Abstract: Method - University of Florida · the bio-rad PV92 Informatics kit students will extract...

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Lucie Dempsey Final Action Proposal Report ICORE 2010

April 2011

Title: DNA Technology and Genomics Lucie Dempsey, Biology Teacher Edgewater High School 3100 Edgewater Drive Orlando, FL 32804 (407) 835-4900 [email protected]

Abstract: The goal of this action proposal is to provide students the opportunity to become familiar with the processes of biotechnology and its real world application in modern day science. By using the bio-rad PV92 Informatics kit students will extract their own DNA from cheek cells, perform PCR to amplify a selected region on their DNA, and through gel electrophoresis will identify the presence or absence of a specific Alu repeat sequence of DNA base pairs in their own genome. As an enrichment activity students can further investigate the field of biotechnology through the virtual game Mission Biotech. Method: This teaching unit will be for my Advanced Placement biology students. The AP biology curriculum includes12 required labs. One of the required labs involves DNA technology such as transformation of E.coli and DNA analysis through gel electrophoresis. This lesson will apply elements of the ICORE summer institute to complete this required AP biology lab, and also to expand students’ knowledge and skills on other real world research applications using biotechnology techniques.

Day 1 – CPET staff to come to Edgewater High School (3.5 hours) I will arrange for students to be on an “in-house” fieldtrip for half of the school day. I have two classes of AP biology students, so one group of students will come to my classroom in the morning and the second group will come to my classroom in the afternoon. Both groups will be doing the same exercises. Activity #1 – Pipetting Activity – Students will learn how to use a micro-pipette through a fun activity which will allow them to follow specific protocol to fill gridded wells to create an unknown illustration.

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Activity #2 DNA Extraction Lab - Students will first extract samples of their own DNA from their cheek cells. The students will be looking to identify a specific Alu repeat (a 300 base pair repetitive sequence of DNA) within a specific region on chromosome 16. Over evolutionary time, up to 1 million copies of the Alu repeat have become randomly inserted throughout the human genome. Some individuals carry this Alu insertion and some do not. Students will ultimately be creating their own DNA fingerprint to determine whether or not they possess this Alu repeat within their own genome.

• Students rinsed their mouths vigorously with Gatorade in order to collect sample cheek cells. After rinsing, students expelled Gatorade from their mouths into a small Dixie cup.

• The students than transferred 1 ml of their saline rinse into a micro test-tube. Students placed their micro test tubes into the centrifuge and spun samples at full speed for two minutes. This procedure pellets cheek cells at the bottom of the micro test tube.

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• Students used a 20 µl micropipette to transfer cells to a screw cap tube containing Instagene. The collected samples were then placed in hot water bath at 56°C for approximately 15 minutes and then at 100°C for 5 minutes. Samples were placed in the centrifuge again for 5 minutes. The resulting supernatant contains the student DNA and can be used for PCR.

Heat was used to Lyse the cells to extract DNA material. The InstaGene matrix consists of a suspension of negatively charged microscopic beads, which bind divalent cations like magnesium (Mg2+). It is important to remove divalent cations from students’ DNA samples because the cations assist enzymes that degrade the DNA template. (When cheek cells are lysed and boiled in the presence of InstaGene matrix, the divalent cations released from the cells bind to the beads, and the heat inactivates the DNA-degrading enzymes. The beads are then pelleted by centrifugation. The supernatant, which contains clean, intact genomic DNA, can be used as the template in the PCR reactions.)

Activity #3 – Students will then use PCR techniques to amplify their extracted DNA samples that they have collected.

• Students were given background information on the Alu sequence • Power point presentation was used to explain the steps involved in the PCR procedure

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• Students transferred 20 µl of their DNA (template) into a PCR tube • Students transferred 20 µl of yellow master mix into the PCR tube • PCR tube was placed in thermocycler for 35-40 cycles of PCR amplification. • Samples were stored in freezer overnight.

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DAY 2 – This lesson will last one class period. Students will analyze samples of their own DNA (from previous day) through the process of gel electrophoresis.

• Students were given brief background on what the technique of gel electrophoresis is and how it works.

• Students used E-Gel Powerbase and Gel for electrophoresis procedure. • 20 µl samples of each of the following : a molecular marker, controls, and student DNA

samples were loaded into the wells of the E-Gel. • Gels were run for 30 minutes. • Results were analyzed by viewing gels with the use of transilluminators.

Well 4 molecular marker Well 5 homozygous positive control Well 6 homozygous negative control Wells 7,8,9 Student DNA

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Well 3 - Molecular Marker Well 4 - Heterozygous control Well 5 - Student DNA – Only student who tested positive (heterozygous) for Alu gene sequence!

