DNA Biotechnology By Peggy Campbell Type of activity: Lab series/project Hands-on Simulation Inquiry Group/cooperative learning

Target Audience: Advanced/AP Biology Genetics Biotechnology

Background Information

This series involves a simulation of restriction enzyme activity and gel electrophoresis and two DNA labs from Carolina Biological.

Notes for teacher:

This series of activities is the culmination of a nine week unit on genetics in which the students review basic concepts, solve Mendelian genetics problems, explore genetics principles like linkage, and learn about the new DNA technology that is now available to researchers. I introduce the students to restriction enzymes and gel electrophoresis through a simulation from BSCS Blue ("Direct Detection of Genetic Disorders") that uses colored paper clips to demonstrate how these techniques may be used to show the presence of the point mutation which results in sickle cell amenia. The paperclips not only allow students to "see" the cutting of the DNA, they also show how larger fragments move slower than smaller ones. Once the students understand these concepts, they are ready to tackle two fairly complex lab activities.

The first lab is the Forensic DNA kit (21-1200 or 21-1205) from Carolina. The students determine which of the two "suspects" has DNA that matches that of the "evidence" left at the scene of a murder. The experiment uses plasmid DNA isolation and restriction analysis of the different plasmids to illustrate how DNA "fingerprints" are made and what information can be obtained from them. After running their "suspect" and "evidence" samples on a gel, the bands are analyzed and (hopefully) the case is solved. An added dimension is to use a camera system for the gels which provides the students with a permanent souvenir of the experience!

The second lab comes about two weeks later, after a complete discussion of viral and bacterial genetics. The purpose of the pBLU Colony Transformation experiment is to demonstrate phenotypic changes in bacteria that have been transformed with an antibiotic-resistant gene and metabolic marker. The students use a rapid method to render E. coli competent to take up plasmid DNA. The cells are then transformed with the pBLU plasmid, which carries genes coding for two identifiable phenotypes: ampicillin resistance and the ability to break down the galactose analog X-gal to produce a visible blue product. Ampicillin is the selective agent in the experiment; only transformed and nontransformed cells grow in the presence of X-gal, but the transformed cells metabolize X-gal to produce blue colonies. I have always had good results, even from students who aren't comfortable in the lab as others, and the results are easily seen and analyzed. It also provides closure to genetics while leading to the next section I cover which is the immune system.

Required of students:
In order to successfully complete this lab series, students must:

• Understand restriction enzymes and the process of gel electrophoresis
• Understand the principles of genetics
• Follow oral and written instructions that are at times fairly complex
• Complete the paper clip simulation from BSCS Blue and answer analysis questions
• Complete the Forensic DNA lab and interpret the gel results
• Complete the Bacterial Transformation lab and interpret the colony results

Preparation time needed:

• BSCS Blue simulation-1 hour to separate the paperclips
• Forensic DNA lab-2 to 3 hours
• Bacterial Transformation lab-1 to 2 hours

Class time needed:

• BSCS Blue simulation-one class period (50 minutes)
• Forensic DNA lab-four class periods
• Bacterial Transformation lab-three class periods

Lesson/Activity:

Materials needed:

1. BSCS Blue Activity-68 paperclips
2. Forensic DNA lab-kit #21-1200/05, inoculating loop, freezer, micropipets, microcentrifuge, hairdryers, electrophoresis chambers and power supplies, large flask, beakers, ice, distilled water, water bath or microwave, 37 degree incubator, and bleach or disinfectant for cleanup.
3. pBLU Colony Transformation lab-kit, beakers, ice, ethanol, plastic wrap, bunsen burners, 42 degree water bath, microwave, 37 degree incubator, and bleach for cleanup.

Procedure:

1. BSCS Blue activity-

a)The following codons are part of the DNA sequence for normal hemoglobin and sickle hemoglobin:

Normal Hemoglobin DNA	GGT CTC CTC	glutamate
Sickle Hemoglobin DNA	GGT CAC CTC	valine

Describe the difference in the genetic code between the two types of hemoglobin and the state the type of mutation which causes the difference.

b)Strands I and II in the figure represent two single strands of DNA. Using paper clips, one team member will make a model of strand I and the other team member will make a model of strand II. Key: black=adenine (A); white=thymine (T); green=guanine(G); red=cytosine(C).

c)The restriction enzyme MstII recognizes the DNA sequence GGTCTCC and cuts the DNA between the first T and the first C of that sequence, reading from left to right. Locate the places on strand I where cuts can be made by MstII and break the strand at those points. Do the same for strand II.

Strand I:

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35

A A G G T C T C C T C T T T T T G G T C T C C T C A G G T C T C C T T

Strand II:

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35

A A G G C T C C T C T T T T T G G T C A C C T C A G G T C T C C T T

Method of Evaluation-Analysis

1. What is the difference between strand I and strand II?
2. Based on that difference, what can you predict about an individual who has strand I in his/her genetic make-up? Strand II?
3. What will be the length of the fragments (how many bases) if strand I is cut with MstII?
   What will be the length of the fragments if strand II is cut with MstII?
4. How could you use MstII to distinguish sickle hemoglobin from normal hemoglobin?
5. Assume that a woman undergoes prenatal diagnosis. The genetic counselor shows the woman and her husband the results of the DNA test. In each case, give the diagnosis for the fetus:

   a) fragment sizes=5,14, 10,6

   b) fragment sizes=5,14,10,24,6

   c) fragment sizes=5,24,6

6. RFLP stands for restriction fragment length polymorphism. Use your knowledge of restriction enzymes and variations in DNA sequences to write a paragraph that explains RFLPs.
7. What tissues can be used for this type of diagnosis?
8. How would different mutations causing the same disorder affect a diagnoses made on the basis of RFLPs?

NOTE: For the kits from Carolina, the procedures are explained in detail in the teacher's manual and the evaluation questions and reinforcement activities are also included.

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