Gel electrophoresis
Gel electrophoresis is a process in which individual fragments of DNA, proteins, nucleic acids, or other nanoparticles are separated by charge and size through an agarose gel with an electric current. Originally developed in the 1950s by Oliver Smithies, gel electrophoresis has been used ever since in the fields of microbiology, biochemistry, and even forensics. In addition to enabling scientists to observe differences in fragment size and isolate individual samples, gel electrophoresis can be used to provide a highly accurate DNA fingerprint that remains unique to each individual. As the study of electrophoresis has developed over time, numerous other electrophoretic methods have also been devised to better suit individual applications.
History
In the early decades of the twentieth century, scientists such as W. B. Hardy began to observe and understand various electrical properties of common biological substances. Proteins, amino acids, and several known enzymes had a predictable charge and would therefore migrate in predictable ways when stimulated with an electric field, allowing the separation of individual types of particles.[1] To describe this technique, the term "electrophoresis" became popular among chemists and biologists, derived from the Greek word "phoresis" which means "being carried."[2] Early forms of electrophoresis used a liquid solution as a migration medium.[1] In 1937, Swedish biochemist Arnes Tiselius, considered the pioneer of electrophoresis, used a liquid medium in a u-shaped tank with an electrode at either end to separate mixtures of substances into individual solutes.[2] Soon after, Michaelis L. Elektrische was able to determine the isoelectric point of several enzymes that had not previously been isolated.[1]
As the use of electrophoretic techniques became increasingly popular, several other forms were developed to perform various functions. Paper electrophoresis, developed by Dionysius von Klobusitzky and Konig, used paper strips saturated in a electrically conductive solution.[1] Substances such as snake venom could successfully be separated into individual, relatively distinct phases based on their charge. In the 1950s Oliver Smithies introduced gel electrophoresis, which used an agarose semi-solid medium that produced distinct bands of individual substances. Originally used for bioanalytical experiments, gel electrophoresis later became one of the most commonly used forms of electrophoresis, with applications from mass spectrophotometry to DNA sequencing.[2] In the 1960s, another new form known as capillary electrophoresis became popular in clinical chemistry.[3]
Process
The first step in gel electrophoresis is the casting of the gel. Most often, the gel consists of agarose (chains of sugar molecules extracted from seaweed) that is dissolved in the buffer TAE (Tris-Acetate-EDTA). Depending on the specific protocol, different agarose percentages are used. These percentages, commonly 0.7%, 0.8%, 1%, and 1.2%, represent a weight-volume ratio for grams of agarose powder and milliliters of liquid TAE.[4] For example, a 1.2% agarose gel would require 1.2 grams of agarose mixed in 100 ml of TAE. Once prepared, the solution may require heating in order to make it pourable for the casting tray. This heating can be done with a microwave. The next step involves preparing the gel casting tray. These trays are often available for purchase and generally range in volume from 30ml to 250ml. Block both open sides of the casting tray using either the dams (provided along with the casting tray) or masking tape. Ensure that no leaks are present and that the tray can be filled sufficiently. One or more gel combs, ranging in number of teeth, must now be placed into the tray. Generally, combs are placed near one edge and optionally near the center. These combs will produce small wells in the gel that will house DNA and dye samples. Using heat protective gloves, pour the warm agarose solution into the casting tray, making sure the solution passes several centimeters up the comb teeth. To prevent wells from cross contaminating, also ensure that the solution does not reach the comb frame.[5] Once poured, the gel will slowly cool and solidify, marked by an opaque color. The gel will have the consistency of a dense jello.[6] Carefully remove the dams from the side of the tray and slowly remove the gel combs, ensuring no tears occur in the gel.[4]
Carefully place the casting tray containing the gel into an appropriately-sized electrophoresis chamber with the wells closer to the negative electrode. During electrophoresis, most dyes and all DNA will migrate toward the positive electrode. Next 1x TAE buffer is poured into the entire electrophoresis chamber, covering the gel in at least 3mm of buffer. TAE buffer allows an electrical charge to flow through the chamber. The samples of DNA or dye are now ready to be dispensed into the gel. Using a 20mm pipetman, carefully extract your first sample from its vial using a disposable tip. Using both hands, carefully position the pipet tip directly above the first well and slowly depress the plunger to release your sample. Use a new disposable tip for each sample. Once all the samples have been loaded and recorded, an electrophoresis power supply is required to administer an electrical charge to the gel. Place the chamber lid on the chamber, ensuring the black wire is connected to the black electrode and the red wire is connected to the red electrode.[5] The amount of voltage varies with the specific experiment. Using the controls on the power supply, set the voltage appropriately and press the start/run button. Initially, small bubbles should be seen around each electrode, indicating that electrolysis is taking place and an electrical charge is successfully flowing through the chamber.[7] Over time, individual bands of DNA or dye will migrate through the gel. Because DNA is negatively charged, it will always migrate toward the positive electrode. Larger strands of DNA will migrate slower as the gel matrix impedes its progress. Smaller strands will migrate more quickly. The resulting patterns of bands will reveal not only the relative sizes of DNA fragments, but will also provide a pattern unique to a specific sample of DNA. This will allow future comparison and identification with unknown samples.[5]
