The Development of Gel Electrophoresis.
The first introduction of a gel electrophoresis was presented in 1950 by Gordon et al and was improved by Wieme by the introduction of the gel being supported on a glass slide. This cut out the need of the paper wicks thus preventing the adsorption of the voltage gradient2. The next major step was in 1953 when the concept of crossed immuno electrophoresis in agar gels. This proved to be a huge landmark in separation techniques. It showed that not only could they separate the complex matrix but were able to also detect directly on the same plate. Thus the beginning of 2D electrophoresis, as not only did it have the zone electrophoretic step, but also by using the correct anti-sera would activate the immuno-detection step by diffusion. The impact of this procedure was immense in biochemistry and molecular biology techniques. There was also a major impact in the study of disease for example the most famous Laurell’s Rocket technique1. This 2D technique showed to be a valuable tool for identification and quantification of proteins in an agarose gel. This procedure has been widely used in biomedical separations especially for the diagnosis of disease and the presence of antibodies in sera. Though these techniques are not as commonly used these days they did lay the foundations for western blotting techniques on 1D sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and 2D gels.
The Introduction of Perfection.
It was during the 1960s that polyacrylamide gels were introduced as they were very efficient in molecular sieving and molecular filtration. Then in 1967 the introduction of sodium dodecyl sulphate (SDS) to the polyacrylamide. This proved to give a very good resolution of membrane proteins. The SDS does have a disadvantage and that is it possesses a strong denaturing effect, although according to Vesterberg1 Hjerten and others showed that the hydrophobic membrane proteins showed less susceptibility to the denaturing effect of the SDS, than the hydrophilic proteins and were easily reactivated by adding an excess of neutral detergent. The SDS-PAGE has proved to be an important and valuable tool for the analysis and isolation of membrane proteins. This electrophoretic method is the most widely used in biomedical laboratories today. Estimation of protein size, assessment of the proteins purity, quantitation of proteins, monitor protein integrity, comparing composition of different samples, analysis of number and size of polypeptides, and for western blotting and also has a 2D capability. Throughout the 1970s techniques were changing and concepts of isotachophoresis and isoelectric focusing were being introduced, though the main aim throughout this decade was to find a way of miniaturising the equipment for analysis of certain biological samples4. Though this concept of smaller materials and the use of thin capillary like tubes was first introduced in 1970 it was not until the 80s capillary electrophoresis was introduced.
The idea of miniaturised equipment for certain samples example ribonucleotides came along well before modern capillary electrophoresis, it was in 1953 when fine silk fibres were used for micro electrophoresis to identify the ribonucleotides in single cells4. The effectiveness of capillary electrophoresis is very similar to a high performance liquid chromatography. This procedure took essential components from HPLC and electrophoresis and was more of an instrumental approach to electrophoresis. Although capillary electrophoresis has a relatively short history it has proved to be very effective for any bioanalysis. In contrast to slab gel electrophoresis capillary electrophoresis takes advantage of two types of driving force, the force causing the electrophoretic migration and the force caused by the electoosmotic flow through the capillary. It is comprised of fused silica capillary’s 20–100 μm in diameter and 20–100 cm long for the separation channel and is encased in a temperature-controlled cartridge. The inlets and outlets are placed in the sample and the buffer vial. Injection is by pressure or electrokinetic means. A sample vial is temporarily pressurised to allow the flow of sample into the capillary in the pressure method and the electrokinetic method uses the electric field to migrate the charged particles into the capillary4.
Key Roles of Electrophoresis in Understanding Molecular Biology.
