Electrophoresis separation methods
Electrophoresis separation methods are analytical techniques that utilize electrical fields to separate and analyze charged molecules. This method is particularly significant in forensic science, where it is frequently employed for tasks such as DNA fingerprinting and drug testing. During electrophoresis, charged molecules, including biological substances like nucleic acids and proteins, migrate through a medium in response to an electric current, allowing for their separation based on size, shape, and charge.
The process typically involves placing samples in a gel, such as agarose or polyacrylamide, which acts as a molecular sieve. As the molecules move through the gel toward the positive electrode, smaller molecules tend to migrate faster than larger ones. The separated molecules can then be visualized using fluorescent dyes. Different types of gels provide varying resolutions; agarose gels are suitable for larger DNA fragments, while polyacrylamide gels offer high resolution for smaller fragments.
Capillary electrophoresis (CE) is a modern adaptation of this technique, allowing for the automated separation of molecules based on their charge-to-size ratio in narrow capillary tubes. CE has gained popularity for applications such as DNA sequencing and forensic toxicology, where it enables rapid screening of biological samples for drugs and impurities. Overall, electrophoresis is a versatile and powerful tool in both biological and chemical analysis.
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Electrophoresis separation methods
DEFINITION: Analytical technique that uses electrical fields to separate and analyze charged molecules.
SIGNIFICANCE: When analyzing biological and nonbiological samples, such as when conducting DNA fingerprinting or drug testing, forensic scientists frequently use electrophoresis separation methods.
Electrophoresis is the migration of molecules in response to electrical fields. Any molecule that possesses an electrical charge—including biological molecules such as nucleic acids (DNA and RNA), proteins, and carbohydrates, as well as the molecules that make up many drugs—can be electrophoretically separated on the basis of size, shape, and electrical charge. Electrophoresis generally involves placing a sample within a supporting medium, applying an electrical current across the medium for a period of time, then visualizing the locations of molecules within the medium.
Electrophoresis of biological molecules such as DNA (deoxyribonucleic acid) commonly employs gels composed of agarose or polyacrylamide. These gels are porous and act as molecular sieves that retard the movement of molecules primarily on the basis of size. Generally, such a gel is in the form of a slab with wells formed at one end of the slab. The gel is placed in an apparatus with a positive electrode at one end and a negative electrode at the other end. The gel is submerged in a buffer solution that contains ions that will conduct an electrical charge. DNA molecules are negatively charged and will migrate through the gel toward the positive electrode. Smaller molecules will migrate more rapidly than larger molecules. The separated molecules can be stained with a fluorescent dye (most commonly ethidium bromide), and their positions within the gel can be compared with the positions of DNA molecules of known size.
Agarose gels have a large range of separation but a relatively low resolving power. With the alteration of the concentration of agarose in the gel, DNA fragments of two hundred to fifty thousand base pairs can be separated, making this method useful for forensic techniques such as short tandem repeat (STR) and Restriction fragment length polymorphism (RFLP) analyses. Polyacrylamide gels have a very high resolving power; DNA molecules that differ in size by a single nucleotide can be differentiated using polyacrylamide gel electrophoresis (PAGE). PAGE is usually used to separate DNA molecules less than five hundred base pairs in length, making it the method of choice for DNA sequencing.
Capillary electrophoresis (CE) is an automated extension of gel electrophoresis in which molecules are separated on the basis of the charge-to-size ratio of the molecules. The molecules do not migrate through a slab gel matrix; rather, the samples are electrophoresed through a conductive liquid or gel medium within a small-diameter (25- to 100-millimeter) capillary tube. CE has been used for DNA sequencing, STR analysis, and analysis of Polymerase chain reaction (PCR) products. Its use in forensic toxicology has received increasing attention, as it enables quick screening of biological fluids for a variety of illicit drugs as well as analysis of drug samples to detect impurities that may be unique to particular drug sources.
Bibliography
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