Lesson 4: Identifying a Potato Killer via PCR and Gel Electrophoresis

Crops

Introduction

“Identifying a Potato killer” is a lesson designed to have students employ DNA extraction, PCR and Gel Electrophoresis techniques in identifying positive and negative leaf samples for the presence of the plant pathogen P. infestans. In this lesson, students will use laboratory technique and inquiry to determine how biotechnology is used to identify pathogens in the module “CSI Dublin: The Hunt for the Irish Potato Killer.” “CSI Dublin: The Hunt for the Irish Potato Killer” is an integrated project incorporating science concepts of earth science, biology, biotechnology and art to allow students to apply their understandings of epidemiology and plant pathology into the historical context of science and human health. This unit plan would be best used after previous units or discussions on DNA, biotechnology, taxonomy, bacteria, viruses, plants, protists, fungi and immunity.

Learning Outcomes

• The learner will employ the scientific method to extract DNA from infected leaf samples.
• The learner will use PCR and Gel Electrophoresis to positively identify P. infestans.
• The learner will appreciate the use of historical samples to solve long-standing mysteries.

Curriculum Alignment – National

• Science in Personal and Social Perspective, Content Standard F “Many diseases can be prevented, controlled or cured by knowledge gained from science.”
• Content Standard A: “As a result of activities in grades 9-12, all students should develop Abilities necessary to do scientific inquiry, Understandings about scientific inquiry”
• Content Standard E: “As a result of activities in grades 9-12, all students should develop Abilities of technological design, Understandings about science and technology”

Curriculum Alignment – State

• North Carolina Standard Course of Study for Biology, Goal 1
• North Carolina Standard Course of Study for Biology, Goal 3.04
• North Carolina Standard Course of Study for Biology, Goal 4.03

Classroom Time Required

This lesson requires 3 to 4 days of class time.
Teacher Preparation: 50 minutes.
Pre-Activity Discussion: 5 to 10 minutes depending on student questions, 1 day if virtual labs are completed in class instead of for homework.
Student Activity Day 1: 60 minutes.
Student Activity Day 2: 2.5 to 3 hours. (See Modifications Section.)
Post-activity discussion: 3 to 5 minutes depending on student questions and clean up.

Prior Knowledge/Vocabulary

Students should understand the steps of the scientific method, DNA Extraction, PCR, Gel Electrophoresis. Teachers may wish to have students undergo a “paper lab” or virtual lab of these techniques in order to understand these processes before they experiment with them in the classroom. Three recommended activities are listed in the Modifications Section.

Materials Needed

• Purchase of “Tracking Phytophthora infestans, the Irish Potato Famine Pathogen Kit” from Carolina Biological Supply Company ($249.00 to $258.00) (See Comments Section)
• Materials Not included in the kit but needed are pipettes, sterile tips, ice containers with crushed ice, micro centrifuge tube racks, minimum of 30, 1.5 ml micro centrifuge tubes
• Student Lab sheets
• CNN Global Challenges Video “A Hunt for the Killer” Can be downloaded at the following web link:http://www.cals.ncsu.edu/plantpath/people/faculty/ristaino/projects/QUICKTIM/Global_512k.mov
• Gel Electrophoresis Equipment including white light illuminators (See Modifications section)
• Thermal Cycler (See Modifications Section)
• Optional: Vortex, forceps, extra gloves, Gel Electrophoresis Models Technology Resources
• Students will need access to computers (with web access) either in class or out of class to complete the virtual labs. (Optional)

Activities

• The First step in the activity is to revisit the concepts of P. infestans and the Great Famine as outlined in the teacher and student guides. A seven minute video from CNN Global Challenges film, "A Hunt for the Killer," is available outlining Dr. Ristaino’s research using historical samples.
• Day 1 activities include DNA extraction and PCR. Students should follow the protocol listed in the Carolina Biological Kit Student Guides. Circulate around the room to ensure students are following directions and adhering to proper safety guidelines.
• Day 2 activity concerns Gel Electrophoresis. Since it takes 45 minutes for a gel to run, students on block scheduling will be able to wash and stain their gels the same day. Students should follow the protocol listed in the Student Guides. Circulate around the room to ensure students are following directions and adhering to proper safety guidelines.
• Assure students that only their analysis questions will be assessed upon accuracy as well as the thought and quality of their responses. Ensure that all students are able to complete the assignment. (See Modifications Section)

Assessment

• The teacher will score the student responses based upon the answers found in the teachers guide.

Modifications

• Many teachers find it difficult to have the time to complete Gel Electrophoresis in the classroom. Carolina Biological supply offers tips on cutting down the time spent in class on this process as well as offers types of faster gels. http://tiny.cc/21zf8
• Before students begin DNA extraction, have them complete this virtual lab available at http://learn.genetics.utah.edu/content/labs/extraction/ Student web quest pages are available for students to complete before this activity. A sample lab using their own cheek cells or other sources is available at http://learn.genetics.utah.edu/content/labs/extraction/howto/
• Before students begin PCR, have them complete this virtual lab available at http://learn.genetics.utah.edu/content/labs/pcr/ Student web quest pages are available for students to complete before this activity.
• Before students begin Gel Electrophoresis, have them complete this virtual lab available at
• http://learn.genetics.utah.edu/content/labs/gel/ Student web quest pages are available for students to complete before this activity.
• If your school does not have the gel electrophoresis chambers, there are “how-to” instructions to build your own from plexi glass located on the Learn.Genetics website at http://learn.genetics.utah.edu/content/labs/gel/gelchamber/
• Many schools do not have access to thermal cyclers. They are available on loan from the NC Science House available at http://www.science-house.org/ or through a local university.
• For ESL and ELL students, they may use an online translator such as Babelfish (http://babelfish.yahoo.com/) to translate entire web pages into their preferred language for information about these biotechnology processes. (See Websites)

Extensions

• Topics: Forensics, Ethics
• The NC DNA DAY website offers a student orientated module on solving a missing mascot using DNA evidence. http://www.ncdnaday.org/Resources2009/Forensics/

Websites

• http://learn.genetics.utah.edu/ This website offers many interactive tutorials and virtual labs on DNA Extraction, PCR, Gel Electrophoresis, essential to concepts of plant pathology.

Comments

• The web link to purchase this kit from Carolina Biological Supply Company is available at http://tiny.cc/dajYv . Requests for kits may be made to Dr. Jean Ristaino at NCSU jean_ristaino@ncsu.edu
• Many Students will find loading the gels difficult which may skew their results. Students may wish to practice with Gel Electrophoresis Models. These durable and reusable gels are excellent for student practice. http://tiny.cc/z88ob

Sources/Credits

• “Tracking Phytophthora infestans, the Irish Potato Famine Pathogen Kit” created by Dr. Jean Beagle Ristaqino, Professor, Department of Plant Pathology, North Carolina State University and Debbie Glenn.
• Carolina Biological Supply Company: http://www.carolina.com

Supplemental Files: 

LP4_DNA Extraction Student Webquest.pdf

LP4_Gel Electrophoresis Student Webquest.pdf

LP4_PCR Student Webquest.pdf

LP 4_DNA Extraction and PCR Student Lab Sheet.pdf

LP 4_Gel Electrophoresis Student Lab Sheet.pdf