Molecular Techniques

Gel electrophoresis

Gel electrophoresis is a basic technique used to separate DNA, RNA or proteins. It is a common starting point for many biotechnology experiments and is often paired with the blotting techniques (see below).

This technique relies on electricity to separate out molecules in an agarose gel, a thick jello-like substance. DNA, RNA and proteins are all electrically charged, so when an electric current is applied to the gel, these molecules will naturally move toward the opposite pole. Because the gel is difficult to travel through, the molecules will travel at different speeds depending on their size. Smaller molecules will be able to move faster and will reach the far end of the gel, while larger molecules will be slowed down and remain near the beginning.

The end result of gel electrophoresis is a gel with the molecules spread out from one end to the other. If they have been coloured, the molecules appear as short bands to the naked eye. The gel can be used in many different ways. Certain regions of the genome will result in a pattern that is unique for every person when run on a gel, which can be used for DNA fingerprinting. Gels are also used to detect the presence or absence of specific DNA or RNA molecules or proteins, when they are combined with the blotting techniques.

The blotting techniques

The Southern, Northern and Western blots are used to detect DNA, messenger RNA (mRNA) and protein, respectively. Blotting refers to the actual technique, where molecules that have been separated on a gel are transferred or blotted onto a type of paper called nitrocellulose. The naming of the different blots originated with the DNA blot, developed by Edward Southern, and the Northern and Western blots followed.

Southern blot

Before the blot itself can be done, DNA that has been cut up with restriction enzymes is separated by gel electrophoresis (see above). For the blotting step, the gel is placed on a sponge which is sitting in a buffer solution. The nitrocellulose paper, where the DNA will be transferred to, is placed on top of the gel and then covered with paper towels and a weight. The transfer of the DNA from the gel to the paper happens by capillary action as the buffer moves toward the dry paper towels. After several hours, the transfer is complete and the paper will have the DNA fragments on it in the same pattern as they were in the gel. The paper can then be incubated with a probe that is specific to a DNA fragment of interest. The probe is radioactively labelled and once the incubation is complete, it can be detected by autoradiography. Controls must be used to ensure that the electrophoresis and the blot were successful. A comparison of the band patterns by autoradiography shows the presence or absence of the DNA of interest.

Northern blot

A Northern blot is done in the same way as a Southern blot, but it uses mRNA instead of DNA.

Western blot

A Western blot also follows the same method as a Southern blot, but is used to detect proteins instead of DNA. After the proteins are blotted onto the paper, antibodies are used to detect their presence. The primary antibody binds to the protein on the paper and the secondary antibody binds to the primary antibody. The secondary antibody is either tagged with a colour or is attached to an enzyme that can produce a colour in order to detect where it is on the paper.

Enzyme-Linked Immunosorbent Assay (ELISA)

ELISA is a technique used to detect the presence of specific antibodies or antigens in a sample. It is a very simple test that can analyse a large number of samples at once, which makes it a very important diagnostic technique.

The ELISA method takes advantage of the natural property of antigens and antibodies to bond together. A plastic dish with many wells in it is coated with an antibody for a particular antigen. Then a different sample is added to each well - for example, blood samples from different people. Several wells will contain positive and negative control samples. If the antigens in the blood match the antibody in the well, they will bind. Those that do not bind will be washed off. A second antibody is then added to the wells, which will only attach to the antigens. This second antibody is attached to an enzyme ("enzyme-linked") that will produce a colour when a solution is added to it. The entire dish can then be read by a scanner that looks for the presence of the enzyme's colour. If a colour is present, it means that sample contained the antigen of interest. If there is no colour, there was no antigen to bind to the first antibody. The control samples are used to make sure the procedure was successful - the positive control should be coloured and the negative control should not.

Polymerase Chain Reaction (PCR)

PCR is a technique used to make thousands of copies of a DNA strand in only minutes, using an enzyme called DNA polymerase. PCR plays an important role in research, diagnosis and forensics.

To use PCR to amplify a DNA strand, the DNA sequences at both ends of the strand must first be known. Scientists can make complementary DNA of these regions, which are known as primers. The primers tell the DNA polymerase where to start copying the DNA and then when to stop. In addition to the primers, a copy of the DNA strand that needs to be copied, nucleotides and DNA polymerase are mixed in a small tube and put in a machine that can closely control the temperature. Specific changes in temperature are essential to the PCR process.

The starting temperature is 96°C, which denatures, or separates the two strands of the DNA. The next step is called annealing, where the primers attach to the DNA strands. This happens at 68°C. Once the primers have annealed, DNA polymerase will extend them by adding nucleotides according to the DNA template at 72°C. Each cycle of these three steps takes less than two minutes and it can be repeated multiple times to produce thousands of copies of the original DNA strand.

PCR can be used for many purposes, including genetic fingerprinting in forensics, paternity testing, mutation detection for disease and cloning genes for research.

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