Materials Included In Kit

Additional Materials Required

Safety Precautions

The materials in this activity are considered nonhazardous. Remind students to follow standard laboratory safety guidelines.

Disposal

The materials in this activity are considered nonhazardous. They may be saved and stored for future use.

Lab Hints

• Enough materials are provided in this kit for 30 students working in pairs or for 15 groups of students. All materials are reusable. Both parts of this laboratory activity can reasonably be completed in one 50-minute class period. The prelaboratory assignment may be completed before coming to lab, and the Post-Lab Questions may be completed the day after the lab.
• Advise students to twist the recombinant plasmid chenille wires together tightly so they will not come apart as the pony beads are moved back together.

Teacher Tips

• Invite students to develop additional methods to simulate recombinant DNA formation.
• Thoroughly discuss the answers to the Prelab Questions before allowing students to begin the activity as it will be nearly impossible without a basic understanding of restriction enzymes.
• Stress to students that Parts A and B use different DNA sequences and restriction enzymes.

Answers to Prelab Questions

1. The restriction enzyme HindIII recognizes the following sequence, 5′ AAGCTT 3′ and makes a cut between the two As. If HindIII is added to the DNA sample shown, draw the resulting DNA fragments that would be generated, where both are labeled 5′ → 3′.
   {10921_PreLabAnswers_Figure_12}
2. If plasmid DNA is cut with the restriction enzyme EcoRI, which recognizes the sequence 5′ GAATTC 3′, should EcoRI also be used to cut the DNA containing the gene of interest, why or why not?

   The same restriction enzyme should be used so the resulting bases will bond with their corresponding base to become base pairs which are then ligated together.

Sample Data

Part A.

Part B. (for chenille wire and pony bead model)

1. The bacterial plasmid—students will turn in the plasmid in circular form.
   {10921_Data_Figure_10}
2. Recombinant DNA plasmid—also in circular form.
   {10921_Data_Figure_11}

Answers to Questions

1. Plasmids such as the one made in this activity are unable to survive on their own, they must be inserted into a host bacteria. Explain why only “small” human genes have been successfully produced using recombinant DNA techniques.

   Bacterial plasmids are usually much shorter than human chromosomal DNA. Therefore, only small genes are capable of being inserted into them.

2. Many humanitarian organizations are attempting to create a super crop which will grow on poor soil, withstand a harsh climate, and combat malnutrition. Describe the steps necessary to insert β-carotene (Vitamin A) into rice.

   Students should generally describe the steps listed in Figure 2.

References

Campbell, N. A. Biology. Benjamin Cummings: San Francisco, CA; 2002; Edition 6, p. 376.

Introduction

How do scientists investigate one tiny gene of interest in a chromosome thousands of base pairs long? Learn how recombinant DNA is formed to make several copies of a certain gene for further study.

Concepts

• Recombinant DNA
• Restriction enzymes
• Gene cloning

Background

The mapping and sequencing of the human genome is one of the greatest scientific accomplishments to date. This would not have been possible without advances in DNA technology, mainly methods used to form recombinant DNA. This is DNA in which genes from two different sources, such as a human and bacteria, are combined in vitro to produce a single new DNA molecule.

One of the many challenges in recombinant DNA technology is that a strand of DNA is extremely long and typically carries many genes. Furthermore, genes may occupy only a relatively small proportion of the chromosomal DNA—the remaining nucleotide sequences are called noncoding regions. For example, a human gene of interest may constitute only 1/100,000 of a chromosomal DNA molecule. The differences between the gene and the surrounding DNA are subtle, consisting only of differences in nucleotide sequence. In order to work with a specific gene, scientists needed to develop a method in which multiple identical copies of DNA on the gene of interest are created. In order to “cut out” the gene and insert it, scientists use special enzymes known as restriction enzymes. Restriction enzymes were discovered in the late 1960s by researchers studying bacteria. These enzymes protect bacteria against intruding DNA from foreign organisms, such as phages or other bacterial cells.

Restriction enzymes work by cutting the foreign DNA at specific recognition sequences, thus rendering the invading DNA useless. Scientists have determined that restriction enzymes cut DNA at specific patterns of nucleotide bases. The majority of restriction enzymes cut at symmetrical sites in which the same 5′ → 3′ sequence of 4–8 nucleotides is found on both antiparallel strands of DNA. (The sequences occur in opposite direction on the double-stranded DNA.) The restriction enzyme HbaI, for example, recognizes and cuts DNA as illustrated in Figure 1.

In order to research a specific gene, the first step is to use a restriction enzyme to isolate the gene of interest. The same restriction enzyme is also added to a purified strain of plasmid or host DNA. Since the restriction enzyme cuts both samples of DNA at the same site, the separate fragments can be joined or ligated together. When the plasmid containing the newly inserted gene is mixed with a bacterial cell, the recombinant DNA plasmid is incorporated into the bacterial host cell, and the resulting cell is known as a recombinant bacterium. The resulting recombinant bacterium is cloned and the clone is tested to make certain the gene functions properly. The recombinant bacterium can be used in various applications such as gene research. For example, a gene for pest resistance may be inserted into plants, or a protein such as human growth hormone made by the gene may be used to treat conditions such as stunted growth. See Figure 2 for a summary of the overall process of recombinant DNA technology. Figure 3 displays how the gene of interest would be inserted into plasmid DNA upon the addition of HbaI restriction enzyme. Hint: Refer to Figure 1 for the recognition sequence of HbaI.

