Day 4: Genetics Selcen Guzey and Tamara Moore Agenda • Genetically modified crops - DNA extraction - PCR Activity 1: PCR with paper clips • Online resources-NIH Activity 2: Genetically modified sugar beet-Debate - Gel electrophoresis GMO Investigator Kit from BioRad • • • • Part I: DNA extraction (30 minutes) Part II: PCR (~4 hours) Part III: Gel electrophoresis (30 minutes) Part IV: Analyzing results (30 minutes) This kid provides an investigation in which you will extract DNA from food samples, amplify genetics sequences using PCR, and use gel electrophoresis to identify the presence or absence of the amplified marker sequences. Pre-lab Discussion • What is a GM crop? A plant that has been genetically modified by the insertion of foreign genetic material (pest resistance, herbicide tolerance, delayed fruit ripening, improved fruit yield, increased nutrient content etc.) • How does one test foods and crops to identify which contain GM genomes? ELISA (Enzyme linked immunosorbent assay) identifies specific proteins produced by GM plants. PCR (Polymerase chain reaction) Identify DNA sequences resulting from genetic modification (35 Promoter and Terminator from Agrobacterium tumefaciens) Part I: DNA Extraction • Each group will extract DNA from a control nonGMO food and a grocery store food item. Control non-GMO food: Certified non-GMO grain from BioRad Grocery store food samples: Cheese puffs, soy-based bacon bits, corn nuts, granola bars, corn cereal, tortilla chips, fresh corn, corn meal, organic cereal, potato chips • Weight your food sample, grind it with water, add a tiny amount of it to a srewcap tube containing InstaGene matrix and boil it for 5 minutes. Part I: DNA Extraction Material Quantity Screwcap tube 2 Beaker of distilled water 1 Food sample 2 Disposable plastic transfer pipets 2 2-20 µl micropipet (for non-GMO food cont) 1 2-20µl pipet tips 1 rack Mortar and pestle 1 Part II: PCR • How might PCR be used to determine if a food sample has been genetically modified? PCR can be used to detect foreign DNA in genetically modified food. PCR allows us to amplify specific sections of DNA and make millions of copies of the target sequence. Part II: PCR • This part of the experiment is to determine whether or not the DNA you extracted from food samples contains or does not contain the target sequences of interest typically found in GM foods. Quantity • In the firstMaterial part, you extracted DNA from a certified nonbath containing 3 1 GMO foodIce and a test food sample. In this part, you will tubes prepare those twomix samples and a positive control (GMO GMO master (red) 1 Plant master mix (green)for 1the PCR. positive templates DNA) GMO positive cont. DNA 1 Test food DNA 1 PCR tubes 6 Foam microtube holder 1 2-20µl micropipet 2-20µl pipet tips 1 1 rack PCR Tubes Tube # DNA Master Mix 1 20µl Non-GMO food control DNA 20µl Plant master mix (green) 2 20µl Non-GMO food control DNA 20µl GMO master mix (red) 3 20µl Test food DNA 20µl Plant master mix (green) 4 20µl Test food DNA 20µl GMO master mix (red) 5 20µl GMO positive control 20µl Plant master mix (green) 6 20µl GMO positive control 20µl GMO master mix (red) 1) You will set up 2 PCR reactions for each DNA sample, which makes 6 PCR reactions in total. One PCR, using the plant primers, is a control to whether plant DNA was successfully extracted from the samples. Plant primers amplify a section of a chloroplast gene (455 bp) 2) The second PCR reaction is to determine whether or not the food samples contains GM sequences. GMO primers identify DNA sequences that are common to (85%) GM plants (203 bp and 225 bp) 3)The non-GMO food is the indicator of false positive and the GMO positive template control is an indicator of false negative. PCR PCR is developed by Kary Mullis in 1983. It relies on thermal cycling, consisting of cycles of repeated heating and cooling of the reaction for DNA replication. DNA template is exponentially amplified. PCR requires several components: • DNA template • Primers that contains sequences complementary to the target region • DNA polymerase (Taq DNA polymerase from Thermus aquaticus) • Nucleotides • Buffer solution How is PCR used? • PCR impacted several areas of genetic research: – PCR used as a medical diagnostic tool to detect