommended for analyzing fragments <1 kb.

The drawback of TBE is that the borate ions in the buffer form complexes with the cis-diol groups of sugar monomers and polymers, making it difficult to extract DNA fragments from TBE gels using traditional methods.
Tris·acetate–EDTA (TAE)
1x working solution composition 50x stock solution components per liter   Amount per liter
40 mM Tris·acetate Tris base 242 g
1 mM EDTA Glacial acetic acid 57.1 ml
0.5 M EDTA, pH 8.0 100 ml

Tris·borate–EDTA (TBE)
0.5x working solution composition 5x stock solution components per liter Amount per liter
40 mM Tris·borate Tris base 54 g
1 mM EDTA Boric acid 27.5 ml
0.5 M EDTA, pH 8.0 20 ml

Gel loading buffer
6x working solution composition 5x stock solution components per 10 ml Amount per 10 ml
0.25% bromophenol blue Bromophenol blue 25 mg
0.25% xylene cyanol FF Xylene cyanol FF 25 mg
40% (w/v) sucrose* Sucrose 5 ml

Pouring the gel
  1. Prepare enough 1x electrophoresis buffer both to pour the gel and fill the electrophoresis tank.
  2. Add an appropriate amount of agarose (depending on the concentration required) to an appropriate volume of electrophoresis buffer (depending on the type of electrophoresis apparatus being used) in a flask or bottle.

    Tip: The vessel should not be more than half full. Cover the vessel to minimize evaporation.

    Tip: Always use the same batch of buffer to prepare the agarose as to run the gel since small differences in ionic strength can affect migration of DNA.

  3.  Heat the slurry in a microwave or boiling water bath, swirling the vessel occasionally, until the agarose is dissolved.

    Tip: Ensure that the lid of the flask is loose to avoid build-up of pressure. Be careful not to let the agarose solution boil over as it becomes super-heated.

    Tip: If the volume of liquid reduces considerably during heating due to evaporation, make up to the original volume with distilled water. This will ensure that the agarose concentration is correct and that the gel and the electrophoresis buffer have the same buffer composition.

  4. Cool the agarose to 55–60°C.
  5. Pour the agarose solution onto the gel tray to a thickness of 3–5 mm. Insert the comb either before or immediately after pouring the gel. Leave the gel to set (30–40 min).

    Tip: Ensure that there is enough space between the bottom of the comb and the glass plate (0.5–1.0 mm) to allow proper formation of the wells and avoid sample leakage.

    Tip: Make sure that there are no air bubbles in the gel or trapped between the wells.

  6. Carefully remove the comb and adhesive tape, if used, from the gel. Fill the tank containing the gel with electrophoresis buffer.

    Tip: Add enough buffer to cover the gel with a depth of approximately 1 mm liquid above the surface of the gel. If too much buffer is used the electric current will flow through the buffer instead of the gel.

Agarose gel electrophoresis allows analysis of DNA fragments between 0.1 and 25 kb (e.g., genomic DNA digested with a frequently cutting restriction endonuclease), while pulse-field gel electrophoresis enables analysis of DNA fragments up to 10,000 kb (e.g., undigested genomic DNA or genomic DNA digested with rare cutting restriction endonucleases). The amount of genomic DNA loaded onto a gel depends on the application, but in general, loading of too much DNA should be avoided as this will result in smearing of the DNA bands on the gel.

Gel loading buffer (see table Gel loading buffer) must be added to the samples before loading and serves three main purposes:

  • To increase the density of the samples to ensure that they sink into the wells on loading.
  • To add color to the samples through use of dyes such as bromophenol blue or xylene cyanol, facilitating loading.
  • To allow tracking of the electrophoresis due to co-migration of the dyes with DNA fragments of a specific size. 

Molecular-weight markers should always be included on a gel to enable analysis of DNA fragment sizes in the samples. See table Commonly used DNA markers in agarose gel electrophoresis for commonly used markers.

Commonly used DNA markers in agarose gel electrophoresis
lHindIII lHindIII–EcoR1  EcoR1 f X174 HaeIII
21,130 21,226 21,226 1353
9416 5184 7421 1078
6557 4973 5804 872
4361 4268 5643 603
2322 3530 4878 310
2027 2027 3530 281
564 1904 271
125 1584 234
1375 194
947   118
831   72
564    
125    

Preparation of samples
  1. Add 1 volume of gel loading buffer to 6 volumes DNA sample and mix.
    Samples should always be mixed with gel loading buffer prior to loading on a gel.

    Tip:
    Do not use sample volumes close to the capacity of the wells, as samples may spill over into adjacent wells during loading.

    Tip: Be sure that all samples have the same buffer composition. High salt concentrations, for example in some restriction buffers, will retard the migration of the DNA fragments.
    Ensure that no ethanol is present in the samples, as this will cause samples to float out of the wells on loading.
Agarose gel electrophoresis
  1. Apply samples in gel loading buffer to the wells of the gel. 
    Prior to sample loading, remove air bubbles from the wells by rinsing them with electrophoresis buffer. 

    Tip:
    Make sure that the entire gel is submerged in the electrophoresis buffer. 

