Restriction Enzyme Road Trip: Enduring Understandings in DNA Biotechnology

Matthew Bostick
Product Development

February 2017

In this activity, students complete a dry lab that employs an entertaining road trip analogy. Using a few simple supplies (meter stick, string, sticky notes, and scissors), students model restriction enzyme digestion and polymerase chain reaction (PCR), then demonstrate their learning by modeling the separation of the resulting “DNA” fragments by gel electrophoresis.

Objectives

• To introduce students to the biotechnology techniques of restriction enzyme digestion of a nucleic acid polymer, polymerase chain reaction, and gel electrophoresis
• To construct a model representing biotechnological methods, concepts, and explanations

Prerequisite knowledge and skills

Students should have prior knowledge of DNA structure and the basic function of enzymes.

Time requirements

Preparation: 15 minutes
Activity: 45 minutes

Materials (for each student group)

• String, 3 m
• Scissors
• 10 Sticky Notes (such as Post-It®)
• Meter Stick
• Pencil(s), Pen(s), or Marker(s) (1 per group or per person)
• Student Guide(s) (1 per group or per person)

Safety

Scissors may cause injuries if used improperly. Ensure that students understand and adhere to safe classroom practices when performing any activity.

Teacher preparation

• Cut string into 3-m lengths for each student group (2 minutes).
• Supply additional materials for each group: sticky notes; scissors; pens, pencils, or markers; meter stick (5 minutes).
• Photocopy the student guide for each student (or group, if desired).

Procedure

1. Begin the lesson with a discussion of DNA structure. Remind students that the human genome contains more than 3 million base pairs, then mention that the string given to each group is approximately 3 m in length.
2. Instruct each group that it will use the string to represent the entire length of a hypothetical road trip. Because everyone gets hungry during a road trip, ask students to brainstorm to come up with 6 different restaurants that they may decide to stop at along the way. Ask students to write the names of their restaurants on the sticky notes, and then to place the notes randomly along the string “highway.” It is advisable to provide students with at least 10 sticky notes, yet limit the restaurants each group can choose to only 6 different chains. This should ensure duplicates of a few restaurants. See example below (Fig. 1).

 
Figure 1  Example of students’ sticky notes placed randomly along their string “highway.”

3. Instruct students to use the scissors and the meter stick to complete the activity as a team, using instructions found in the student guide.

Answers to student guide questions

1. In the road trip analogy, what would the scissors, string, sticky notes, string (trip) fragments, distance map, and mile markers represent if we were doing gel electrophoresis? Use the background reading in the student guide to help you answer the question.
   The scissors represent restriction enzymes, the string fragments represent DNA strands, the sticky notes represent different nucleotide sequences that may be recognized by restriction enzymes, the distance map represents the agarose gel, and the mile markers represent the DNA size standards used to estimate the relative lengths of DNA fragments.
2. In gel electrophoresis, what causes certain fragments of DNA to migrate farther than others?
   Differences in the size of the fragments allow variable length DNA fragments to migrate at different rates during gel electrophoresis. Smaller fragments will migrate farther than larger fragments.

Extension activities

We suggest the Rainbow Road Trip activity as a precursor to using advanced restriction enzyme or forensic science kits such as:

• Exploring Electrophoresis and Forensics Classroom Kit (item #211014)
• Sickle-Cell Anemia Protein Electrophoresis 8-Station Kit (item #211032)
• PCR Forensics Simulation 8-Station Kit (item #211214)
• Restriction Enzyme Cleavage of DNA 8-Station Kit (item #211149)
  

Name                                                                                                                      Student Guide
Date                                                        

Rainbow Road Trip: Introduction to
Restriction Enzymes and Gel Electrophoresis

Introduction

During this activity, you will use a road trip analogy to model a genetic engineering technique that “cuts” DNA with molecular scissors known as restriction enzymes. Bacteria produce these special enzymes as a defense mechanism against any viral “foreign” DNA that may be invading the cell. These enzymes slice invading foreign DNA into fragments that are no longer functional, thus stopping the invader in its tracks.

Geneticists, medical researchers, crime scene investigation units, and evolutionary biologists may use these special enzymes to cut DNA into fragments of varying lengths. There are many different restriction enzymes and, in general, each kind cuts at highly specific sites along the DNA macromolecule. Scientists can use these fragments to answer some very interesting questions.

Background

Considered by many to be the father of modern genetics, Augustinian monk Gregor Mendel crossbred pea plants to make some amazing scientific discoveries during the 1800s. Mendel designed experiments to study the hereditary nature of observable pea plant characteristics, such as plant height and flower color. He demonstrated that invisible “factors” within breeding organisms could produce visible traits in offspring in highly predictable ways.

We now call these “invisible factors” genes. We use biotechnology techniques to make the DNA molecule, and even the sequences of nucleotide bases (adenine, thymine, cytosine, and guanine) that code for specific genes, indirectly visible. How do scientists isolate just one specific segment of human DNA, a macromolecule that is over 3 billion base pairs long?

