Gel Electrophoresis
Gel Electrophoresis | Multimedia | Activities
DNA gel electrophoresis is a method scientists use to separate and analyse DNA fragments based on size. The DNA can be extracted from cells and digested by restriction enzymes, taken from a polymerase chain reaction, or from a set of plasmids. When the DNA is run through the gel, the final result is a sequence of bands corresponding to different sizes of DNA.
Molten agarose is poured into a mould, and a comb is inserted in the liquid agar to form wells. Agarose gel has a number of pores. The size of these pores determines how quickly DNA can move through the gel. Larger pieces of DNA cannot move as quickly as small pieces through the pores, so the larger pieces end up closer to the start of the gel.
The gel is then placed into a gel electrophoresis tank filled with buffer. The buffer keeps pH levels constant, and acts as an electrical conductor for the gel.
Next, DNA (usually in a solution) is added to the wells of the gel, as well as a tracking dye and a reference lane. The tracking dye allows the scientist to track the movement of the DNA in the gel, so that the DNA does not run off the end of the gel. The reference lane contains various pieces of DNA with known sizes, so that scientists can compare the unknown DNA pieces to the known DNA in the reference lane, in order to develop a sense of size for the unknown DNA
Finally, the power supply is turned on. DNA is negatively charged, and because of this, will move towards the positively charged end of the gel electrophoresis tank, being repelled by the negative charged end.
DNA is not visible on the gel, so a dye must be applied in order to be able to see the DNA bands. The most common dye, and the one used in the Gene Technology workshop, is methylene blue. The gel is covered in methylene blue, which binds to the DNA. The excess dye is washed off in warm water, and the DNA bands become clearly visible.
Another option is to add ethidium bromide to the DNA before running the gel. The ethidium bromide intercalates between bases, and glows under UV light. Once the gel has been run, it can be placed under a UV light to identify the bands of DNA. This second method is useful for detecting minute amounts of DNA, but carries more safety hazards than the methylene blue method. The methylene blue method is usually sufficient for most gels, especially in a secondary school setting.
Molten agarose is poured into a mould, and a comb is inserted in the liquid agar to form wells. Agarose gel has a number of pores. The size of these pores determines how quickly DNA can move through the gel. Larger pieces of DNA cannot move as quickly as small pieces through the pores, so the larger pieces end up closer to the start of the gel.
The gel is then placed into a gel electrophoresis tank filled with buffer. The buffer keeps pH levels constant, and acts as an electrical conductor for the gel.
Next, DNA (usually in a solution) is added to the wells of the gel, as well as a tracking dye and a reference lane. The tracking dye allows the scientist to track the movement of the DNA in the gel, so that the DNA does not run off the end of the gel. The reference lane contains various pieces of DNA with known sizes, so that scientists can compare the unknown DNA pieces to the known DNA in the reference lane, in order to develop a sense of size for the unknown DNA
Finally, the power supply is turned on. DNA is negatively charged, and because of this, will move towards the positively charged end of the gel electrophoresis tank, being repelled by the negative charged end.
DNA is not visible on the gel, so a dye must be applied in order to be able to see the DNA bands. The most common dye, and the one used in the Gene Technology workshop, is methylene blue. The gel is covered in methylene blue, which binds to the DNA. The excess dye is washed off in warm water, and the DNA bands become clearly visible.
Another option is to add ethidium bromide to the DNA before running the gel. The ethidium bromide intercalates between bases, and glows under UV light. Once the gel has been run, it can be placed under a UV light to identify the bands of DNA. This second method is useful for detecting minute amounts of DNA, but carries more safety hazards than the methylene blue method. The methylene blue method is usually sufficient for most gels, especially in a secondary school setting.
Applications of Gel Electrophoresis
Gel electrophoresis is common in gene technology, and serves many different purposes. One of the most important is its use in forensic science, DNA fingerprinting is a method of comparing DNA samples. DNA can be extracted from blood, hair, skin or other human cells found at a crime scene or obtained from suspects. Both the original piece of DNA and the suspects’ DNA are analysed using gel electrophoresis. On the gel, several positions on the chromosome, called loci, are tested. Each locus is compared to the same locus on other people’s DNA. It is possible that two people can have identical sequences at one locus, so typically between 7 and 12 loci are tested to ensure a match.
Example of Gel Electrophoresis used in forensic science
Another application of gel electrophoresis is in paternity testing. A child’s genetic markers will be a mixture of his or her mother and father’s. Any bands present in the child’s DNA, but not in the mother’s DNA, must belong to the father. By comparing the bands, the child’s father can be determined.
Example of Gel Electrophoresis used in Paternity Testing
Research scientists often use plasmids to study genes or small sections of DNA. Plasmids are made by digesting a strand of DNA using a particular restriction enzyme, and isolating a region of interest. An existing plasmid, that often carries a gene for antibiotic resistance, is digested using the same restriction enzyme. The sticky ends on both the DNA region of interest can combine with the sticky ends of the plasmid, forming recombinant DNA plasmids. Plasmids can be inserted into bacteria, and expressed alongside the normal DNA genome. This same plasmid can be isolated from the bacteria, cut with the same restriction enzymes and run through a gel. This is done in order to ensure that the region of interest is present in the plasmid. Typically, there will be two dark areas on the gel. The large streak will be the original plasmid, the smaller band will be the region of interest.
Example of Gel Electrophoresis used in Plasmid Testing
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Example of Gel Electrophoresis used in forensic science
Another application of gel electrophoresis is in paternity testing. A child’s genetic markers will be a mixture of his or her mother and father’s. Any bands present in the child’s DNA, but not in the mother’s DNA, must belong to the father. By comparing the bands, the child’s father can be determined.
Example of Gel Electrophoresis used in Paternity Testing
Research scientists often use plasmids to study genes or small sections of DNA. Plasmids are made by digesting a strand of DNA using a particular restriction enzyme, and isolating a region of interest. An existing plasmid, that often carries a gene for antibiotic resistance, is digested using the same restriction enzyme. The sticky ends on both the DNA region of interest can combine with the sticky ends of the plasmid, forming recombinant DNA plasmids. Plasmids can be inserted into bacteria, and expressed alongside the normal DNA genome. This same plasmid can be isolated from the bacteria, cut with the same restriction enzymes and run through a gel. This is done in order to ensure that the region of interest is present in the plasmid. Typically, there will be two dark areas on the gel. The large streak will be the original plasmid, the smaller band will be the region of interest.
Example of Gel Electrophoresis used in Plasmid Testing
The movies on this page require the Macromedia Flash player browser plug-in. It can be downloaded for free from the Macromedia web site. External Link: Get Flash Player.