DNA and Electrophoresis Simulation

Introduction

In this dramatic simulation, every student in your class will be a key part of a giant DNA model and participate as a fragment resulting from the action of a simulated restriction enzyme. Students will also physically experience how fragment size can affect the ability to move through a simulated electrophoresis gel.

Concepts

• DNA structure
• Restriction enzymes
• Gel electrophoresis
• Restriction fragment

Materials

Safety Precautions

The hooks on the ends of the DNA base pairs can be dangerous if misused. Some students will be walking backward during the simulation. Do not have unusually dangerous items in the pathway of the simulated gel.

Disposal

All materials may be saved for future use.

Prelab Preparation

1. Plan your classroom DNA model based upon the number of students in your class. If you have an odd number of students, then the odd numbered person can serve as the restrictive enzyme and the class timer. If you have an even number, then you can serve as the enzyme and timer.
2. Use Figure 1 to select the number of matched base pairs (A-T, C-G) so that every member of the class will have a complement.

No. of students

3. Set up an “obstacle” course to simulate the electrophoresis gel. The design need not involve much more than moving lab tables so that they are not all in a straight line. The large fragments will have some difficulty moving fast even without many obstacles. A few restricted areas will slow the fragments down.

Procedure

1. Move students to the hallway to assemble the initial DNA molecule. (If this is not possible, consider alternatives that will work in your specific school setting. An obstacle course in a gymnasium might be appropriate.)
2. Use the appropriate sequence from Figure 1 for the number of students in your classroom to select bases and organize the starting DNA molecule.
3. Explain to the class that each student represents a single nucleotide. Have students extend their left arm straight out in front of them. (This is the phosphate group.) Next, extend their right arm out to the side to make a 90° angle to their left arm. (This is one of the nitrogen bases.) Their body is the deoxyribose.
4. Arrange students into the DNA molecule in a pattern similar to that shown in Figure 2. Discuss the key DNA elements and characteristics as the molecule is assembled. Begin with one “side”, then have remaining students match up. Each student should place his left hand on the left shoulder of the student in front of him; matching bases are linked together.

5. Students will realize very quickly that for the components of the model to be used consistently the two lines must be facing in opposite directions. (The antiparallel structure of DNA.) The colored base pairs must be matched and hooked precisely following the sequence selected from Figure 1.
6. When the DNA molecule is completely assembled the restriction enzyme should move into the DNA structure at the appropriate restriction site and move down the middle of the molecule until the end of the restriction site is found. The restriction enzyme breaks the phosphate–sugar bond, some base connections, and then another phosphate–sugar bond before it exits the DNA and moves to the next restriction site.
7. The HpaII restriction enzyme cuts the DNA sequences so that there are “sticky-ends.” (If blunt ends are desired, an alternate enzyme can be used.) “Sticky-end” cuts make for interesting fragments when moving through the obstacle course.
8. Once the DNA is cut the fragments need to be loaded into a well (the classroom door frame is a good “well” simulator). Each fragment (in turn) needs to “run” the length of the gel (the length of the classroom or obstacle course). The rule for the run is that the molecular fragment must stay intact (e.g., hands on shoulders, base pairs hooked) while moving as quickly as possible through the obstacle course. (Remember some students will be walking backwards. Caution students to be careful.)
9. One fragment should go through the gel at a time and the time required for running the course should be timed and recorded. All students making up the fragment must cross the “finish” line or get to the end of the gel before stopping the timer.
10. Record the time and the fragment size on a chart on the classroom chalkboard. Run all fragments, compare times, and discuss the simulation and the results in detail.

Teacher Tips

• This kit contains enough materials for a class demonstration with as many as 31 students. All materials are reusable. There are 30 reusable DNA base pair simulators (one for every student) that can be reused indefinitely.
  
  The color scheme for the base pairs is as follows:

  Adenine = Green
  Thymine = Red
  Guanine = Blue
  Cytosine = Yellow

• The basic structure of DNA should be taught prior to this demonstration. Discuss/review all DNA structure concepts as the gel electrophoresis simulation is being conducted.
• A large card with the restriction enzyme cutting pattern might be helpful for the person serving as the restriction enzyme.
• The small fragments can move more quickly and thus further in a gel compared to larger fragments which will move more slowly and thus a shorter distance in a gel. The analogy for the speed of molecular fragments passing through an obstacle course is very helpful for students in visualizing why fragments separate in an electrophoresis gel. Given the same “running time,” different size fragments will end in different locations in the gel.
• If a large ticking timer is available it can help dramatize the time it takes for the fragments to negotiate the obstacle course.

Discussion

This activity assumes a basic knowledge of the structure of DNA. If this knowledge is not known, consult any basic biology textbook. In this activity students simulate the action of restriction enzymes and the creation of restriction fragments.

Restriction enzymes, or restriction endonucleases, are proteins that recognize and bind to specific DNA sequences and cut the DNA at or near the recognition site. A nuclease cuts the phosphodiester bonds of the DNA backbone and an endonuclease cuts somewhere within the DNA molecule. The restriction enzymes commonly used in the laboratory generally recognize specific DNA sequences of 4 to 6 base pairs. These recognition sites are palindromic in that the 5'-to-3' base sequences of each of the complementary DNA strands is the same. Most of the enzymes make a cut in the phosphodiester backbone of DNA at a specific position within the recognition site, resulting in a break in the DNA, and thus producing restriction fragments.

Following are a few examples of restriction enzymes and their recognition sequences.

Notice that the “top” and the “bottom” strands read the same from 5′ to 3′. Also notice that some of the enzymes introduce two staggered cuts in the DNA molecule while others cut each strand at the same base pair location. Those that cut at the same place are said to produce “blunt ends.” Those that result in ends with single-stranded protrusions are called “sticky-ends.”

References

Special thanks to Sue Whitsett, Fond du Lac High School, Fond du Lac, WI, for providing this activity.