Gel electrophoresis is an essential tool for the characterization of proteins and fragments of DNA and RNA.
This technique exploits the differences in size and charge in different molecules in a sample. Each sample to be separated, plus a control, are loaded into wells at one end of a porous gel submerged in an ionic buffer medium. When an electrical charge is applied across the gel, test and reference molecules migrate across the medium at different rates. Various dyes such as fluorescent ethidium bromide, bromophenol blue, and xylene cyanol F make protein molecules, DNA, or RNA visible for easy evaluation of relative speeds during the process.
How Gel Electrophoresis Works
In gel electrophoresis, size matters.
The porous gel used in electrophoresis acts like a molecular sieve. It separates larger molecules from smaller ones. Bulkier, larger molecules take longer to migrate across the gel, while smaller molecules quickly move toward the cathode. The mobility of the molecules is also dependent on their charge.
There are some very specific laboratory skills required for doing gel electrophoresis. Buffer solutions need to be made for a specific molarity and a specific pH. Samples to be analyzed are loaded into preformed wells in the gel with a pipette, in equal aliquots. The voltage of the electrical current is set for expeditious but controlled separation of the putative target molecules. Placement of the samples and control in the gel must take into consideration the simple fact that positively charged molecules will migrate toward the cathode and negatively charged molecules will migrate toward the anode.
The speed at which molecules travel through the gel is known as electrophoretic motility. Simply stated, smaller molecules have greater electrophoretic motility than larger ones. Small size and strong charge increases electrophoretic motility, while large size and weak charge decreases it.
In experiments from Modern Biology, ethidium bromide serves as the dye to reveal the relative movement of the samples and the control. Illuminating the gel with UV light makes the separate bands visible. Electrophoresis bands have to be measured immediately, because they will diffuse over time. It is good technique to photograph the bands for future reference.
What Kinds of Analyses Involve Gel Electrophoresis?
Some of the key applications of gel electrophoresis are:
- DNA fingerprinting, also known as DNA profiling or genetic profiling. DNA taken from crime scenes is compared to DNA taken from suspects. This process can reveal that DNA from a crime scene is identical to the DNA of a suspect, but it can also show relationships between the person giving the sample and the person whose DNA was found at the scene of the crime.
- Analyzing the results of a polymerase chain reaction, also known as PCR. PCR takes a small strand of DNA and amplifies it. This makes genome sequencing possible. It also is useful for DNA profiling, testing for parentage, and detecting the DNA of pathogens, especially viruses.
- Real-time sex determination from bone fragments. Sometimes only bone remains after a criminal assault or in an ancient corpse. PCR and gel electrophoresis together can give a quick determination of the sex of the victim by their bones.
- Analyzing genes associated with a particular illness. Some pathogens, like the bacteria that cause tuberculosis, for example, are difficult to grow in the laboratory from sputum and fluid samples taken from patients. The combination of PCR and gel electrophoresis gives laboratories the ability to identify pathogens with small numbers of organisms, which can be dead or alive.
- Identifying toxins produced by pathogenic microorganisms. Pertussis, also known as whooping cough, is an example of a disease that generates toxins that bind to white blood cells so they cannot fight the infection. PCR and gel electrophoresis rapidly identify the toxin, so healthcare providers can rapidly identify the disease.
- DNA profiling for taxonomic studies to distinguish different species. For centuries, species of plants and animals were defined on the basis of easily observable, external characteristics. Modern taxonomy includes a consideration of DNA sequences, made possible by PCR plus gel electrophoresis.
- Genetic modification. PCR and gel electrophoresis are essential tools of both genetic modification and cloning. PCR creates numerous copies of a genetic sequence to be inserted into a GMO organism, those copies separated out by gel electrophoresis.
- The study of evolutionary relationships. Biologists know the genetic relationships of mastodons and modern elephants and Neanderthals and modern humans through the use of PCR and gel electrophoresis. The DNA in the bodies of Ice Age creatures is usually highly degraded. But PCR can be used to make copies of identifiable strands of DNA that can be separated out with gel electrophoresis.
As you can see, gel electrophoresis and PCR are inseparable laboratory skills. There has never been a more exciting time to teach biology, and there has never been a time when so much was expected of biology teachers. With products from Modern Biology, you can take your students beyond the mere retention of vocabulary words and testable facts to understanding scientific method and critical thinking.
