Biotechnology Hands-on Investigations Nancy Dow Jill Hansen Tammy Stundon May 11, 2013

1. Biology Partnership (A Teacher Quality Grant) Biotechnology Hands-on Investigations Nancy Dow Jill Hansen Tammy Stundon May 11, 2013

2. Pre-test???? Q and A board What is Biotechnology? How do we use Biotechnology?

3. Florida Next Generation Sunshine State Standards SC.912.L.16.10 Evaluate the impact of biotechnology on the individual, society and the environment, including medical and ethical issues. Clarification Students will evaluate examples and/or explain the possible impact of biotechnology on the individual, society, and/or the environment. Content Limits Items may assess current issues but will not require knowledge of specific biotechnologies or specific medical issues. Items assessing the possible impacts of biotechnology will not assess monetary impacts.

4. Crossing -over • Occurs during Prophase I of Meiosis • Between chromatids of homologous pairs • Increase genetic variation DNA • Function • Components • Location

5. U.S. Department of Energy Genomics:GTL Program http://www.ornl.gov/hgmis

6. Biotechnology • Genetic engineering • def: use of technology to alter the genes of viruses, bacteria, and other cells for medical or industrial purposes to better the quality of life • Altering genes of unicellular organisms and plants and animals

7. Genome: composed of DNA, is our hereditary code (the “blueprint”) • Molecular biology: the study of genes and the molecular details that regulate the flow of genetic information from DNA to RNA to proteins, from generation to generation. Biotechnology uses this knowledge to manipulate organisms’ DNA to help solve human problems.

8. Caught by a Kiss! Using enzyme amylase Forensic Science! Crime Scene Isolate DNA evidence from saliva Ready to use what you know in the ‘real world’?

9. Gel Electrophoresis • Technique used to separate DNA bands based on size • Comparison of sample bands to markers allows… • Visible confirmation of desired product • Quantification of sample DNA

10. How does it work? • Electrolysis:the splitting of water using electricity • Electrophoresis: a method of separating charged molecules in an electrical field; DNA has an overall negative charge Used to separate DNA fragments by size

11. Why Run a Gel? DNA fingerprints are useful in several areas of society. They are used by professionals in human health and the justice system. 1. Diagnosis of inherited disorders 2. Developing cures for inherited disorders 3. Forensic or criminal 4. Personal identification

12. Extract DNA, suspend in buffer • Restriction enzymes search and isolate unique sequences found in your genome. Everyone has a different number of these sequences found at different locations • PCR replicates your unique sequences so we have enough to visualize • Result is several different pieces of DNA of differing sizes and amounts

13. How do we visualize DNA?

14. Steps of Gel Electrophoresis Prepare agarose gel (just like jello – but not as tasty) Pour into casting tray with comb and allow to solidify Add running buffer, load samples and marker Run gel at constant voltage until band separation occurs View DNA on UV light box and document results

15. ElectrophoresisEquipment Power supply Gel tank Cover Electrical leads  Casting tray Gel combs

16. 1% agarose 2% agarose Agarose Gel • A porous material derived from red seaweed • Acts as a sieve for separating DNA fragments; smaller fragments travel faster than large fragments • Concentration affects DNA migration • Low conc. = larger pores better resolution of larger DNA fragments • High conc. = smaller pores better resolution of smaller DNA fragments

18. Loading Dye Practice loading your gels now! • DNA samples are loaded into a gel after the tank has been filled with buffer, covering the gel • Contains a dense substance, such as glycerol, to allow the sample to "fall" into the sample wells • Contains one or two tracking dyes, which migrate in the gel and allow monitoring of how far the electrophoresis has proceeded.

19. Who Dunnit?

20. Virtual Gel - Guided Inquiry

21. POLYMERASE CHAIN REACTION(PCR) • 1983 • With PCR, you can target and make millions of copies (amplify) a specific piece of DNA (or gene) out of a complete genome. - PCR impacted several areas of genetic research: - as a medical diagnostic tool to detect specific mutations that may cause genetic disease - in criminal investigations and courts of law to identify suspects - in the sequencing of the human genome

22. PCR Amplification PCR intro • PCR makes use of the same basic processes that cells use to duplicate their DNA (replication) • Complementary DNA strand hybridization • DNA strand synthesis via DNA polymerase

23. Recipe for PCR Amplification 1. DNAsample: containing the intact sequence of DNA to be amplified 2. Master mix: √ Free nucleotides (dNTPs): raw material of DNA (A,T,C,G) √ DNApolymerase (Taq polymerase): enzyme that assembles the nucleotides into a new DNA chain √ Primers: pieces of DNA complementary to the template that tell DNA polymerase exactly where to start √ Fluorescent dye: “lights up” when it binds to complete DNA strands 3. Thermocycler: used to rapidly heat and cool DNA samples to facilitate DNA amplification.

24. Steps of PCR 1. Temperature separates the strand ↑ temperature 2. Primers attach like Bookends ↓ temperature 3. Taq takes free nucleotides & adds them to the ends of the primer ↑ temperature Step 3 is also called Elongation

25. Results of PCR With 30 cycles the DNA is amplified exponentially Results in 1.1x1012 sets of precise-length DNA PCR Song

26. Manipulating the DNA Bt CORN Bacterium (Bacillus thuringiensis) Bt crystal gene Plant cell Corn genome 1 2 3 Corn plant destroyed by insect pests Bacterial gene coding for Bt crystals, which are poisonous to the insect pests, is inserted directly into the corn plant’s DNA. Bt crystals—toxic to insect pests— are now produced by the corn plant itself, reducing the amount of pesticides the farmer must use.

27. Manipulating the DNA GOLDEN RICE Genes in the pathway for beta-carotene production are introduced into the white rice genome. Almost 10% of the world’s people suffer from vitamin A deficiencies—leading to 250,00 cases of blindness each year. The addition of beta-carotene-producing genes to white rice has increased its vitamin A content almost 25-fold. Daffodil Bacteria White rice Beta-carotene Golden rice

28. Manipulating the DNA GENETICALLY MODIFIED CROPS IN THE UNITED STATES Corn Cotton Soybeans 85% 76% 55% 45% 24% 15% Proportion of crops that are not genetically modified Proportion of crops that are genetically modified Commonly called GM Foods

29. GMO Computer lab • Selective breeding • Transgenic manipulations • Guess what’s coming to dinner “

30. GMO Debate Your scientists were so preoccupied with whether or not they could, they didn't stop to think if they should.” Dr. Ian Malcolm, Jurassic Park • Should we or shouldn’t we…… Jurassic Park Mr. DNA clip

31. Follow up • Q & A • Post Test

32. DNA Science: by David A. Micklos and Greg A. Freyer • Walkthrough with Quiz • Virtual Lab