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Pre-Lab Instruction – (4 class periods)

• Virus structure (Students made models of viruses) • Lytic and Lysogenic Cycles • Discussion of Restriction Enzymes…what they are and their function in bacteria • DNA scissors activity • Discussion on how restriction enzymes can be used in genetic engineering and also its use in

DNA fingerprinting/gel electrophoresis. • Structure and function of DNA

Post –lab Instruction ( 5 class periods)

• Replication, Transcription, Translation – powerpoint and discussion • Control of gene expression in prokaryotic and Eukaryotic cells – powerpoint and discussion • Genetic variation in prokaryotes: mutation, transformation, transduction, conjugation.- student

groups were assigned one topic to research and then share with the class.

Enrichment: Mission Biotech Game

• Four weeks prior to CPET visit and Lab Activities….students were given an opportunity to come in after school for 5 days to play Mission Biotech on UF laptop computers for extra credit.

• Students were given pre-tests prior to playing game on following topics: a) Knowledge about Science related to Biotechnology b) Ideas about Technology and Science c) Mission Biotech Content

• Students were given post-tests (same as pretest) after completing all learning activites….Mission Biotech, Lab activities (DNA extraction, PCR, Gel electrophoresis) , and molecular biology lessons.

Assessment: Students were given a quiz on Molecular Biology content

Mission Biotech Pre and Post Test

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RESULTS:

This unit of instruction was successful and the learning objectives were achieved. Students expressed that as a result of both the lab experiences and the mission biotech game that they did become familiar with the processes of biotechnology and its real world application in modern day science. Students expressed that they had a better understanding of the concepts as a result of participating in these activities, especially the half day field trip activities. The students suggested that more hands on lab experiences similar to the Alu lab should be incorporated into the curriculum. These lab activities “made learning fun!”

In addition, the average score on the quiz was 78%. I did not give a pre-test for this unit; however, the group of students that participated in the Mission Biotech Enrichment activity did take a “Survey of Knowledge about Science related to Biotechnology” pretest and posttest. I am literally finishing up this unit of instruction on the very day that this final action proposal report is due; and as a result I unfortunately will not have the data analysis back from Mission biotech to include in this report. I did however, assess the pre and post tests myself and the students showed an average of a 20% increase in their scores between the pre and post tests.

OUTCOMES:

As a result of my participation in the ICORE summer institute, I have been able to provide my students with hands on lab experiences that would not have been possible otherwise. There is no money in our school budget for lab equipment or even consumable supplies. Without the ICORE visitation and equipment locker my students would not have been able to participate in these real world hands on lab activities.

On a personal level my participation in the ICORE summer institute has been a career changing experience. In the last twenty years, knowledge in biological sciences has increased dramatically. Many topics, such as biotechnology, were not taught before then. I have often found myself in the position of having to teach students about concepts that were not part of the curriculum when I was in college. Lacking formal education in these new areas, I felt that I was not adequately prepared to share this cutting edge information with my students. The Summer Institute has been the answer to my prayers. I had the opportunity to participate, with other motivated teachers, in the most exciting educational experience I have ever had. We were taught some of the most modern techniques in biotechnology with the finest equipment available. This was

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truly a pivotal experience in my professional life. It has allowed me, quite literally, to move my teaching practice forward into the 21st century. There is so much more to learn, but, I have resources and information to share with my students and fellow teachers. My AP Biology students are learning and connected to innovative techniques that are being applied in the real world. Next year, I am piloting a biotech course at my high school. Other AP Biology teachers in the county have expressed interest in the Summer Institute and one colleague will be attending this summer. My professional learning community has expanded to include university and business professionals in the field. I am part of a grant proposal to participate in aligning a biotechnology curriculum between high school and community colleges. This is education as it is meant to be, a broad community of professionals at many levels coming together to educate our youth and prepare them for careers in science and technology.

MODIFICATIONS:

The unit was successful and met the objectives of providing students the opportunity to become familiar with the processes of biotechnology and its real world application in modern day science. Next year I would like to plan ahead and do a better job of scheduling the activities so that they follow a more logical sequence. For example:

• Students should have molecular biology content PRIOR to lab activities • Offer Mission Biotech as enrichment AFTER students participate in hands on lab

activities. • I had also planned to do the Southern Blot simulation, but ran out of time. I

would like to include this activity next year.

The greatest challenge in teaching the Advanced Placement biology course is getting through the material in a timely manner. Every year when March arrives I realize how much material I still have left to cover before the AP exam in early May, so whatever concepts I have not yet taught by that point are rushed. This is how I felt about this particular unit on molecular biology and biotechnology. Next year I would like to do a better job pacing the course material and not feel rushed covering this unit.

Arranging for the half day field trip and the CPET visitation was without a doubt the best part of this unit!! Not only would I have CPET come out again to do these lab activities next year, but I would like to see if I could incorporate more visitations and also plan ahead to better utilize resource opportunities like borrowing equipment from the equipment locker and use the simulation lab activities offered by CPET.

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