5.3: DNA Gel Electrophoresis

One of the commonly used techniques to compare genomes is DNA gel electrophoresis. This technique is used to separate DNA fragments of different lengths resulting from a restriction enzyme digestion (using an enzyme to cut the DNA, essentially a DNA scissor). It takes advantage of the fact that a DNA molecule, or fragment, has an overall negative charge, and is thus attracted to a positive electric field. In addition to a digested DNA sample, DNA gel electrophoresis requires a gel electrophoresis chamber, which consists of a water-tight apparatus, with a positive electrode on one end and a negative electrode on the other, containing an agarose gel.

The agarose gel, which is typically the shape of a thin slab, is a semi-solid gelatin matrix that has tiny holes that DNA fragments can move through. The gel is placed into the chamber with the wells closest to the negative electrode. The chamber is then partially filled with a clear electrophoresis buffer, which contains ions that allow it to carry electrical current. The gel has several empty spaces on one end called loading wells into which the DNA samples are loaded. Once the DNA samples are loaded into the wells, the chamber is then connected to a power supply and voltage is applied. Because of DNA’s negative charge, fragments will migrate through the gel toward the positive end of the chamber. The smaller the DNA fragment, the faster it will migrate (run) through the gel matrix, resulting in the separation of the fragments based on size. Once electrophoresis is complete, the smallest DNA fragment in each well will be

closest to the positive electrode, and the largest DNA fragment will be closest to the loading well (the negative electrode). DNA fragments of the same size will collect at the same location in their lane, creating a band that can be seen. Any given band within a gel may have millions of DNA fragments of equal length.

a) Shown are DNA fragments from seven samples run on a gel, stained with a fluorescent dye, and viewed under UV light; and b) a researcher from International Rice Research Institute, reviewing DNA profiles using UV light. (credit: a: James Jacob, Tompkins Cortland Community College b: International Rice Research Institute)

Materials needed

1. Micropipette
2. Pipette tips
3. Gel electrophoresis chamber
4. Agarose gel
5. Practice loading gel
6. Distilled water
7. Pigment samples
8. Electrophoresis buffer
9. Power Supply

Activity

In this lab activity you will do the following

• Load a agarose gel with various samples using a micropipette
• Run the gel in an electrophoresis chamber
• Analyze the results

Practice Gel Loading

When working with DNA samples, you are measuring and dispensing volumes in microliters (μl). In order to accurately measure such tiny volumes requires a special device called a micropipette. Prior to working with your actual samples and your actual agarose gel, you will practice with the loading dye and a ‘practice gel’. After watching a demonstration of how to use a micropipette and load a gel, perform the following:

1. Cover your practice loading gel with water (to simulate gel electrophoresis buffer).
2. Load a tip onto your micropipette.
3. Draw up 10μl of practice loading dye:
   1. With your thumb, depress the micropipette plunger down to the first stop.
   2. Place the micropipette tip into the practice loading dye.
   3. Draw up practice loading dye by slowly releasing the plunger.
   4. Pull the tip out of the practice loading dye—you now have exactly 10μl of dye in the micropipette tip.
4. Carefully load the practice loading dye into a well of the practice loading gel:
   1. Place the tip of the micropipette into the well, being careful not to pierce the bottom of the gel with the tip (this would cause your sample to ‘leak’ out of the bottom of the gel).
   2. Dispense the dye into the well by slowly depressing the plunger to the second stop.
   3. Without releasing the plunger, pull the tip out of the well (if you release the plunger too soon, you will suck your sample back up).
   4. After the plunger is out of the well, slowly release the plunger.

5. Each group member should repeat steps 3-4 several times so that everyone is comfortable using a micropipette and loading a gel.
6. When finished, carefully remove the tip from the micropipette and dispose of it in the biohazard container.
7. Rinse your practice gel with cold tap water to remove the dye and return it to your table.

Gel Electrophoresis Activity:

1. Take the agar gel that has been set and solidified.
2. Remove the cover of the chamber by sliding it off while holding the chamber firmly.
3. Place the gel in the center of the electrophoresis chamber, oriented such that the wells are closest to the negative (black) electrode of the chamber.
4. If necessary, add more electrophoresis buffer to the chamber so that the top of the gel is covered by approximately 1cm of buffer.
5. Carefully load 10μl of each sample into the wells of the gel.
6. Place the cover on the gel electrophoresis chamber and connect the electrode terminals to the power source.
   1. Make sure that the negative (black) and positive (red) color-coded indicators on the cover and chamber are properly oriented.
   2. Likewise, make sure that the electrodes are plugged into the correct electrode terminals on the power source. Before proceeding to the next step, ask the instructor to inspect your setup.
7. Plug in and turn on the power source and set it to 75V. You should be able to see bubbles forming at each end of the chamber.
8. Run your gel for approximately 60 minutes.
   1. Caution: do NOT allow your gel to run too long, or else the fragments will literally run out of the end of the gel!
   2. Carefully monitor the progress of your samples every 5 minutes.
   3. Typically, the gel is done and should be turned off when the blue ‘fast dye’ has run approximately 2/3rds down the gel.
   4. When you think the gel has finished running, let your instructor know.
9. After electrophoresis is complete, turn off and unplug the power source, then carefully remove the cover from the chamber.
10. Using the figure below, draw a picture of your results (using color pencils) and answer the questions.
11. Follow your instructor’s instructions on how to clean and reset your gel electrophoresis experiment.