Uses
Gel electrophoresis has several uses in microbiology, forensics, and biochemistry. Primarily, this technique is used to isolate certain strands of DNA, proteins, or nucleic acids.[8] In criminal forensics, gel electrophoresis can be used to create a DNA fingerprint that is unique to each individual.[9] (This process is discussed in depth in the next section.) In microbiology, electrophoresis is used to separate DNA fragments by size. Restriction enzymes are added to an organism's DNA sample, which splices the DNA at specific base sequences. Since the splicing sites of each restrictive enzyme are known, biochemists can treat a DNA sample and subject the resulting fragments to electrophoresis. The desired fragment can now be removed from the gel and analyzed independently.[8] This process can also assist biologists in cloning. In order to purify DNA from a cell culture or test the efficacy of available restriction enzymes, gel electrophoresis is employed. After the DNA has been isolated, this section of the gel can be cut out and the DNA extracted for cloning or genetic engineering.[10] Virology, the study of viruses and other pathogens, also uses gel electrophoresis to identify specific strands of viruses based on their distance of migration through the gel.[11] Additionally, electrophoresis can be used to determine the presence of a knockout gene or genetic insert in an organism's genome, which are natural or artificial additions or deletions of a specific DNA strand.[12]
The separation of proteins through gel electrophoresis involves slightly more complex methods. Proteins vary widely in size and charge, unlike nucleic acids and DNA, and may not migrate through the gel at predictable rates or may not migrate at all. To combat this problem, proteins are often denatured, which removes its three dimensional shape native in cells.[13] The proteins are coated in a detergent, which applies a negative charge to each protein. The most common protein detergent is sodium dodecyl sulfate (SDS), which applies a negative charge proportional to protein size.[14] This allows biochemists to effectively isolate certain types of proteins.[13]
DNA Fingerprinting
DNA fingerprinting is used to differentiate between the DNA of two individuals and determine if an unknown biological sample belongs to a known individual.[9] This can aid in criminal investigations or can be used to identify a type of microbe in a dish sample.[11] The first step in producing a DNA fingerprint is to obtain a DNA sample. Each individual's genome contains variable number tandem repeats (VNTRs), which are repeating patterns of DNA base sequences found in non-coding regions of the DNA.[9] Because these sequences do not code for any proteins, mutations occur much more frequently in these regions, further distinguishing these sections from person to person.[15] The number and type of VNTRs in each person remains unique. To increase the amount of an individual's DNA to work with, a process known as polymerase chain reaction (PCR) is performed. During PCR, target sequences of DNA are duplicated several times, the number of times dependent on the individual's unique DNA. Then restriction enzymes will be introduced into the DNA samples, splicing DNA into uniquely sized strands. These strands of different lengths are referred to as restriction fragment length polymorphisms (RFLPs).[9]
Gel electrophoresis is then used to separate out the DNA lengths in a patterns of bands. Often this pattern will be compared with a sample of known origins in order to determine the origins of the unknown sample. For example, a blood sample found at a crime scene may be compared with a known DNA fingerprint from a suspect. If they match, the DNA is said to be from the same individual.[11] In modern DNA fingerprinting, additional steps are taken to ensure that each individual has a unique DNA fingerprint. The series of patterns from the agarose gel may be drawn out of the gel with a nylon membrane, which will be treated with chemicals to divide the DNA strands into single-stranded DNA similar to RNA. Radioactive probes are then injected, which contain their own base sequences that only bind to their corresponding sites on the DNA samples. Under UV light or photographic film, these radioactive probes will glow, revealing another layer of unique patterns that correspond not only to fragment length, but also specific fragment sequence.[9]
Video
Gel electrophoresis is a relatively simple procedure with a variety of applications.
References
- ↑ 1.0 1.1 1.2 1.3 IDT. Gel Electrophoresis Integrated DNA Technologies. Web. Published 2005.
- ↑ 2.0 2.1 2.2 Abdel-Latif, MonzirElectrophoresis Monzir-Pal.net. Web. Accessed 20 April 2015.
- ↑ Precissi, Juliet. Capillary Electrophoresis UC David Chem Wiki. Web. Accessed 20 April 2015.
- ↑ 4.0 4.1 Author Unknown. How To Make an Agarose Gel for Eletrophoresis University of Michigan. Web. Accessed 20 April 2015.
- ↑ 5.0 5.1 5.2 Author Unknown. Agarose Gel Electrophoresis AddGene. Web. Accessed 20 April 2015.
- ↑ Mayrand, Sandra. Introduction to Gel Electrophoresis University of Massachusetts. Web. Accessed 20 April 2015.
- ↑ Owen. Agarose Gel Electrophoresis Colorado State University. Web. Updated 15 January 2000.
- ↑ 8.0 8.1 Gel Electrophoresis Biotechnology Learning Center. Web. Published 20 November 2007.
- ↑ 9.0 9.1 9.2 9.3 9.4 DNA Fingerprinting California State University, Northridge. Web. Accessed 26 Aprl 2015.
- ↑ Agarose Gel Electrophoresis Cloning Virginia Tech. Web. Accessed 26 April 2015.
- ↑ 11.0 11.1 11.2 Freeman, Mary. What Can Gel Electrophoresis Be Used For? eHow. Web. Accessed 26 April 2015.
- ↑ Phillips, Theresa. Knockouts Biotech Syle About.com. Web. Accessed 26 April 2015.
- ↑ 13.0 13.1 Berg, Jeremy. The Purification of Proteins National Central for Biotechnology Information. Web. Published 2002.
- ↑ Campbell.SDS-PAGE Davidson College. Web. Published 2001.
- ↑ Goltsov.Associations between mutations and a VNTR in the human phenylalanine hydroxylase Gene US National Library of Medicine. Web. Published September 1992.
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