1. Genetically Modified Foods
The essential questions?
What ethical issues arise from genomic manipulation?
What are the societal implications?
How do scientist manipulate DNA and the genome of an organism?
Project ARISE: Advancing Rhode Island Science Education
Funding provided by a Science Education Partnership Award from the National
Center for Research Resources

2. Genetically Modified Foods
Student will know:
Students who complete this unit should have a better understanding of the
technology used to develop GM foods and any potential risks and benefits
of genetically modifying organisms.
Project ARISE: Advancing Rhode Island Science Education
Funding provided by a Science Education Partnership Award from the National
Center for Research Resources

3. Genetically Modified Foods
Students should ask:
Do we have enough information on GM foods to make an informed decision to
support or reject GM foods?
Project ARISE: Advancing Rhode Island Science Education
Funding provided by a Science Education Partnership Award from the National
Center for Research Resources

4. Genetically Modified Food
ARISE August 3, 2009

5. Outline
How to make a GM organism
Techniques
Homework for tonight

6. Have you ever eaten genetically modified food?
Can you tell the difference between a genetically modified organism and a non-GM organism?
Do GM foods taste any different? Could they?

7. What is genetic modification?
Does genetic modification only happen in plants?
No, the first gene was transferred into bacteria.
What are some reasons for genetic modification?
Express recombinant insulin in bacteria
What are some of the benefits and some of the disadvantages of GM foods?

8. How long have humans been genetically modifying organisms?
Teosinte
What about in the lab? How long have scientists been modifying organisms?
How is modern technology used to genetically modify organisms?

9. Why would we want to modify an organism?
Better crop yield, especially under harsh conditions
Herbicide or disease resistance
Nutrition or pharmaceuticals, vaccine delivery
“In 2004, approximately 85% of soy and 45% of corn grown in the U.S. were grown from Roundup Ready® seed.”
OER open education resources

10. Roundup Ready Gene
“The glyphosate resistance gene protects food plants against the broad-spectrum herbicide Roundup®, which efficiently kills invasive weeds in the field.  The major advantages of the "Roundup Ready®” system include better weed control, reduction of crop injury, higher yield, and lower environmental impact than traditional weed control systems. Notably, fields treated with Roundup® require less tilling; this preserves soil fertility by lessening soil run-off and oxidation.”

11. How to make a GM organism
Clone gene into vector (i.e. plasmid) with restriction enzymes and other molecular techniques
Transform into organism or into biological vector (agrobacteria or virus)
Infect plant with bacteria
Select for transformants with herbicide

12. What we are doing today Extract DNA from plant or food product
Use the technique of PCR to copy a region of DNA found in Round-Up Ready foods
Tomorrow we will analyze these products with gel elecrophoresis

13. Many of the same techniques are used to make a genetic modifications as to detect one
Polymerase Chain Reaction (PCR)
Restriction enzymes
Gel electrophoresis
Transformation

14. PCR
Invented in 1983 by Kary Mullis (Nobel Prize in 1993 for its discovery)
Uses primers to exponentially amplify a specific region of DNA
Components needed for the reaction:
DNA
Primers to region of interest
DNA polymerase (Taq – used to synthesize the DNA)
dNTPS (the building blocks of the copied DNA)
Buffer (with appropriate salts to ensure the enzyme works properly)

15. PCR Three steps of the reaction:
Denaturation: High heat (94-98o) to separate the strands of DNA
Annealing: (50-60o – depends on the primers) this step allows the primers to bind to the denatured DNA strands
Elongation (74o) – DNA polymerase synthesizes the new strand
This step is dependant on the length of the product to be amplified (1min/1kb of DNA)
Check products with gel electrophoresis and sequencing

16. PCR: Cycles

17. PCR:

18. PCR: Thermocycler

19. PCR: Applications Used to test for gene products for disease diagnosis
Used to amplify small amounts of material
Forensics
Fossil Records
Used for recombinant DNA technology
Used for virus detection

20. Resources Potential Problems
No amplification due to wrong buffer conditions
No amplification due to lost enzyme activity
Primers are wrong
Online Resources:
Click on Techniques
PCR is found in amplifying

21. Restriction Enzymes

22. Restriction Enzymes
Restriction enzymes are also called restriction endonucleases
They cut double stranded DNA at sequence specific sites
They can produce “sticky ends” or “blunt ends” depending on the enzyme
Sticky Ends
Blunt Ends
Sticky Ends
Blunt Ends

23. Restriction Enzymes
1978 Nobel Prize in Medicine was awarded to Daniel Nathans and Hamilton Smith for the discovery of restriction endonucleases
Restriction enzymes were discovered in E.coli as a defense mechanism against bacterial viruses (bacteriophages)
The recognition sites are usually 4-12 nucleotides long
Sequences are palindromic (GAATTC)
There are hundreds of restriction enzymes currently being used

24. Restriction Enzymes
What is better for making recombinant DNA: Sticky ends or blunt ends?

25. Restriction Enzymes Student activity Potential Problems:
Wrong buffer
* activity
Online resources
Click on Techniques
Restriction enzymes are found in cutting and pasting

26. Gel Electrophoresis
Gel electrophoresis is used to separate nucleic acids (DNA and RNA) or proteins for analytical use
DNA and RNA are separated using agarose
Proteins are separated using polyacrylamide
The gel is a matrix (cross-linked polymers) that allow products to be separated
Separation is based on the size (based on charge) of a product as it moves through a charged field
What is the charge on DNA and proteins? What is the shape? Why is this important?
How would you connect the electrodes?

27. Gel Electrophoresis
The negative charge is at the top (closest to the samples) and the positive charge is at the bottom
Samples are negatively charged and will travel towards the positive charge
DNA and RNA are negative because of their sugar-phosphate backbone
Proteins are denatured to give a constant shape and given a charge through the negative loading buffer used
Samples are diluted in a loading buffer that helps the samples stay in the wells

28. Gel Electrophoresis Applications Separating restriction digests
Analyzing/purifying PCR products
Sequencing
Protein analysis

29. Gel Electrophoresis

30. Gel Electrophoresis

31. Gel Electrophoresis
Sample agarose gel stained with ethidium bromide (EtBr)

32. Gel Electrophoresis Student activity
Practice loading a gel with 20uL Kool Aid or food coloring
Run gel and see color separation
Discuss what it means for the colors to separate

33. Gel Electrophoresis

34. Gel Electrophoresis Online Resources: http://www.dnai.org/b/index.html
Potential Problems
Connecting the charges backward
Not enough loading dye
Running the gel too hot
Handling EtBr
Online Resources:
Click on Techniques
Gel electrophoresis is found in sorting and sequencing

35. Homework Activate your Brown ID: http://activate.brown.edu
Read “Teaching High School Science Chapters 1 and 2
Read “GM foods & Teaching Critical Thinking”
Read GM Foods Unit and list three concerns or questions regarding the unit.

36. Restriction Enzymes

37. Restriction Enzymes: Applications
Restriction enzymes are commonly used in laboratories to create recombinant DNA
Harvest DNA products for other applications
DNase a general nuclease used to eliminate DNA in RNA samples

38. Gel Electrophoresis