Figure 1. Side view of a gel electrophoresis chamber.
Objectives
Activities
Discussion
Questions
Glossary
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Gel electrophoresis is a technique used to separate DNA fragments based on size, yielding a unique “fingerprint.” At this workstation you will learn these techniques and how they are applied.
Objectives: top
1. Discuss the purpose of
using gels to visualize DNA.
2. Load gels.
3. Correlate the size of DNA fragments to their respective movements through
the gel.
4. Correlate the proteins sizes and shapes to their respective movements through
the gel.
5. Explain the use of restriction enzymes.
6. Analyze a gel and make a diagnosis regarding sickle cell anemia.
Activities: top
1. View computer animation.
2. Use restriction enzymes to cut DNA.
3. Load gels with DNA.
4. View gels at different stages of migration.
5. Watch demonstration of reading gels using ultraviolet light.
6. View gels with normal and mutated genes.
Discussion: top
Gel electrophoresis uses positive and negative charges to separate charged particles (see Figure 1). Electricity travels through a buffer solution in the electrophoresis chamber. The buffer allows the electric current to flow from the cathode (negative electrode) to the anode (positive electrode). DNA is negatively charged; therefore it travels toward the positive electrode.
Melted agarose gel is initially poured into a casting tray and a comb is added. After the agarose gel has cooled and solidified, the comb is removed resulting in the formation of wells. The gel is placed inside the electrophoresis chamber containing the buffer solution and DNA is then loaded into the wells. After loading the samples of DNA, the power supply is turned on allowing the DNA to separate.
Figure 1. Side view of a gel electrophoresis chamber.
Gel electrophoresis is a
technique used to separate DNA fragments based on size, yielding a unique “fingerprint.”
Because the DNA is too large to fit through the pores of the agarose gel, the
DNA must be digested, or cut into smaller fragments, using a restriction enzyme.
A restriction enzyme recognizes a particular sequence of bases on the DNA helix
and breaks the DNA at that point. These recognition sites are palindromic in
that the 5’ -to- 3’ base sequences on each of the two strands are
the same.
Below two common restriction enzymes:
| EcoRI: | 5' GAATTC 3' | HindIII: | 5' AAGCTT 3' |
| 3' CTTAAG 5' | 3' TTCGAA 5' |
Under appropriate conditions, a given restriction enzyme will cleave a piece of DNA into a series of fragments. The resulting fragments are small enough to maneuver through the pores of the agarose gel. Fragments that are smallest in size are able to travel the farthest from the well; whereas large fragments travel only a short distance from the well (See Figure 2).
Figure 2. Relationship between length of DNA fragment and migration distance from the well.
DNA is often stained because it is colorless. When staining DNA fragments, it is common to expose the gel to single stranded, radioactive DNA probes that have a known sequence of bases. This probe will pair with portions of the DNA fragments that contain its complementary base sequence. This allows scientists to identify the relative lengths of the DNA fragments and to only observe bands containing a complimentary sequence for the radioactive probe. Once the bands are exposed, the unknown samples can be compared to known substances in order to determine similarities or differences in their composition (see Figure 3).
Figure 3.
Comparison of known gel results for normal hemoglobin (AA), sickle cell disease
(SS) and sickle cell trait (AS). S represents the molecular size marker. What
results are present in the lanes marked with a question mark?
Questions: top
1. What is the net charge
of a DNA molecule? Does this cause DNA to travel toward the positive or negative
electrode? Why?
2. What is used to cut DNA?
3. Describe how DNA fragments migrate through a gel.
4. What is the composition of the loading buffer and what purpose does it serve?
5. What is the name of the dye that is used to monitor the rate of migration
of DNA?
6. What would happen to the DNA if the electrodes were reversed?
7. What happens to the DNA when the agarose gel is placed in a solution containing
ethidium bromide?
8. What is the purpose of the ultraviolet light?
9. What is the purpose of running molecular weight standards?
10. What is the approximate size of the fastest and the slowest fragments of
DNA on this gel?
Glossary: top
Agarose gel: A porous matrix that slows the movement of charged molecules of DNA in an electrical field, causing larger fragments to move more slowly than smaller fragments.
Anode: A positive electrode.
Cathode: A negative electrode.
Electrophoresis: The movement and separation of charged molecules in solution in an electrical field.
Restriction enzyme:
An enzyme that cuts double-stranded DNA at specific sequences.