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 EducationFunding 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 EducationFunding 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 EducationFunding provided by a Science Education Partnership Award from the National Center for Research Resources

Genetically Modified Foods

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?

• What about in the lab? How long have scientists been modifying organisms?

• How is modern technology used to genetically modify organisms?

Teosinte

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.”

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

• 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

PCR: Cycles

PCR:

PCR: Thermocycler

• 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

PCR: Applications

• 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

Resources

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

Sticky Ends

Blunt 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

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

• 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

Restriction Enzymes

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

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• 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 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

Restriction Enzymes: Applications

Gel Electrophoresis