Questions

1. What would happen if you positioned the agarose gel backwards within the chamber, with the sample wells on the positive electrode end of the electrophoresis chamber?
2. Describe why larger (longer) fragments move through an agarose gel more slowly than smaller (shorter) fragments?

Group Discussion Activity

Your professor will separate you into small groups. You will be given a genomic scenario to discuss and questions to answer. The following are the possible scenarios.

Scenario 1: Amy and the breast cancer test

This scenario deals with a 15 year old girl (Amy) who has a family history of breast cancer. Her paternal aunt has been diagnosed with the disease and tests have shown that she is a carrier of the BRCA1 mutation associated with breast cancer. There is a dispute within the family about whether Amy’s father should be tested to find out whether he is also a carrier of the BRCA1 mutation, and may have passed the risk to Amy. Her mother does not want the test to take place. The decision is Amy’s. The group has to decide whether Amy should ask her father to take the test.

Scenario 2: Jill and the schizophrenia risk

This scenario is set in the future and assumes that people will be able to find out this information through a recreational genotyping kit. Summary: Scenario 2 deals with a woman (Jill) who has no knowledge of her father’s family history. She has a husband and a young baby and pays to be genotyped to get a better understanding of her genetics. She does not know that the test will reveal that she has a rare chromosome deletion, associated with a higher than normal risk of developing schizophrenia.

Scenario 3: Sam and the insurance company

This scenario is set in the future and assumes that insurance companies will be able to use genetic information to determine insurance premiums. Summary: Scenario 3 deals with a middle-aged man (Sam) with a family history of heart attacks. He wishes to purchase insurance but the insurers ask him to be genotyped. Sam’s brother (who has had heart problems himself) is keen for Sam to be tested. He feels that knowing if Sam is at high risk of heart disease will help him to take steps to avoid severe heart problems. Sam fears he will be refused insurance if he takes the test and is not certain he wants to know the results. His wife is firmly against genotyping. The group has to decide if Sam should take the test.

Scenario 4: Heather, her risk of Alzheimer’s and her reluctant twin

This scenario is set in the future and assumes that recreational genotyping has become commonplace. Summary: This scenario presents a young woman (Heather) who has an identical twin. Heather wishes to start a family with her husband and has decided to get herself genotyped. Her twin objects to the test and does not wish to know the results. The results show that the twins are carriers of two copies of the gene variant ApoE-e4, which puts them at high risk of developing Alzheimer’s disease. The group has to decide whether Heather should tell her sister or parents about the results. As the twins are identical, whatever is revealed about the genotype for one twin will match the genotype of the other. If the twins were non-identical the genetic similarities would only be as close as that of any other pair of siblings.

Scenario 5: Pete’s potential adverse drug reaction

This scenario is set in the future. A test for the genes linked to this drug reaction does currently exist, but is not currently widely available to patients. Summary: This scenario is centered on (Pete) whose doctor would like to prescribe the antibiotic flucloxacillin, since it is particularly effective for his type of infection. However, in rare cases flucloxacillin can cause a serious liver injury, and Pete is offered a test to determine whether he has a particular genotype associated with this severe reaction. Pete is concerned about the cost but his wife is keen for him to take the safe option. His teenage son is concerned about misuse of the data if it becomes part of his medical records. The group has to decide whether Pete should take the test.

Scenario 6: Should a baby have its genome sequenced?

This scenario is set in the future. It assumes that we are at a point where whole genome sequencing can be carried out cheaply and quickly. Summary: In this scenario expectant parents are asked whether they would like to have their child’s genome sequenced after it is born. The information would form part of the child’s ID card.

Scenario 7: Andy’s unexpected paternity results

This scenario is set in the future and assumes that recreational genotyping has reached the point that it is affordable for most people to purchase a genotyping kit as a gift. Summary: This scenario involves Andy, who buys a genotyping kit online. He finds it fun and therefore decides to buy one for his father. Looking through their results together, Andy notices that there are different markers on the Y chromosomes of him and his father, indicating that he is not Andy’s biological father. The group has to decide whether Andy should tell his father what he’s found.

Scenario 8: Should researchers share incidental findings?

This scenario is set in the present day. Summary: This scenario is about a researcher (Lin) who is working on a study into childhood developmental disorders. She discovers that one of the children in the study has a mutation associated with the cancer retinoblastoma. The study has a strict policy not to share incidental findings with participants. The group has to decide whether Lin should tell the child’s parents.

For review only as this link will not be included in the final draft.

CLICK HERE! To view the supporting materials for the listed scenarios