Introduction:
Electrophoresis is a common molecular laboratory technique by which a mixture of charged molecules is separated according to size under the influence of an electric field. It is used to identify, quantify, and purify nucleic acid fragments. Samples are placed into wells of an agarose or acrylamide gel and exposed to an electric field, which causes the charged sample to migrate to the opposite electrode. Shorter DNA fragments travel faster, whereas longer fragments stay closer to the gel’s origin, resulting in size separation.
Principle:
Many key biological compounds, including as amino acids, proteins, nucleotides, and nucleic acid, have ionizable groups and hence exist in solution as electrically charged species, either anions (-) or cations (+) at any given pH. These charged particles (cations) migrate to the cathode (negative electrode) or to the anode (positive electrode) under the influence of an electrical field, depending on the nature of their net charge.
In an electric field, any charged ion or molecule will move. The degree of migration is influenced by the net charge, size, and shape of the object, as well as the electric current applied. It can be represented by following equation:
E * q
v = —————
f
v = velocity of migration of the molecule.
E = electric field in volts per cm
q = net electric charge on the molecule
f = frictional coefficient
f varies with size but not with shape for molecules with similar conformations. Thus, a molecule’s electrophoretic mobility (µ) is proportional to its charge density (charge\mass ratio).
Factors affecting electrophoresis:
Electrophoresis is affected by several factors, including the ion or molecule’s properties, the environment (buffer) in which the molecule or ions are examined, and the applied electrical field. During electrophoresis, these parameters have a significant impact on the migration rates of molecules in the sample.
Strength of electric field
The space that surrounds electrically charged particles is described by an electric field. Which is defined by the electric force per unit charge.
This electric field, which is radially outward from a positive charge and radially inward from a negative point charge, exerts a force on other charged objects. Voltage, current, and resistance in the electric field all have an impact on ion mobility.
Fig: Motion by electrophoresis of a charged particle
Charge on the molecule
The rate of migration is accelerated by a net increase in the charge.
Voltage
The voltage supplied has an effect on the travel time of the molecules being separated. The faster DNA travels through the gel, the higher the voltage.
Overly high voltages, on the other hand, may cause the gel to melt or the DNA bands to smear or distort.
Nature of support medium
The rate of compound migration is determined by the type of support medium used. The type of gel used influences molecular sieving.
The medium could influence the electrophoretic rate through adsorption, molecular sieving, and electro-osmosis. Adsorption produces sample tailing, which affect the rate and resolution of the separation. It is always highly recommended to select an inert medium.
Current
The current and time are directly proportional to the distance traveled by the ions.
Size and shape of molecule
The migration rate of the sample being separated is influenced by its size and shape.
Rate of migration is inversely proportional to the increased in size and shape of molecule.
Ionic strength and pH of buffer
The pH of the supporting medium is stabilized by buffer, which impacts the migration rate of a molecule. In comparison to typical buffers, zwitterionic buffers have been found to be far more capable of resisting prolonged electrolysis.
In organic molecules, the degree of ionization is influenced by pH. The rate of migration of an organic compound is affected by ionization, which increases as the pH of the compound rises, and it’s quite the opposite for organic bases.
Electroendosmosis
Electroendosmosis (also known as electro-osmotic flow) is a phenomenon that can impact electrophoretic separation. The presence of charged groups on the surface of the support medium causes this event. When voltage is given to the electrolyte near the capillary walls, cations in the electrolyte migrate to the cathode, dragging electrolyte solution along with them. This results in a net electroosmotic flow from the cathode to the anode.
Fig: Electroosmotic flow through a glass capillary
Types of electrophoresis:
Based on direction
- Horizontal electrophoresis
- Vertical electrophoresis
Fig: Vertical and horizontal electrophoresis
Based on buffer
Continuous electrophoresis
A continuous buffer system has only a single separating gel and uses the same buffer in the tanks and the gel. e.g. Agarose Gel Electrophoresis
Discontinuous electrophoresis
In a discontinuous system a non-restrictive large pore gel, called a stacking gel, is layered on top of a separating gel. e.g., SDS PAGE Electrophoresis.