Experiment Overview

The purpose of this activity is to build a simulated recombinant bacterium using manipulatives. Keep in mind the DNA sequences in chromosomes and plasmids are much longer than in this simulation. It is not unusual to find a chromosome containing 20,000 base pairs (20 Kbp).

Materials

Prelab Questions

1. The restriction enzyme HindIII recognizes the following sequence, 5′ AAGCTT 3′ and makes a cut between the two As. If HindIII is added to the DNA sample shown, draw the resulting DNA fragments that would be generated, where both are labeled 5′ → 3′.

   5′ AAGCTT 3′
   3′ TTCGAA 5′

2. If plasmid DNA is cut with the restriction enzyme EcoRI, which recognizes the sequence 5′ GAATTC 3′, should EcoRI also be used to cut the DNA containing the gene of interest, why or why not?

Safety Precautions

This laboratory activity is considered nonhazardous. Please follow all laboratory safety guidelines.

Procedure

Part A.

1. Scientists are interested in studying the gene that makes insulin. This gene may be isolated from the chromosome with the use of the BamHI restriction enzyme. BamHI recognizes the sequence, 5′ GGATCC 3′, and cuts between the G and G. On the Recombinant DNA Worksheet, show the DNA fragments that would be generated upon the addition of BamHI to the chromosomal DNA.
2. The bacterial plasmid shown on the worksheet also has a restriction site capable of being cut by BamHI. Show where the plasmid DNA would be cut upon the addition of BamHI.
3. Complete the cloning of the insulin gene by drawing the recombinant DNA molecule that would be obtained by joining the chromosomal DNA fragment into the cut plasmid. Be sure to insert the gene of interest in the correct place in the bacterial plasmid and label each step.

Part B.

1. Obtain 2 blue chenille wires, 2 red chenille wires, 31 white pony beads, 31 green pony beads, 22 blue pony beads, 22 pink pony beads and two twist ties.
2. Each color pony bead represents a different nitrogenous base. A is white, T is green, C is blue and G is pink. (Optional) Using a permanent marker, label the beads with their corresponding nitrogenous base.
3. Take one blue chenille wire and string the corresponding pony beads onto the wire as displayed in Figure 4, with the 5′ end to the left and the 3′ end to the right. Once finished, lay the newly made chenille wire flat on the work surface.
   {10921_Procedure_Figure_4}
4. Obtain a red chenille wire to make the complementary strand. Using the wire and beads made in step 3 as a template, string the complementary bases onto the red chenille wire. Note: Before beginning the construction of the complementary strand, fill in the correct letters on the beads in Figure 5. You should now have two strands next to each other, one in the 5′ → 3′ direction and the other in the 3′ → 5′ direction.
   {10921_Procedure_Figure_5}
5. Cut the two twist ties into three pieces. Wrap the twist ties around the two chenille wires every six base pairs to keep the two rings together (see Figure 6). Note: Three twist tie pieces will be needed and there should be four base pairs remaining at the end.
   {10921_Procedure_Figure_6}
6. To simulate a plasmid DNA molecule, wrap the two chenille wires into a circle so the 5′ end attaches to the 3′ end and vice versa, as shown in Figure 6.
7. Using the remaining two chenille wires, string the DNA sequences shown below—one wire for the template strand and one wire for the complementary strand. Note: The template strand should be strung on the blue chenille wire and the complementary strand on the red chenille wire. The wires represent chromosomal DNA.

   5′ TTCGAATTCCCTGAATTC 3′ (Template strand)
   3′ AAGCTTAAGGGACTTAAG 5′ (Complementary strand)

8. The restriction enzyme EcoR1 recognizes the DNA sequence 5′ GAATTC 3′ and cuts between the G and A. Using the chromosomal DNA constructed in step 7, which contains the gene of interest, find the location(s) where EcoR1 will cut this piece of DNA. Mark the locations on each strand in step 7.
9. Obtain the DNA strands made in step 7. Having found where EcoRI cuts the DNA strands in step 8, remove the beads that do not contain the gene of interest. Using scissors, shorten each chenille wire containing the gene of interest so that there is 1" of wire remaining at each end (see Figure 7).
   {10921_Procedure_Figure_7}
10. Find the location where EcoRI will cut the original plasmid. Untwist the ends of the chenille wires and twist ties and move the beads down the wire, leaving a gap where EcoRI cuts the plasmid DNA on each wire.
11. Cut the plasmid DNA in the middle of each gap, leaving approximately 1" of chenille wire on each end.
12. Insert the gene of interest into the correct location in the plasmid DNA.
13. Twist the ends of the chenille wires tightly to connect the gene of interest with the plasmid, ligating them together.
14. Examine the recominant DNA molecule and submit the model to the instructor along with the completed worksheet.

Student Worksheet PDF

10921_Student1.pdf