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

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

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

Genetically Modified Food ARISE August 3, 2009

Outline How to make a GM organism Techniques Homework for tonight

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? http://www2.cast.ilstu.edu/ksmick/250/250lablink.htm

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? http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/A/Avery.html http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/R/RecombinantDNA.html#cloning

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?

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 http://www.oercommons.org/courses/detecting-genetically-modified-food-by-pcr/

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.”

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 http://www.pbs.org/wgbh/harvest/

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

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

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)

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

PCR: Cycles

PCR:

PCR: Thermocycler

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

Resources Potential Problems No amplification due to wrong buffer conditions No amplification due to lost enzyme activity Primers are wrong Online Resources: http://www.dnai.org/b/index.html http://www.genome.gov/10000202 Click on Techniques PCR is found in amplifying

Restriction Enzymes

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

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

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

Restriction Enzymes Student activity Potential Problems: Wrong buffer * activity Online resources http://www.dnai.org/b/index.html Click on Techniques Restriction enzymes are found in cutting and pasting

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?

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

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

Gel Electrophoresis

Gel Electrophoresis

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

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

Gel Electrophoresis

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: http://www.dnai.org/b/index.html Click on Techniques Gel electrophoresis is found in sorting and sequencing

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.

Restriction Enzymes

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

Gel Electrophoresis