Gel Electrophoresis
Gel electrophoresis is a tool that separates different lengths of DNA. In forensics it is a routinely used in DNA Typing, drug analysis, explosive residues, gunshot residues, and forgery analysis. Because DNA's backbone contains many phosphate groups, fragments are negatively charged. This occurs because at neutral pH the phosphates in DNA readily give up their hydrogen ions to become negatively charged. When placed in a gel and subjected to an electric field DNA will migrate towards the positive anode and move away from the negative cathode.
Agarose is a commonly used material for making gel slabs. Agarose is a large polymer of sugar found in marine algae. The powdered seaweed is about 200nm in diameter. When mixed with a buffer solution, heated, and allowed to cool, DNA samples can be loaded into small wells. Each tiny well positioned at the cathode end of the gel box can hold between ~ 5-10µl of DNA. Submerged in buffer of Tris-Acetate (TAE) or Tris-Borate (TBE), the samples move towards the positive anode, at rates that depend on the length of their strands. The electrical field can be controlled by a power supply which can vary the voltage running to the electrode on each end of the gel box. Typically, a 10 x 40 cm gel requires 100-600 v to separate the DNA strands. 30
Because DNA fragments will move at speeds defined by their size (length), small fragments migrate at a faster rate than long fragments, thus at the end of gel run the DNA sample that was loaded into the gel well is separated into bands. If a DNA ladder has been run along with the sample, the relative length of each band can be measured. DNA ladder provide known fragments to compare samples against. An example of a DNA ladder is Lambda phage DNA. Lambda phage is digested using restriction enzymes, its resultant fragments are identify and thus later used as a genetic marker or DNA ladder. To view the fragment after its run dyes are loaded either before or after the run to enhance the viewing of the DNA fragments.
In order to visualize the banding pattern of DNA on a gel, dyes are often added to the gel. One type of commonly used dye is a fluorescent dye called Ethidium Bromide. EtBr binds between the bases of the DNA. When viewed under an ultraviolet illuminator, the fluorescent dye can be visualized and photographed. The drawback of using EtBr is that the chemical is carcinogenic and thus must be carefully handled.
Other gels made of polyacrylimide are available and are different than agarose gels because of their pore size. Polyacrylamide gels have a pore size of ~100-200 Å versus agarose whose pore size runs between ~1500 - 2000 Å. 31 In a high school laboratory class, agarose serves the purpose better because gel powders are relatively inexpensive.
Another electrophoresis method is capillary electrophoresis (CE). Capillary electrophoresis instruments are now routinely used in modern forensic DNA analyses. The advantages of CE are that it requires minute amounts of DNA, a result is available within minutes rather than hours, there are no gel slabs to cast and resolve, and the process is fully automated. CE has another major advantage because a sample can be separated with a single base difference. CE results are available electronically, making the photographing gel samples unnecessary. This printout or visual of a CE result is called an electropherogram. 32
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