Charged molecules move through a gel, when an electric current is passed across it based on their size (figure 1).
It is a preparative technique, usually done prior to mass spectrometry, PCR, cloning or RFLP.
In 1984, David Schwartz and Charles Cantor described the modified form of gel electrophoresis known as pulsed field gradient gel electrophoresis (PFGE) technique for separating large fragments of DNA (several Mbs).
PFGE gained tremendous advantage for restriction-site mapping of human chromosomes.
This course covers general principle, different types of gels used in electrophoresis, apparatus used, method involved and applications of gel electrophoresis.
High molecular weight substances, such as nucleic acids and proteins are separated by this method using gel as a supporting media.
The term gel refers to the matrix, which is used for separation of the target molecules.
In most cases, the gel is a cross-linked polymer, whose composition and porosity is chosen based on the specific weight and composition of the compound to be analyzed.
Molecular sieving property of the gel allows separation of large molecular weight compounds with same charge, but differing in size and shape.
Different types of gels
In an appropriate buffer solution, the branched chains of amylopectin component of starch intertwine and form semi-rigid gel.
Structurally and physiologically active proteins can be separated using starch gel utilizing its molecular sieving property.
Agar/ Agarose gels
Agar or agar agar (Figure 2) is a gelatinous substance derived from the red algae. It is a non-toxic, cheap and complex powdery mixture.
When extracted from the seaweed, it is in the form of agarose and agaropectin.
Agarose is uncharged, therefore is desirable for an electrophoretic matrix. Charge on agarose leads to electro-osmotic flow through the gel.
Agarose is a linear polymer made up of the repeating monomeric unit of agarobiose (Figure 3).
Agarobiose is a disaccharide made up of 1,3-linked-β-D-galactopyranose and 3,6-anhydro-L-galactopyranose.
Polyacrylamide is a polymer (-CH2CHCONH2-) formed by joining of the acrylamide subunits and comonomer, N, N’-methylenebisacrylamide.
Apparatus and Method
Power pack provides stable current with control on current output as well as voltage.
The unit consists of electrodes, buffer reservoirs, a support for the electrophoresis medium and a cover (Figure 5).
Stainless steel electrodes are generally used, but platinum electrodes is used where the buffer on the steel electrodes causes corrosion.
Gels can be run as horizontal or vertical slabs made of perspex units. Slabs carry large number of samples making them commercial and suitable for an ideal separation.
Preparation of Gel
Gel is prepared by mixing agarose or poylacylamide with suitable buffer in a beaker. There are several methods for preparing the gel. These methods might differ in,
- buffering system used
- sample size to be loaded
- total volume of the gel (typically thickness is kept to a minimum, whereas length and breadth are varied as needed)
- whether gel is prepared horizontally or vertically
A comb is inserted in the gel and gel is allowed to cool. Later, comb is removed from the gel that leaves behind slots in which samples are added for the analysis.
Sample Application and Visualization
Samples are applied by micro-syringe or pipette into the slots prepared by combs. Small volume of sample is loaded in the well.
Buffers used, in which samples are dissolved are usually sucrose or glycerol. They increase the density of sample (causing the sample to sink into well).
The migration of samples in a gel is monitored by a marker dye (bromophenol blue) and ethidium bromide, EtBr (Figure 7), which is added to the sample before loading it.
EtBr strongly binds to the DNA molecule by intercalating between the bases (Figure 8). It is fluorescent so it absorbs invisible UV light and transmits the energy as visible orange light.
Ethidium bromide is commonly used in molecular biology laboratories to stain electrophoresis gels.
The compound forms fluorescent complexes with nucleic acids and these can be viewed under UV light.
EtBr can act as mutagen, carcinogen, or teratogen in certain animal and microorganisms test systems.
Although EtBr has not been thoroughly evaluated in humans, based on current toxicity data and its interaction with DNA it should be handled with considerable caution.
Applications of Gel Electrophoresis
- Gel electrophoresis is used in the study of evolutionary relationships by analyzing genetic similarity among populations or species.
- It is used to study the structure and function of proteins.
- It is used in separation of DNA fragments for DNA fingerprinting to investigate crime scenes (Figure 9).
- Paternity testing using DNA fingerprinting is done by gel electrophoresis.
- Blotting techniques use gel electrophoresis for analysis of macromolecules.
- It is a preparative technique, usually done prior to mass spectrometry, PCR, cloning or RFLP.
- Study of evolutionary relationships by analyzing genetic similarity among populations or species is done by this method.
Steps Involved in DNA Fingerprinting
An electric current is applied to the gel from a power source. Negatively charged DNA moves toward the positive side.
Larger fragments move slower and are located near the top whereas smaller fragments move faster and are near the bottom.
Bands are stained but different shades indicate the amount of DNA each band contains. DNA bands are studied carefully to establish a link between biological evidence and a suspect in a criminal investigation