Gel electrophoresis

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Gel electrophoresis
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DNA electrophoresis apparatus. An agarose gel is placed in this buffer-filled box and electrical current is applied via the power supply to the rear. The negative terminal is at the far end (black wire), so DNA migrates towards the camera.
Classification Electrophoresis
Other Techniques
Related Capillary electrophoresis
SDS-PAGE
Two-dimensional gel electrophoresis
Temperature gradient gel electrophoresis

Gel electrophoresis is the separation of deoxyribonucleic acid, ribonucleic acid, or protein through an electric charge.[1] It is usually performed for analytical purposes, but may be used as a preparative technique to partially purify molecules prior to use of other methods such as mass spectrometry, PCR, cloning, DNA sequencing, or immuno-blotting for further characterization.

Separation

"Gel", refers to the matrix used to separate the molecules. In most cases the gel is a crosslinked polymer whose composition and porosity is chosen based on the weight and composition of the target of the analysis. When separating proteins or small nucleic acids (DNA, RNA, or oligonucleotides) the gel is usually made with different concentrations of acrylamide and a cross-linker, producing different sized mesh networks of polyacrylamide. When separating larger nucleic acids (greater than a few hundred bases), the preferred matrix is purified agarose (a component of agar which is a red seaweed extract). In both cases, the gel forms a solid but porous matrix that looks and feels like clear Jell-O. Acrylamide, in contrast to polyacrylamide, is a neurotoxin and needs to be handled using Good Laboratory Practices (GLP) to avoid poisoning.

"Electrophoresis", refers to the electromotive force (EMF) that is used to push or pull the molecules through the gel matrix; by placing the molecules in wells in the gel and applying an electric current, the molecules will move through the matrix at different rates, towards the anode if negatively charged or towards the cathode if positively charged (note that gel electrophoresis operates as an electrolytic cell; the anode is positive and the cathode is negative).

Visualization

File:AgaroseGel.jpg
DNA agarose gel. The first lane contains a DNA ladder for sizing, and the other four lanes show variously-sized DNA fragment that are present in some but not all of the samples.

After the electrophoresis runs, when the smallest molecules have almost reached the anode, the molecules in the gel can be stained to make them visible. Ethidium bromide, silver, or coomassie blue dye can be used. Other methods can also be used to visualize the separation of the mixture's components on the gel. If the analyte molecules fluoresce under ultraviolet light, a photograph can be taken of the gel under ultraviolet light. If the molecules to be separated contain radioactive atoms, an autoradiogram can be recorded of the gel (as in the example shown here).

If several mixtures have initially been injected next to each other, they will run parallel in individual lanes. Depending on the number of different molecules, each lane shows separation of the components from the original mixture as one or more distinct bands, one band per component. Incomplete separation of the components can lead to overlapping bands, or to indistinguishable smears representing multiple unresolved components.

Bands in different lanes that end up at the same distance from the top contain molecules that passed through the gel with the same speed, which usually means they are approximately the same size. There are molecular weight size markers available that contain a mixture of molecules of known sizes. If such a marker was run on one lane in the gel parallel to the unknown samples, the bands observed can be compared to those of the unknown in order to determine their size. The distance a band travels is approximately inversely proportional to the logarithm of the size of the molecule.

Applications

Gel electrophoresis is used in forensics, molecular biology, genetics, microbiology and biochemistry. The results can be analyzed quantitatively by visualizing the gel with UV light and a gel imaging device. The image is recorded with a computer operated camera, and the intensity of the band or spot of interest is measured and compared against standard or markers loaded on the same gel. The measurement and analysis are mostly done with specialized software.

Nucleic acids

In the case of nucleic acids, the direction of migration, from negative to positive electrodes, is due to the natural negative charge carried on their sugar-phosphate backbone.[2] Double-stranded DNA fragments naturally behave as long rods, so their migration through the gel is relative to their radius of gyration, or, for non-cyclic fragments, roughly size. Single-stranded DNA or RNA tend to fold up into molecules with complex shapes and migrate through the gel in a complicated manner based on their tertiary structure. Therefore, agents that disrupt the hydrogen bonds, such as sodium hydroxide or formamide, are used to denature the nucleic acids and cause them to behave as long rods again.

Gel electrophoresis of large DNA or RNA is usually done by agarose gel electrophoresis. See the "Chain termination method" page for an example of a polyacrylamide DNA sequencing gel.

Proteins

File:SDSPAGE.png
SDS-PAGE autoradiography. The indicated proteins are present in different concentrations in the two samples.

Proteins, on the other hand, can have different charges and complex shapes, therefore they may not migrate into the gel at similar rates, or at all, when placing a negative to positive EMF on the sample. Proteins therefore, are usually denatured in the presence of a detergent such as sodium dodecyl sulfate/sodium dodecyl phosphate (SDS/SDP) that coats the proteins with a negative charge.[1] Generally, the amount of SDS bound is relative to the size of the protein (usually 1.4g SDS per gram of protein), so that the resulting denatured proteins have an overall negative charge, and all the proteins have a similar charge to mass ratio. Since denatured proteins act like long rods instead of having a complex tertiary shape, the rate at which the resulting SDS coated proteins migrate in the gel is relative only to its size and not its charge or shape.[1]

Proteins are usually analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), by native gel electrophoresis, by quantitative preparative native continuous polyacrylamide gel electrophoresis (QPNC-PAGE), or by 2-D electrophoresis.

History

A 1959 book on electrophoresis by Milan Bier cites references from the 1800s.[3] However, Oliver Smithies made significant contributions. Bier states: "The method of Smithies ... is finding wide application because of its unique separatory power." Taken in context, Bier clearly implies that Smithies' method is an improvement.

See also

References

  1. 1.0 1.1 1.2 Berg JM, Tymoczko JL Stryer L (2002). Molecular Cell Biology (5th ed. ed.). WH Freeman. ISBN 0-7167-4955-6.
  2. Lodish H, Berk A, Matsudaira P; et al. (2004). Molecular Cell Biology (5th ed. ed.). WH Freeman: New York, NY. ISBN 978-0716743668.
  3. Milan Bier (ed.) (1959). Electrophoresis. Theory, Methods and Applications (3rd printing ed.). Academic Press. p. 225. OCLC 1175404. LCC 59-7676.

External links

ar:فصل كهربائي للهلام ca:Gel d'electroforesi de:Gelelektrophorese id:Elektroforesis gel nl:Gelelektroforese ur:ھلامہ برقی رحلان



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