Kansas College and Career Ready Standards

Science

• HS-LS4-2. Construct an explanation based on evidence that the process of evolution primarily results from four factors…(2) the heritable genetic variation of individuals in a species due to mutation and sexual reproduction.

• HS-LS3-2. Ask questions to clarify relationships about the role of DNA and chromosomes in coding the instructions for characteristic traits passed from parents to offspring.

• HS-LS3-3. Apply concepts of statistics and probability to explain the variation and distribution of expressed traits in a population.

Learning Objectives

• Students will analyze DNA using gel electrophoresis technique.
• Students will use restriction enzymes to cut model sections of DNA.
• Students will model restriction enzyme digestion and gel electrophoresis using paper based techniques.
• Students will gain real world lab experiences utilizing pipettes and electrophoresis equipment.

Materials

• Yield by Design, Hybrid Seeds PowerPoint
• Yield by Design Student Worksheet
• Copies of Restriction Enzymes and Electrophoresis and DNA Sequences (pg. T10-11)
• Restriction Enzymes and Electrophoresis Graph (pg. S1-2)
• Copies of Hybrid Electrophoresis Lab Analysis Worksheet (pg. S3)
• Colored pencils, markers or highlighters
• Scissors
• Minione Crime Scene Genetics Kit
• Minione Electrophoresis Lab Station, including electrophoresis apparatus, variable volume micropipette (2-20 uL) and micropipette tips
• Solo cup labeled as trash for disposing of used tips
• 100 mL graduated cylinder
• 250 uL Erlymyer flask
• Prepared TBE buffer (provided in lab kit)
• Aliquoted samples of DNA

Safety Considerations

As with all labs, make sure your lab area is clean and free of debris and other unnecessary equipment.  Ensure the lab table is dry before plugging in any electrical equipment, and clean any spills  immediately. When removing gel, make sure you unplug the apparatus from the outlet before you take it out of the tank.

Procedures for Instruction

Length of Time for Preparation: 30-45 minutes

Length of Time for Classroom Teaching: 60 minutes

Preparation Procedure

• Prep will include pipetting out samples into individual micro centrifuge tubes for each lab group.
• Mixing electrophoresis buffer as per the directions provided.
• Obtain a microwave and place it in an easily accessible location in the lab area.

Background Information

A hybrid, as a biological term, often refers to an organism of two different parents, each exhibiting different traits. This term had been so popularized in the agriculture community by its use in variety names that it has become synonymous with “variety” in some situations. To be clear, a crop variety is characterized as a classification group that is below the level of species and subspecies and that includes certain phenotypic and consequently genetic characteristics. When using the word “hybrids” to describe new varieties of plants, confusion can be created.

Most varieties today are created through a combination of traditional breeding techniques, as well as genetic engineering practices. All new varieties must go through a rigorous process of development and testing prior to the process of applying for deregulation status, which allows the variety to be sold in both the United States and abroad. This process can take upward of 12 to 14 years and is the culmination of the efforts of thousands of individuals.

A fair understanding of DNA as the molecule of heredity is necessary for students to comprehend this lab. Understanding the basic structure of DNA, as well as the processes that create proteins, are essential to understanding how different DNA can cause different traits to be seen and expressed in organisms. Since most of the traits that are included in new varieties do not carry distinct visual phenotypic differences, students must be able to understand the underlying differences in DNA before they can appreciate the subtle differences in the varieties of crops.

Classroom Discussion

Macro vs. Micro Differences

• Post the picture of a man for 10 seconds. Tell students to take a good look at the man because you will have to identify him later. You can take notes to help you remember.

• • Remove the picture. Ask: What kinds of notes did you take?
    • Allow students to respond. Possible responses might include: eye color, hair color, facial hair no facial hair, skin color, etc.
  • Show the group pictures. Which one of these is the same man of you saw before?

• Post the picture of a corn plant for 10 seconds. Tell students to take a good look at the plant because you will have to identify it later. You can take notes to help you remember.
  • Remove the picture. Ask: What kids of notes did you take?
    • Allow students to respond, possible responses might include: height of plant, number of leaves, color of plant, name of plant, etc.
  • Show the crop photo. Which one of these is the same as the one you said before?

• Review answers. The man was easy to spot. You have visual clues that you have been trained to spot your entire life! The plant might not be so easy to spot. We see a corn plant and just see a corn plant. It’s hard for us to visually distinguish plants of different varieties from each other. They visually look the same.
• What caused the man’s features to be the way they are?
  • Hopefully students’ responses will be DNA.
• What caused the plant’s features to be the way they are?
  • Hopefully, again, the same answer will be DNA.
• So, how do I tell if they are the same or not? I can test a man’s DNA to compare his traits to another person. This is how we catch some criminals.
• Can I also test a plant’s DNA to compare to other plants?

Activity: Comparing DNA…

• We know that DNA is made up of four basic nitrogen bases, and the order of those nitrogen bases determines our traits. How can we compare those bases? Letter by letter would take forever! What’s a faster way to do it? Restriction enzymes!

