8 Comparing DNA

By Shelley Casey

Provided by Kenan Fellows Program.

In this simulation, students will use electrophoresis and DNA fingerprinting to look for the presence of a gene that is able to supply resistance to the Chestnut blight (Cryphonectria parasitica). The control in this case is a segment of DNA from a Chinese chestnut tree that is known to contain a gene for resistance.

This lesson is designed to simulate a gel electrophoresis and generate a DNA fingerprint. Students will learn that often more than one restriction enzyme is required to achieve a correct analysis of the DNA data. The DNA samples provided are meant to show the gene for chestnut blight resistance in both Chinese chestnut trees and American-Chinese hybrids as a means for locating a possible nucleotide sequence to map in order to create transgenic trees in the future.

Learning outcomes

Students will be able to recognize that different restriction enzymes cut DNA at specific sites producing segments of various lengths. They will be able to compare these DNA lengths on a simulated gel and identify which genes most resemble each other.

Teacher planning

Time required

45 minutes

Materials needed

If you wish to conduct the optional demonstration, you will need the following:

• gel electrophoresis chamber
• power source
• gel

Technology resources

None required.

Student handouts

Pre-activities

Students should already have knowledge of DNA including function, location, and base pairing. You may wish to briefly review this information. It also is helpful if they already understand that restriction enzymes are used to cut DNA at specific restriction sites. Remind students that PCR is a process that could be used to make copies of a segment of DNA in order to run the DNA through a gel for comparison.

Activities

1. Hand out copies of the Comparing DNA lab sheet and DNA Segments to each student. Have students silently read the first page of the lab sheet.


This liquid-filled box and attached power supply are used for gel electrophoresis.

2. If equipment is available, demonstrate the steps of an electrophoresis as described in the reading. The gel may be passed around for students to feel and this allows them to see the wells into which the DNA is placed. It is not necessary to a use buffer and DNA, just show how the gel is placed in the chamber and that one electrode is positive and one is negative. If this equipment is not available, show a picture of the equipment and explain the process using the diagram provided. Be sure to point out that the DNA has already been cut with the restriction enzymes when it is placed in the gel. Also, the smaller pieces of DNA will be at the bottom, or positive end, of the gel. If a completed gel is available, show this to students so they know what their paper will look like when they have completed the activity.

About the photograph
Gel electrophoresis showing six DNA tracks. In the first row (left), DNA with known fragment sizes was used as a reference. Different bands indicate different fragment sizes (the smaller, the faster it travels, the lower it is in the image); different intensities indicate different concentrations (the brighter, the more DNA). DNA was made visible using ethidium bromide and ultraviolet light.
3. Direct students to the DNA Segments handout. Point out that the way the first restriction enzyme cuts is at the bottom of the page. Instruct them to follow Procedure #1 for the control only.
4. Once students have drawn their lines representing the restriction enzyme cuts, have them move on to Procedure #2. Allow time for the class to finish this step, then go over the correct answers before proceeding. The correct base pair lengths for this control are as follows: 4, 22, 19, 21, 17, and 14. If students do not have these correct, show them how to obtain these numbers. Another student may help with this.
5. Show students how to mark their papers labeled “gel for comparison.” A dark mark should be made parallel to its corresponding number. Make sure they only place the numbers listed above under the column labeled control. These marks represent the DNA bands that would appear on an actual gel.
6. Have students complete the cuts and markings for the remaining trees on the EcoRI sheet.
7. Direct students to the second sheet labeled DNA segments for comparison. This sheet uses a different restriction enzyme since both tree 2 and tree 3 will appear to be a match for the control. Using a second enzyme will eliminate one of the choices. Make sure students understand that a different restriction enzyme will recognize a different DNA sequence — located at the bottom of the page.
8. Instruct students to complete the second set of cuts and markings just as they did the first and proceed to the analysis questions.
9. Explain to students that the tree that matches the control most, will be the tree that is least likely to be killed by the blight fungus. This is the tree whose DNA would be used for further research.

Assessment

Draw a sample completed gel on the board and ask students the following questions:

• At which end of the gel does the DNA get loaded? (the end with the wells)
• What must be done to the DNA before it can be loaded into the gel? (it must be cut with restriction enzymes and dyed so it can be seen)
• Which end of the gel will be positive and which end will be negative? (the end where the DNA is loaded should be negative and the opposite end should be positive)
• Where will the shortest pieces of DNA appear on the gel (the bottom), the longest (the top)?
• Which two DNA samples (on the examples you have drawn) appear to be the most alike and why? (this will depend on your drawing)

Also, check the remainder of the handout for correctness (Tree 2 matches the control)

Modifications

For students with reading difficulties, it may be helpful to have students in the class read sections of the first page aloud while everyone else follows along. Also, you may want students to work in groups of two to allow stronger students to help those that may be confused.

Supplemental information

Kits for performing a gel electrophoresis such as those from Carolina Biological and Bio-Rad may be used. The kits contain lambda DNA, but you can have students imagine it’s chestnut tree DNA.

Critical vocabulary

restriction enzyme

any of the enzymes that cut nucleic acid at specific sites and produce fragments of various lengths.

buffer

an ionic compound that resists changes in its pH

PCR

a technique to amplify a single or few copies of a piece of DNA across several orders of magnitude, generating millions or more copies of a particular DNA sequence.

gel electrophoresis

a technique used for the separation of DNA using an electric current applied to a gel matrix and is generally only used after amplification of DNA via PCR.

control

a standard against which other conditions can be compared in a scientific experiment.

Comments

This lesson is a continuation of the unit on American chestnut biotechnology. Gel electrophoresis is a vital part of mapping an organism’s genome in order to find blight resistance and ultimately produce genetically engineered trees.

• Next: What is genomics?

References

• Brown, Betty et al. Get a Clue. DESTINY, UNC-Chapel Hill’s
  Traveling Science Learning Program: 2006.

Contents: Restoring the American chestnut

• Next: What is genomics?
• Previous: Low-tech PCR

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