    Tip: To load samples, insert the pipet tip deep into the well and expel the liquid slowly. Take care not to break the agarose with the pipet tip. 

    Tip: Once samples are loaded, do not move the gel tray/tank as this may cause samples to float out of the wells. 

    Tip: Be sure to always include at least one lane of appropriate molecular-weight markers. 

  2. Connect the electrodes so that the DNA will migrate towards the anode (positive electrode). 

    Tip:
    Electrophoresis apparatus should always be covered to protect against electric shocks. 

  3. Turn on the power supply and run the gel at 1–10 V/cm until the dyes have migrated an appropriate distance. This will depend on the size of DNA being analyzed, the concentration of agarose in the gel, and the separation required. 

    Tip:
    Avoid use of very high voltages which can cause trailing and smearing of DNA bands in the gel, particularly with high-molecular-weight DNA. 

    Tip: Monitor the temperature of the buffer periodically during the run. If the buffer becomes overheated, reduce the voltage. 

    Tip: Melting of an agarose gel during the electrophoresis is a sign that the buffer may have been incorrectly prepared or has become exhausted during the run.
     
    Tip: For very long runs, e.g., overnight runs, use a pump to recycle the buffer. 
Pulse-field gel electrophoresis
  1. Apply samples in gel loading buffer to the wells of the gel.

    Tip: Pulse-field gel electrophoresis uses high voltages, so TBE buffer, which has greater buffering capacity than TAE buffer, should be used.

    Tip: Prior to sample loading, remove air bubbles from the wells by rinsing them with electrophoresis buffer.

    Tip: Make sure that the entire gel is submerged in the running buffer.

    Tip: To load samples, insert the pipet tip deep into the well and expel the liquid slowly. Take care not to break the agarose with the pipet tip.

    Tip: Once samples are loaded, do not move the gel tray/tank as this may cause samples to float out of the wells.

    Tip: Be sure to always include at least one lane of appropriate molecular-weight markers.

  2. Connect the electrodes so that the DNA will migrate towards the anode (positive electrode).

    Tip: Electrophoresis apparatus should always be covered to protect against electric shocks.

  3. Turn on the power supply and run the gel at 170 V with a switch interval of 5–40 s until the dyes have migrated an appropriate distance. This will depend on the size of DNA being analyzed, the concentration of agarose in the gel, and the separation required.

    Tip: Monitor the temperature of the buffer periodically during the run. If the buffer becomes heated, reduce the voltage.

    Tip: Melting of an agarose gel during the electrophoresis is a sign that the buffer may have been incorrectly prepared or has become exhausted during the run.

    Tip: For very long runs, e.g., overnight runs, use a pump to recycle the buffer.
Staining

To allow visualization of the DNA samples, agarose gels are stained with an appropriate dye. The most commonly used dye is the intercalating fluorescent dye ethidium bromide, which can be added either before or after the electrophoresis (see table Comparison of ethidium bromide staining methods). Alternatives include recently introduced commercial dyes such as SYBR Green.

Tip: Stock solutions of ethidium bromide (generally 10 mg/ml) should be stored at 4°C in a dark bottle or bottle wrapped in aluminum foil.

Addition of ethidium bromide prior to electrophoresis — add ethidium bromide at a concentration of 0.5 µg/ml to the melted and subsequently cooled agarose, that is, just before pouring the gel.

Mix the agarose–ethidium bromide solution well to avoid localized staining.

Addition of ethidium bromide after electrophoresis — soak the gel in a 0.5 µg/ml solution of ethidium bromide (in water or electrophoresis buffer) for 30–40 minutes.

Tip: Rinse the gel with buffer or water before examining it to remove excess ethidium bromide.

Tip: Staining buffer can be saved and re-used.

Note: Ethidium bromide is a powerful mutagen and is very toxic. Wear gloves and take appropriate safety precautions when handling. Use of nitrile gloves is recommended as latex gloves may not provide full protection. After use, ethidium bromide solutions should be decontaminated as described in commonly used manuals (1, 6).

Visualization

Ethidium bromide–DNA complexes display increased fluorescence compared to the dye in solution. This means that illumination of a stained gel under UV light (254–366 nm) allows bands of DNA to be visualized against a background of unbound dye. The gel image can be recorded by taking a Polaroid photograph or using a gel documentation system.

Tip: UV light can damage the eyes and skin. Always wear suitable eye and face protection when working with a UV light source.

Tip: UV light damages DNA. If DNA fragments are to be extracted from the gel, use a lower intensity UV source if possible and minimize exposure of the DNA to the UV light.

Comparison of ethidium bromide staining methods
Addition of ethidium bromide prior to electrophoresis Addition of ethidium bromide after electrophoresis
Faster and more convenient procedure Slower procedure requiring additional step
Allows monitoring of migration during electrophoresis Does not allow monitoring of migration during electrophoresis
Requires decontamination of gel tanks and comb No decontamination of gel tanks and comb necessary
More ethidium bromide is required Usually less ethidium bromide is required
Electrophoretic mobility of linear
DNA fragments is reduced by ~15%
No interference with electrophoretic mobility

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