• Give each student a sequence of DNA, a pair of scissors, and the first restriction enzyme.
  • There are four different sequences of DNA. Working in groups, have each member of the group use a different sequence.
• Using colored pencil/marker underline when you see the exact set of 5 letters (nitrogen bases).
• Tell the students to cut their sequence right after they see the matching code on their DNA. Everyone’s DNA is different so yours’ will not be the same as your neighbors!
• Have the students hold up their cut or not cut DNA. Are the fragments the same size?
• Why are the fragments different? The letters were at different places or weren’t there at all. What if we keep doing this with more restriction enzymes? We will end up with multiple fragments based on where they would cut your sequence!
• Use the other three restriction enzymes to cut your sequences.
• Now go find someone with the same DNA as you.
  • Match up with members of the other lab groups. How can we be sure that their DNA is the same? Look at the size of the DNA fragments
  • Count up the number of base pairs in each fragment. Write that number on the back. Compare with your matching DNA. You have the same sized fragments because you had the same DNA.

*** More activities will be completed during the lab process, there is a 15-20-minute window where the process of electrophoresis is explained. This can be done before or during the lab, whichever is your preference as a teacher.

Procedure for Lab

• Peel up one corner of the gel cup. Microwave for 20 seconds.
• Be aware of the steam coming out of the cup, pick up the cup and swirl it several times, making sure that all the solid agar has liquefied.
• Let the cup cool slightly. Continuing to swirl the cup will help with even cooling.
• Set up the gel tray by inserting the clear gel tray into the gel mold. Place the six-sided comb into the top position of the mold.
• Pour in the warm gel. Each gel cup will create one tray of gel. To fill both sides of the gel mold, you will need two cups. This would be enough for two lab groups.
• Set the gels aside to cool and solidify. The gel takes a slightly cloudy appearance once set. You should be able to touch the gel and feel a resistance. Do not press too hard or you will put a divot in your gel.
• Measure out approximately 150 mL of TBE buffer. Note: buffer may precipitate out. Make sure you are stirring the buffer well before use. All precipitate may not dissolve, but this should not affect results.
• Once the gel has solidified pull the comb straight up to reveal the wells. Then, pull up on the clear plastic gel tray, keeping the gel on top of the gel plastic gel Wipe any excess liquid off the bottom of the gel tray and the sides. Be careful when handling the gel. The gel tray only has sides, no front or back, so the gel may slide off the front if you are not holding the gel parallel to the ground.
• Place the gel into the electrophoresis tank, matching up wells with the lines on the black plate on the bottom of the tank. The well should be oriented at the negative side of the tank, as is indicated by the negative sign (-) at the top of the tank. The gel will only slide into the tank in one way. It should slide all the way to the bottom and rest on top of the black plate. The gel must be on the gel tray and not touching the black electrodes on either side of the tank.
• Pour the TBE buffer into the tank. Pour in one side of the tank This will force any air from under the gel out so that no bubbles are present. 150 uL should just barely cover the gel in the tank. If the gel slightly floats up, simply press down on the gel until it is resting on the black plate once again.
• Place the tank into the electrophoresis power supply. Make sure that the gold rivets are touching the electrodes in the tank. The tank should slide in easily, do not force a tank in. It is designed to slide in only one direction. You should hear a “click” if it’s inserted properly.
• Once the gel is in the tank, plug in the power supply and turn on the black light. The larger lightbulb button will provide sufficient light to see the wells.
• Pipette in the samples, 10 uL in each well, making sure to change pipette tips as you move from one sample to another. Make sure to keep the pipette plunger depressed as you remove the pipette tip from the gel. Otherwise, you will draw the sample back up into the pipette instead of leaving it in the gel.
• Once all samples are loaded, depress the smaller lightbulb button, and place the orange viewing hood on top of the tank (this will only work in one direction). Then, press the power button light. The light indicator should turn green to indicate the apparatus is working. You should also be able to see bubbles developing on the electrodes in the tank. Do not remove the viewing cover until you are finished with the lab.
• Check on the gel every 5 minutes for a total of 15-20 minutes. Within the first 5 minutes, you should be able to see the bands migrating down toward the positive electrode in the tank. Continue to watch and check the gel every 5 minutes until 20 minutes have elapsed.
• Once 20 minutes has elapsed, a photo can be taken of the gel using the viewing hood. The hole in the top of the viewing hood is at the perfect focal length for student devices. The photo will also include a ruler for assistance in analysis later either by hand or software analysis tools, such as Logger Pro, can be utilized if desired. Pictures should be taken to preserve the results. As time passes, the bands will disperse in the gel, and results will fade.
• Once good pictures have been taken, your lab is complete. TBE buffer can be reused; however, sufficient concentrate has been provided for new buffer every time the lab is run. Gels can be disposed of in the trash. Make sure students remove the clear plastic gel trays before they throw out the gels. Return the plastic tray to the gel mold tray.

During wait time: Finish Restriction Enzyme and Gel Electrophoresis Activity.