Electrophoresis has played an important role in our understanding of molecular biology throughout the years of this young field of science. In the 1970s refinement of electrophoretic techniques made it possible to map the structure and sequence of DNA. Danna and Nathens5 established the length of fragments of SV40 DNA which had been produced by the new restriction endonuclease derived from Hemophilus Influenzae. The techniques also made it possible to illustrate the difference in migration of the linear and circular DNA of various different phages. Electrophoresis used in this field have also aided our understanding of gene technology and it is easy to say that without these tools along with the use of allied techniques such as blotting science would not of been able to start the biggest investigations to date the mapping of the human genome1. With the introduction of capillary electrophoresis it has made it possible to separate much smaller proteins and has advanced our knowledge in the structure and sequence of our DNA and has also been a successful tool in the field of gene technology by giving us the understanding of how the genes work and can be manipulated. The importance of chromosomal, nucleic acids electrophoresis is that it has enabled us to determine their sequence and understand the biological variability among all life forms, such as animals, humans, micro organisms and plants.
Key Roles of Electrophoresis in Understanding the Diagnosis of Disease.
Increased attentions have been focused towards proteins and their roles in diseases and their uses in the diagnosis of a disease. For example the techniques of electrophoresis have advanced our knowledge in the aetiology and pathology of HIV and its development to AIDS. As a result it has enabled us to understand the treatment of such diseases1. The developments of these techniques and others such as immuno-electrophoresis have shown great importance to studies in humans for the effects of drugs and chemicals enabling us to treat the disease with the right combination of drugs and therapy. Electrophoresis has also played a key role in the fight against cancer as we are able to cure some forms of the disease as we now understand its genetic code we are able to treat it more successfully. The techniques and the knowledge we have gained in the benefit of disease diagnosis due to electrophoresis have supplied us with a standard and by comparing patients results with the known standard enables us to diagnose rapidly. In research proteomics electrophoresis has been a successful piece of armoury to investigators in the investigation of protein expression changes that occur in a biological system, such as during disease development and progression, stress or drug exposure, or during normal cell and tissue development. This has provided important information on the pathways involved in these processes and can lead to the identification of new protein markers for the diagnosis or the treatment of diseases due to changing protein expression patterns. By performing a haemoglobin electrophoresis it has now made it possible to diagnose sickle cell disease in newborns as well as a fully grown adult6. Although solubility testing methods identify sickle haemoglobin, these tests are not as accurate as an electrophoresis test as they miss haemoglobin C and other genetic variants which would be seen in electrophoresis.
Conclusion.
Electrophoretic methods and techniques are now the most widely utilised analytical separations in molecular biology and disease diagnosis. This is due to its accuracy and benefits we get from the knowledge we obtain as of these methods. I have shown here a summary of the major developments in the history of electrophoresis, from its pioneering days of Arne Wilhelm Kaurin Tiselius and his theory of moving boundary electrophoresis through out the decades to present day and the development of capillary electrophoresis. I have pointed out the key roles and discoveries made in molecular biology and I have also presented a couple of examples on how these techniques have aided us in the understanding disease and diagnosis. Although methods of today are increasingly diversified and are used in many different fields of science with the right application electrophoresis will further mankind’s understanding and give a greater insight into different species genetics and further diagnosis and treatment of diseases such as AIDS and cancers. It will also provide us with the capability of the mapping of the human genome completely and with such knowledge will enable us to further investigate and possibly cure genetic disorders and terminal illnesses.
Barry Hollinshead.
WORD COUNT 1956
References.
- Olof Vesterberg. 1989. History of Electrophoretic Methods. Journal of Chromatography, 480, 3-19.
- Pier Giorgio Righetti. 2005. Electrophoresis: The March of Pennies, the March of Dimes. Journal of Chromatography A, 1079, 24-40.
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Nobel winners.com. Arne Wilhelm Kaurin Tiselius. Available: , last accessed 15/02/2006.
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Herb Schwartz. Andras Guttman. Separation of DNA by Capillary Electrophoresis. Available: , last accessed 10/02/2006.
- Brody J. R. Kern S.E. 2004. History and Principles of Conductive Media for Standard DNA Electrophoresis. Analytical Biochemistry, 333, 1-13.
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Doris L. Wethers, M.D., Sickle Cell Disease in Childhood: Part I. Laboratory Diagnosis, Pathophysiology and Health Maintenance. American Academy of Family Physicians. Available: , last accessed 17/02/2006.