While Mendel inferred genetic variation within a pea plant’s phenotype, or physical expression of traits such as flower color, molecular geneticists have developed techniques that allow scientists to examine genetic variation at the level of the DNA that makes up genes. One of these techniques is known as restriction enzyme digestion. In this method, a specific region of the genome is isolated and then copied using a technique known as polymerase chain reaction, or PCR. Copies of DNA from this region may then be “cut” into fragments of different sizes using an enzyme known as a restriction enzyme.

Just as you pull over at a specific exit along a highway once you recognize the road sign of your favorite fast food restaurant, restriction enzymes recognize short and very specific sequences along a strand of DNA, and then “pull over” to cut the DNA at specific locations within those sequences. For instance, the restriction enzyme Sma1 will recognize the DNA nucleotide sequence CCCGGG and then make a cut between the cytosine and guanine nucleotides as shown in Fig. 1.

Notice that the resulting double stranded DNA fragments are now of different lengths after the cut has been made:

Figure 1  The restriction enzyme Sma1 will recognize the DNA nucleotide sequence CCCGGG
and then make a cut between the cytosine and guanine nucleotides.

Researchers may then separate these variable-length fragments based on their relative sizes, using a technique known as gel electrophoresis. In electrophoresis, a sample of DNA cut by a specific restriction enzyme is loaded into a well on the edge of a gelatinous medium (composed of the polysaccharide agarose). The gel along with the DNA is then placed within an electrical field. The phosphate group (PO₄^3-) in the “backbone” of DNA is a negatively charged molecule, thus the DNA molecule carries a net negative charge. Because opposites attract, DNA fragments will “migrate” to the positive end of the electrical field.

However, these variable-length DNA fragments will not migrate through the agarose “gel” at the same rate. Larger fragments of DNA, such as the 15 base pair fragment in the previous example, will diffuse through the gel medium at a much slower rate than relatively smaller fragments, such as an 11 base pair fragment. An example of gel electrophoresis of the fragments resulting from the Sma1 restriction enzyme digestion modeled above would look like the illustration in Fig. 2.

Figure 2  DNA sample cut with Sma1 restriction enzyme.

These differences in migration rate cause the fragments of DNA to separate from one another over time. Fragments of the same size collect in the same location on the gel, forming distinct bands. Typically, a “size standard,” composed of DNA fragments of known nucleotide base pair lengths, is run on the same gel as the sample, to confirm or determine the size of the DNA fragments in the samples.

The resulting patterns produced by restriction enzyme/gel electrophoresis analysis may then be useful to geneticists and evolutionary biologists for many reasons. For instance, researchers may be able to use the band patterns to detect mutations in DNA or even to determine familial relatedness among DNA samples from different individuals.

Materials

• String
• 10 Sticky Notes Scissors
• Pencil, Pen, or Marker
• Meter Stick

Procedure

1. It’s time to go on a road trip! Imagine that the length of the string given to your group represents the entire distance of your road trip. Use the following scale for your road trip: 1 meter of string = 100 “miles” of highway driving. Measure the length of your string and convert it to “miles” of highway. Record the distance of your hypothetical road trip.
   
   A. Total Length of String Highway (in meters) =                                                                                      meters
   
   B. Total Length of String Highway (in miles) = ____________________________________________ miles
   (scale: 1 meter of string = 100 highway miles)
2. Because everyone gets hungry during a road trip, your group will brainstorm to come up with 6 different restaurants that you may find at exits along the highway. Write the names of these restaurants on the sticky notes, then place them randomly along the string “highway.” You will have 10 sticky notes to write on, but you are limited to only 6 different restaurants to choose from. Therefore, you will have duplicates of a few of your restaurants. See Fig. 3 below as an example.

Figure 3  Example of sticky notes placed randomly along a string “highway.”

3. The student in your group with the first birthdate of the calendar year gets to choose one restaurant chain to stop at along the trip. This student will take scissors and cut the string highway every time this particular restaurant’s sticky note road sign appears along the string.
   
   For instance, if the student chose to stop at Taco Hut in the Fig. 3 example above, then the student would cut the string twice, producing 3 total fragments. See Fig. 4 below.

Figure 4  Example of cuts to a string “highway.”

The cuts in the Fig. 4 example would produce the following 3 highway fragment lengths:



4. Arrange your group’s highway fragments in order of decreasing length on your desk or work station.
5. Measure the length of each of your highway fragments. Remember that the conversion is 1 meter = 100 “miles.” Record your fragment lengths in the data table below.
   
   

6. Now it is time to organize your group’s distances between restaurant stops on the distance map below (Fig. 5). Use the distance size standard “mile marker” bars on the left of the map to assist you. In the provided example found in Fig. 4 above, the trip was broken up into 3 fragments using the hypothetical Taco Hut exits, or cuts. Use your pencil, pen, or marker to mark your group’s trip fragment lengths in miles on the distance map.

Figure 5  Distance map.

Questions

1. In the road trip analogy, what would the scissors, string, sticky notes, string (trip) fragments, and mile markers represent if we were doing a restriction digest followed by gel electrophoresis? Use the background reading in this student guide to help you answer the question.
2. In gel electrophoresis, what causes certain fragments of DNA to migrate farther than others?