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...and shape of the molecules, relative hydrophobicity of the samples, and on the ionic strength and temperature of the buffer in which the molecules are moving. After staining, the separated macromolecules in each lane can be seen in a series of bands spread from one end of the gel to the other To completely understand the separation of charged particles in gel electrophoresis, it is important to look at some simple equations relating to electrophoresis. When a potential difference (voltage) is applied across the electrodes, it generates a potential gradient (E), which is the applied voltage (V) divided by the distance (d) between the electrodes. E = V / d When the potential gradient E is applied, the force on a molecule bearing a charge of q coulombs is Eq newtons. F = Eq It is this force that drives a charged molecule towards an electrode. There is also a frictional resistance that slows down the movement of this charged molecule. This frictional force is a measure of the hydrodynamic size of the molecule, the shape of the molecule, the pore size of the medium in which electrophoresisis taking place and viscosity of the buffer. The velocity (v) of a charged molecule in an electric field is given by the equation: v = Eq / f where f is the frictional coefficient. In electrophoresis, the force moving the macromolecule (nucleic acids or proteins) is the electrical potential, E. The electrophoretic mobility (???) of an ion is the ratio of the velocity of the particle, v, to the electrical potential. ??? = v / E Electrophoretic mobility is also equal to the net charge of the molecule, Z, divided by the frictional coefficient, f. ??? = Z / f When a potential difference is applied, molecules with different overall charges will begin to separate due to their different electrophoretic mobilities. Even molecules with similar charges will begin to separate if they have different molecular sizes, since they will experience different frictional forces. Some forms of electrophoresis rely almost totally on the different charges on molecules to effect separation, while other methods exploit differences in molecular size and therefore encourage frictional effects to bring about sepThe current in the solution between the electrodes is conducted mainly by the buffer ions with a small proportion being conducted by the sample ions. Ohm's law expresses the relationship between current (I), voltage (V), and resistance (R): R = V / I This equation demonstrates that it is possible to accelerate an electrophoretic separation by increasing the applied voltage, which would result in a corresponding increase in the current flow. The distance migrated will be proportional to both current and time. However, increasing the voltage would ignore one of the major problems for most forms of electrophoresis, namely the generation of heat. During electrophoresis the power (W, watts) generated in the supporting medium is given by W = I^2 R Most of this power generated is dissipated as heat. Heating of the electrophoretic medium can have the following effects: An increased rate of diffusion of sample and buffer ions leading to broadening of the separated smaples. The formation of convection currents, which leads to mixing of separated samples. Thermal instability of samples that are rather sensitive to heat. This may include denaturation of proteins or loss of activity of enzymes. A decrease of buffer viscosity, and hence a reduction in the resistance of the medium. Biotechnology is the manipulation of the biological capacity of cells and their components. For thousands of years people have used biotechnology by using yeast to make flour into bread and grapes into wine. We are now using biotechnology to study the basic processes of life, diagnose illnesses, and develop new treatments for diseases. Some of the tools of biotechnology are natural components of cells. Restriction enzymes are made by bacteria to protect themselves from viruses. They inactivate the viral DNA by cutting it in specific places. DNA ligase is an enzyme that exists in all cells and is responsible for joining together strands of DNA. Restriction enzymes can be used to cut DNA at specific sequences called recognition sites. They then rejoin the cut strands with DNA ligase to new combinations of genes. Recombinant DNA sequences contain genes from two or more organisms. Using this technique, researchers have gained the ability to diagnose diseases such as sickle cell anemia, cystic fibrosis, and Huntington's chorea early in the course of the disease. Many researchers are also applying the techniques of biotechnology to find new treatments for genetic diseases. DNA technology has triggered research advances in almost all fields of biology. The new techniques opened up the study of the molecular details of eukaryotic gene structure and function. Today, hundreds of useful products are produced by genetic engineering, the manipulation of genetic material for practical purposes. It has become routine to combine genes from different sources--often different species--in test tubes, and then