GEL ELECTROPHORESIS TECHNIQUE

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The term “electrophoresis” refers to the movement of a solid particle (e.g., nucleic acid molecules) through a polymer matrix or gel under the influence of electric field. Electrophoresis is a molecular biology technique that is used to separate nucleic acid molecules (DNA and RNA) and other macromolecules such as proteins mainly on the basis of their charge to mass ratio as they migrate through a gel in an electric field. Gel electrophoresis technique is a molecular biology technique that is used to separate nucleic acid molecules (DNA and RNA) according to their sizes and conformation or charges. It is generally used in the molecular biology laboratory for the separation and purification of nucleic acid fragments. The process occurs in an electrophoretic tank or chamber laden with specialized type of gel (e.g., agarose gel) through which controlled electric charge is allowed to pass through.

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A dye (e.g., ethidium bromide) is added to the gel so that the nucleic acid fragments can be visualized under ultraviolet (UV) light. The size range of nucleic acid fragments that can be separated using agarose gel is usually in the range of 0.2 kb to 20 kb. Gel electrophoresis is an important technique that is used to analyze the products of a PCR reaction, and it allows separated fragments of nucleic acid molecules or protein molecules from a given microorganism or cell to form characteristic pattern of bands known as fingerprints when they are passed through a gel under the influence of electric charges.

Gel electrophoresis is the technique of separating charged molecules such as DNA in an electric field. Fragments of separated nucleic acid molecules move through gel in electric fields according to their different sizes, and this serves as the basis for the utilization of electrophoresis to identify the individual fragments of a particular DNA. It is worthy of note that after isolating a piece of DNA from an organism, and cutting same into different fragments using restriction enzymes; there is need to study the individual fragments, and this can only be made possible through electrophoresis which gives a detailed analysis of each fragment of the nucleic acid. Deoxyribonucleic acid (DNA) is a negatively charged nucleic acid molecule because of its phosphate groups.

When the separated DNA fragments is placed in a gel and allowed to move through an electric field (with positive and negative ends), the DNA molecule tends to move towards the positive terminus (i.e., the anode) of the electric charge than the negative terminus (or cathode) because of its notable negative charge (Figure 1). Smaller molecules of DNA migrate through the gel faster than the larger molecules because of the sieving nature of the gel used for gel electrophoresis technique. The separated DNA fragment is allowed to run for a specific amount of time, and the DNA fragments are visualized under UV light after the addition of ethidium bromide (EtBr) which makes the bands visible. DNA is a colourless macromolecule, and EtBr is used in gel electrophoresis to make the different bands of the nucleic acid (DNA) visible. EtBr is mutagenic and can cause cancer; thus it should always be handled with utmost care.

Figure 1. Agarose gel electrophoresis apparatus (arrowhead) for the separation of DNA fragments and protein molecules.

Agarose gel is used for the separation of DNA fragments while acrylamide or polyacrylamide is the gel matrix used for the separation of protein molecules. While agarose is used in horizontal gel apparatus as shown here in Figure 1, polyacrylamide is mainly used in vertical gel apparatus applied in advanced separation techniques such as blotting. Thegel electrophoresis apparatus is used for separating nucleic acids based on their mobility under the influence of an electric field in an electrophoresis tank. DNA is negatively charged, and when an electric current is applied during electrophoresis, the DNA molecules will move from the cathode (coloured black) end towards the anode (coloured red) end of the electrophoresis tank.

The EtBr intercalate between the nitrogenous bases of the double stranded DNA molecule, and this causes the DNA molecule to fluoresce or produce an orange colour when the gel carrying the DNA fragments is photographed or illuminated with UV light. Electrophoretic technique is the most versatile method of analyzing, identifying and purifying the fragments of nucleic acid molecules (DNA and RNA) and proteins; and it is unique because it separates macromolecules according to their sizes and charges. Agarose and polyacrylamide are the two notable gels used in electrophoresis experiment. While agarose gel is used in most simple electrophoresis techniques (e.g., separation of nucleic acid molecules), polyacrylamide gel is mainly used in advanced electrophoresis such as those that has to do with protein separation. Several electrophoresis techniques are available and they include:

  • Agarose gel electrophoresis,
  • Pulse field gel electrophoresis (PFGE), and
  • Polyacrylamide gel electrophoresis (PAGE) amongst others.

FURTHER READING

Alberts B, Bray D, Lewis J, Raff M, Roberts K and Watson J.D (2002). The molecular Biology of the Cell. Fourth edition. New York, Garland, USA.

Cooper G.M and Hausman R.E (2004). The cell: A Molecular Approach. Third edition. ASM Press.

Dale J (2003). Molecular genetics of bacteria. Jeremy W. Dale and Simon Park (4th eds.). John Wiley & Sons Ltd, West Sussex, UK. Pp. 312-313.

Lewis R (2004). Human Genetics: Concepts and Applications. Sixth edition. McGraw Hill Publishers, USA.

Robert L. Nussbaum, Roderick R. McInnes and Huntington F. Willard (2001). Genetics in Medicine. Philadelphia, USA. Saunders publishers.

Sambrook, J., Russell, D.W. (2001). Molecular Cloning: a Laboratory Manual, 3rd edn. Cold Spring Harbor Laboratory Press, New York.

Tamarin Robert H (2002). Principles of Genetics. Seventh edition. Tata McGraw-Hill Publishing Co Ltd, Delhi.     

Twyman R.M (1998). Advanced Molecular Biology: A Concise Reference. Bios Scientific Publishers. Oxford, UK.

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