Based on support media
- Paper electrophoresis
- Cellulose acetate electrophoresis
- Agar electrophoresis
- Agarose electrophoresis
- Starch electrophoresis
- Polyacrylamide gel electrophoresis (PAGE)
Based on origin
- Native or non-denaturing gel electrophoresis (e.g., Agarose gel electrophoresis)
- Non-native or denaturing gel electrophoresis (e.g., SDS-PAGE)
Based on dimension
- 1 dimension electrophoresis
- 2-dimension electrophoresis
Material required for electrophoresis:
- Electrophoresis chamber
- Agarose gel
- Gel casting tray
- Running Buffer
- Staining agent (dye)
- A comb
- DNA Ladders/Markers
- Sample/Starting Material/Template
- Loading buffer
- DNase/RNase-Free distilled Water
- UV transilluminator/ Gel documentation system
Fig: Components required for electrophoresis
1. An electrophoresis chamber and power supply.
2. Gel casting trays, available in a variety of sizes and composed of UV transparent plastic.
3. Sample combs, around which molten medium is poured to form sample wells in the gel.
4. Tris-acetate-EDTA (TAE) or Tris-borate-EDTA (TBE) are commonly used as electrophoresis buffers.
5. Loading buffer, which contains glycerol/sucrose to allow the sample to “fall” into the sample wells, and one or two tracking dyes (Bromophenol blue/xylene cyanole), which migrate in the gel and that monitoring or tracing in order to know how far the electrophoresis has proceeded.
6. Staining: DNA molecules are easily visualized under an ultraviolet light when electrophoresed in the presence of the staining dye such as SYBR Gold, SYBR Green I, SYBR Green II, and SYBR Safe, ethidium bromide, Eva Green, silver-stain. Alternatively, nucleic acids can be stained after electrophoretic separation by soaking the gel in a solution of ethidium bromide.
7. Transilluminator (an ultraviolet light box), which is used to visualize stained DNA in gels.
Fig: Band of DNA under UV transilluminator
Workflow:
In many molecular biology experiments, gel electrophoresis is being used. To achieve optimal separation and analysis of nucleic acid samples, nucleic acid electrophoresis must be set up in a number of approaches.
Selecting and preparing gels
- Agarose gels
- Polyacrylamide gels
- Buffer choice in gel preparation
Preparing standards and samples
- Nucleic acid ladder selection
- Sample and ladder preparation
- Loading dye and buffer choice
Running electrophoresis
- Running buffer choice
- Voltage
- Run time
Visualizing samples in the gel
- Fluorescent stains
- UV shadowing
Documenting gels
- Fluorescent imaging
- Autoradiography
Fig: Key steps in electrophoresis
Applications:
In the field of biotechnology, agarose gel electrophoresis is a commonly used method.
- The principal function of electrophoresis has been the separation of biological molecules, which includes molecules with lower relative molecular weights, such as amino acids, as well as molecules with larger relative molecular masses, such as proteins and polynucleotides, including RNA and DNA molecules.
- Separation of DNA fragments for use in DNA fingerprinting at crime scenes.
- The main applications of electrophoresis have been in the separation of biological molecules, which includes molecules with relatively lower relative molecular masses such as amino acids, and also molecules of higher relative molecular masses such as proteins and polynucleotides (including RNA and DNA molecules).
- It is a powerful separation method widely used to examine DNA fragments generated by restriction enzymes, and it is a practical analytical approach for estimating the size of DNA molecules in the range of 500 to 30,000 base pairs, among the numerous types of Molecular methodology.
- To investigate the genes linked to a specific disease.
- For the isolation and manipulation of cloned DNA fragments, nucleic acid electrophoresis is commonly performed at the laboratory bench.
- In the current era of genome sequencing, nucleic acid electrophoresis has become very important.
- Separating DNA fragments for DNA fingerprinting at crime scenes.
- In taxonomic and evolutionary investigations, DNA profiling is used to discriminate between populations or species.
- Analysis of macromolecules using blotting (e.g., Southern blotting, northern blotting and western blotting) techniques.
References:
- Pulimamidi Rabindra Reddy and Nomula Raju (2012). Gel Electrophoresis and Its Applications, Gel Electrophoresis – Principles and Basics, Dr. Sameh Magdeldin (Ed.), ISBN: 978-953-51-0458-2.
- Green MR, Sambrook J (2012) Analysis of DNA. In: Molecular Cloning: A Laboratory Manual (4th ed). Cold Spring Harbor: Cold Spring Harbor Laboratory Press. pp 81–156.
- Thermo Fisher Scientific Inc (2010) Nucleic Acid Detection and Analysis. In: Molecular Probes Handbook: A Guide to Fluorescent Probes and Labeling Technologies. pp 349–360.
- Wilson, K., Hofmann, A., Walker, J.M. and Clokie, S. eds., 2018. Wilson and Walker’s principles and techniques of biochemistry and molecular biology. Cambridge University Press.