transfer this recombinant DNA into living cells where it can be replicated and expressed. E. coli is often used because it is easy to grow and its biochemistry is well understood. The most important achievements resulting from recombinant DNA technology have been advances in our basic understanding of eukaryotic molecular biology.For example, only through the use of gene-splicing techniques have the details of eukaryotics gene arrangement and regulation been opened to experimental analysis. DNA technology has launched an industrial revolution in biotechnology, the use of living organisms or their compinents to perform practical tasks. Biotechnology based on the manipulation of DNA in vitro (outside of living cells) is distinct from earlier phases in that it is more precise. DNA technology is in the process of revolutionizing biological research, human medicine, criminal law, and agriculture. Gel Electrophoresis is one of the staple tools in molecular biology and is of critical value in many aspects of genetic manipulation and study. One use is the identification of particular DNA molecules by the band patterns they yield in gel electrophoresis after being cut with various restriction enzymes. Viral DNA, plasmid DNA, and particular segments of chromosomal DNA can all be identified in this way. Another use is the isolation and purification of individual fragments containing interesting genes, which can be recovered from the gel with full biological activity. Gel electrophoresis makes it possible to determine the genetic difference and the evolutionary relationship among species of plants and animals. Using this technology it is possible to separate and identify protein molecules that differ by as little as a single amino acid. The protein molecules in a sample of fish muscle tissue and plant grain endosperm tissue can be separated according to their individual molecular mass and compared to samples that have been treated with a reducing agent, such as 2-mercaptoethanol. Complex proteins (composed of two or more polypeptide chains) can be broken down into their respective polypeptide fractions. A reducing agent breaks the disulfide bonds that hold the polypeptide together. It is widely known that each individual has a DNA profile as unique as a fingerprint. Actually, over 99% of all 3 billion nucleotides in human DNA which we inherit from each parent are identical among all individuals. However, for every 1000 nucleotides that we inherit there is 1 site of variation or polymorphism, in the population. These DNA polymorphisms change the length of the DNA fragments produced by the digestion of restriction enzymes . The resulting fragments are called restriction fragments length polymorphisms (RFLP's--"riflips"). Gel electrophoresis can be used to separate and determine the size of the RFLPs. The exact number and size of fragments produced by a specific restriction enzyme digestion varies from individual to individual. DNA fingerprinting has proved valuable, not only for convicting felons and exonerating the innocent, but also for establishing maternity or paternity and proving family relationships. More exotic uses include the identification of missing children in Argentina, soldiers killed in war, and even the body of Nazi physician Joseph Mengele, the so-called "Angel of Death." The fundamental techniques involved in genetic fingerprinting were discovered serendipitously in 1984 by geneticist Alec J. Jeffreys of the University of Leicester in Great Britain while he was studying the gene for myoglobin, a protein that stores oxygen in muscle cells. He found that the myoglobin gene contains many segments that vary in size and composition from individual to individual and that have no apparent function. Jeffrey called these segments minisatellites because they were small and they surround the part of the gene that actually serves as a genetic bluprint. The minisatellites accountsfor less than 1 percent of hte total DNA of a human. Jeffreys isolated several of these minisatellite genes and inserted each into bacteria, which produced large amounts of the DNA segments. These segments could then be purified and labeled with radioactive isotopes to produce genetic probes that are the key tool in producing genetic fingerprints. Steps to Producing a Genetic Fingerprint: The first step is to obtain a sample of of DNA from such substances as blood, semen, hair roots, or saliva. Using newly developed biochemical techniques to multiply the amount of DNA present, researchers can work with as small a sample as one hair root. The individual cells from the sample are split open, and the DNA is separated from the rest of the cellular debris. The DNA is then treated with specilaized proteins called restriction enzymes, which cleave the DNA into smaller fragments by cutting at specific sites. Since the minisatellites from any two individuals have different compositions, they are cleaved at different sites, producing fragments of different lengths. The DNA fragments are then applied to one end of a thin, jellylike substance called an agarose gel, and an electric current is passed through th...