Sorting things is a good first step to getting to know more about them. Organising them into groups is therefore very important. Most natural objects have certain attributes : for instance pebbles taken from a stream all have different sizes and weights. We can use a sieve to sort them according to their sizes. As we sieve them, the smallest pebbles pass through first.
When using a sieve, you simply shake gently to help the pieces travel in the required direction. In biology, you use specific 'sieves', and specific 'gentle shakes' to sort biological components. Molecular biologists sort DNA molecules according to their size using the principle of the sieve. Instead of encouraging pebbles to go through a sieve by shaking it, molecular biologists persuade different sized DNA molecules to move through a gel (their version of the sieve) by passing an electric current through it. Because this gel acts like a sieve, smaller molecules move through it much quicker than larger ones.
An important principle of physics is that negatively charged molecules (cations) are attracted towards a positive electric charge (anode). If we dissolve sodium chloride in water, it ionises to form sodium ions (Na+) and chloride ions (Cl-). If we then apply a current, the positively charged sodium ions will move towards the negatively charged cathode, whilst the negatively charged chloride ions migrate towards the anode. In the same way, bigger molecules like DNA and proteins will move in an electric field. Their speed will depend on the ratio between their charge and their mass. Shape also is important, especially in semi solid media like gels.
Do you remember the structure of DNA? Let's have a look at a nucleotide. Note that at neutral pH, each nucleotide has a negative charge. Therefore, in water, and at pH7, DNA is negatively charged, and will move in an electric field toward the positive pole (anode).
The structure of DNA, in general, is a long filament. In the case of circular DNA, tensions can appear, and we will see the consequences of that a bit later on.
If all DNA molecules had the same shape, then the speed of migration would depend on the ratio charge / mass. Because this ratio is identical whatever the length of the DNA, they will migrate at a relative speed depending on its mass. Small molecules will move faster, because they are not impeded so much by the structure of the medium they are in. In a gel, bigger molecules travel more slowly than smaller ones, because the network of the gel slows them down.
Electrophoresis is the technical term for using an electric current to separate DNA molecules in a gel according to their size. The gels that molecular biologists use contain millions of invisible pores. To move through the gel, DNA has to pass through this network of pores. For this reason, smaller DNA molecules can move through the gel much faster because they don't have to squeeze through the gel's pores as the larger DNA molecules have to.
One type of gel that molecular biologists use a lot to separate DNA is agarose gel. Agarose comes to the researcher in the form of a dried powder (it is actually extracted from seaweed, just like the agar that microbiologists use to grow bacteria on). They boil the agarose in a buffer solution, then pour the liquid agarose into a tray. When the liquid agarose cools to about 40°C, it solidifies into a gel.
The gel is laid in a tank which is connected to a power supply and a voltage is applied. This is illustrated in the figure above.
The pictures below are illustrations of different kinds of tanks.
Diagram 2: Loading and electrophoresis of DNA on an agarose gel. The agarose gel is set horizontally. Note the representation of the blue dye that migrates at the same speed as 100 bp DNA. (blue line).
When the sample is loaded in a well at one side of the gel (diagram 2A), and the current applied, the DNA starts to migrate in the direction of the anode (diagram 2B). It is therefore very important to put the leads in the right position! If the gel has been properly made,. i.e. the buffer is the same in the gel and in the tank, the DNA migrates solely according to its size. Fragments of the same size migrate together, and there is no way the fragments are going to move side ways. If the solution contains DNA of only two different sizes, then the DNA will migrate and form two bands (diagram 2C). A blue dye is present in the sample that migrates as the same speed as the smallest fragments of DNA (100bp). It helps determinating when the smaller fragments have reached the end of the gel, and therefore to decide when to stop the electrophoresis. On the diagram, the dye is represented by the blue line accross the gel. Note very small fragments of DNA can run faster than the dye, and therefore go out of the gel.
The longer the current is applied, the further the DNA is going to migrate. A dye is added to the DNA mixture first to help locate the smaller fragments (which move fastest), and prevent all the sample from going out of the end of the gel at the anode end.
DNA is not visible at the wavelength our eyes are used to. We use a dye called "ethidium bromide, EtBr" to stain the gel. Ethidium bromide sticks to DNA and is visible under UV light. It is possible to see the bands on a EtBr stained gel by putting the gel on a UV transilluminator. The gel appears black with pink bands of DNA. A picture is taken using a special red filter. Bands appear white or grey on the picture. Coloured dyes like Azure Blue can also be used to stain DNA.
Biolab lets you run a gel in a split second and see the photo of what the gel will look like. Use it to run your DNA.