• You will be modeling the process of electrophoresis in this kinesthetic activity.
• Divide your students into groups, one group of 6, a few groups of 3 to 4, and a few groups of 2 to 1.
• Make a “lane” or line for your students to walk down with a few obstacles. Three to five chairs work well, depending on the space you have available. Place the chairs directly in the “lane” or path you want the students to take.
• Have the groups take turns trying to get from one end of the lane to the other in a very short amount of time. The trick is you cannot lose members of your group. Having them lock elbows or hold hands maintains this. They must all stay together. You only make it as far as your last member. Give each group 5 seconds to walk down the lane. Time can be adjusted to fit the number of students.
• Mark how far each group got with a piece of tape or meter stick, indicating the number of group members on it.
• You will find that the groups with the smallest number of individuals will make it farther down the There is less to move, literally one person vs. five or six.
• Explain to your students that the same thing happens in electrophoresis. The larger the DNA fragment, the slower it is moves down the lane.
• The gel is represented by the chairs in their path. Everyone had the same number of chairs to navigate around. The gel is the same no matter what lane we are talking about.
• The larger the fragment, the shorter the distance it can travel from the wells.
• The smaller the fragment, the farther the distance it can travel from the wells

Now, have students finish their modeling activity by filling out the Restriction Enzyme and Electrophoresis Graph of their own DNA fragments from the Restriction Enzyme Digestion earlier. Make sure that students are using a writing utensil that is bright and easy to see on their graph. Afterward, students can answer the questions at the bottom of the graph sheet on a separate piece of paper or simply on the back of their graph.

Teachers Resources

Teacher Tips

Students should have experience with pipetting before you attempt this lab. Proper technique is critical due to the small amounts of samples being used in each well. If the samples are released outside of the well, no bands will be produced for that particular sample. Reminding students about the functionality of both the hard and soft stop for the pipette will help prevent accidental release. It is also possible for students to puncture the gel and deposit their samples underneath the gel. This, too, will not produce any results. Steady hands and patience are critical for the best results. It is encouraged to complete the Hitting the Bullseye pipetting lab prior to this lab.

Teacher Resources

• West Coast Seeds  What are Hybrid Seeds Exactly? 
  Easy to understand definition of hybrid seeds.
• Khan Academy Restriction enzymes & DNA ligase
  Article explaining the use of restriction enzymes in gene insertion.
• LabXchange  Restriction Enzyme Digest
  Restriction enzyme digest simulation provides an opportunity to practice a restriction digest in a virtual lab setting.
• Science News for Students  The mixed-up world of hybrid animals
  Great explanation of hybrids. While it is specifically talking about animals, it will be great background information.
• Amoeba Sisters  Gel Electrophoresis
  This video introduces gel electrophoresis and how it functions to separate molecules by size.

Lab Analysis and Conclusion

See Hybrid Electrophoresis Lab Analysis Sheet for student questions:

• Photograph and visually analyze the gel.
• Have students take a picture of the completed gel through the window on the top of the UV hood. Compare the bands seen in the completed gel. Recall that the sequence of DNA we are looking for is for one particular gene.
• Have students complete the questions on the Lab Analysis Worksheet. At this point, students can upload their pictures into analysis software, such as Logger Pro, and apply measurements and base pair lengths to each particular band, if you wish to go that deep.

Science and Agriculture Careers

Understanding the basics of genomic analysis is the key to truly understanding the vast diversity of seed varieties being developed on an ongoing basis. Since the vast majority of hybrid plants cannot be distinguished from their non-hybrid peers, it is critically important for students to understand the differences between the two. Primarily, that this difference is not visual but genetic. Analysis, such as this, takes basic laboratory skills and an understanding of laboratory equipment that is used across the world. Several different careers in agriculture are fundamental in the development of new varieties of crops. Botanists, horticulturalists, biochemists, biological engineers, climatologist, ecologists, food scientists, geneticists, microbiologist, plant pathologists, and an army of lab technicians are all involved in the development of every new variety that hits the market. Without their valuable hard work and dedication, we would not be able to provide for the growing demands we see in agriculture.

To learn more about agriculture careers, visit https://agexplorer.ffa.org/.

Sources

• Daniel Herschlag. RNA Chaperones and the RNA Folding Problem. J. Biol. Chem. 1995 270: 20871-.doi:10.1074/jbc.270.36.20871

• Castiglioni, , Warner, D., Bensen, R. J., Anstrom, D. C., Harrison, J., Stoecker, M., Abad, M., Kumar, G., Salvador, S., D’Ordine, R., Navarro, S., Back, S., Fernandes, M., Targolli, J., Dasgupta, S., Bonin, C., Luethy, M. H., … Heard, J. E. (2008). Bacterial RNA chaperones confer abiotic stress tolerance in plants and improved grain yield in maize under water-limited conditions. Plant physiology, 147(2), 446-55.

Disclaimer

Any educator electing to perform demonstrations is expected to follow NSTA Minimum Safety Practices and Regulations for Demonstrations, Experiments, and Workshops, which are available at https://static.nsta.org/pdfs/MinimumSafetyPracticesAndRegulations.pdf, as well as all school policies and rules and all state and federal laws, regulations, codes and professional standards. Educators are responsible for abiding appropriate legal standards and better professional practices under a duty of care to make laboratories and demonstrations in and out of the classroom as safe as possible. If in doubt, do not perform the demonstrations.