specific mutations that may cause genetic disease – PCR used in criminal investigations and courts of law to identify suspects – PCR used in the sequencing of the human genome PCR Denaturation: DNA is heated to separate strands (94 °C for 1 minute) Annealing: The mixture is cooled and primers bind to strands (60 °C for 1 minute) Elongation: DNA polymerase adds nucleotides to strands, producing 2 complementary strands (72 °C for 2 minutes) The procedure is repeated again! PCR Simulation http://learn.genetics.utah.edu/content/labs/pcr / Generation of precise-length fragments Original long template DNA molecules are never fully duplicated After each cycle, two intermediate-length strands are produced (they cannot exponentially amplified) The precise-length strands generated from the intermediate strands are amplified exponentially. PCR with Paper Clips • In this activity you will be performing PCR using paper clips. Each colored paper clip represents a particular nucleotide in the DNA: Blue: Adenin Red: Thymine Green: Guanine Yellow: Cytosine Online Resources NHI • http://ghr.nlm.nih.gov/ • http://www.ncbi.nlm.nih.gov/education/ • Human molecular genetics book: http://www.ncbi.nlm.nih.gov/books/NBK7580 / • BLAST: Basic Local Alignment Search Tool http://blast.ncbi.nlm.nih.gov/Blast.cgi Breast Cancer Susceptibility 1 and 2 Genes For every 1000 women…. 120 (12%) will get breast cancer 6 of them inherited mutations in BRCA1 or BRCA 2. BRCA 1 and BRCA 2 mutations account for about 5% of all breast cancer. This may seem like a small number, but a woman who inherits a cancer causing BRCA1 or BRCA2 mutation, her risk of developing breast cancer is up to 85%. The remaining cases are caused by mutations in other genes, environmental exposures, and other unknown factors. Breast Cancer Tests How can we diagnose BRCA 1 and BRCA 2 mutations? The doctor would isolate DNA from the patient’s blood and sequence the BRCA 1 and 2 genes to determine whether known cancer- causing mutations are present. National Center for Biotechnology Information (NCBI) 1) Go to http://www.ncbi.nlm.nih.gov/ 2) Type BRCA1 into the Search box 3) Click the “All resources” 4) Click the “Map viewer” 5) Select home sapiens and click go 6) Choose chromosome 17 7) Click BRCA1 8) Reference sequences under the table of contents 9) Click the FASTA BLAST Can be used to: -Compare 2 or more DNA sequences to each other -Compare 2 or more protein sequences to each other -Compare a single sequence to many sequences found in databases BLAST • Go to the NCBI blast website http://blast.ncbi.nlm.nih.gov/Blast.cgi • Select nucleotide blast • Click to “Align two or more sequences” • Copy reference sequence for BRCA1 • Copy the DNA sequence from the patient • Click BLAST • Click formatting options, find the Alignment view and choose “Query-anchored dots with identities” • Click reformat GM Crops • Bt corn, Bt cotton, and Bt potato (resistant to the Bacillus thuringiensis (Bt) toxin) • Roundup Ready soybeans and sugar beet (tolerant to the herbicide-glyphosate) • Golden rice (synthesize beta-carotene) • Flavr Savr tomato (slow the ripening process) • UH Rainbow papaya (resistant to plant virus disease) How do you genetically modify a crop? 1) Identify a protein that has the potential to improve a crop. Bt crops has a gene from Bacillus thuringiensis (Bt). They produce delta-endotoxin that is lethal to corn borers, a common pest on corn plants. 2) Isolate the gene that codes for the protein 3) Engineer the gene so that the crop plant’s cells can read it correctly- removing introns, adding promoter (CaMV35) and terminator from Agrobacterium tumefaciens. How do you genetically modify a crop? Inducing the gene into the plant: 1) Cells are removed from the parent plant, they grow on a special medium-Single cells, plant cells without cell walls and pieces of leaves can often be used to generate a new plant on culture media given the required nutrients and hormones. 2) The gene then transferred into the plant cells by: - electric shock/electroporation - heat shock -gene gun -Agrebacterium tumefaciens This bacterium causes crown gall disease by inserting some of its DNA to a plant cell. 3) The transformed cells are isolated and then induced with plant hormones to differentiate and grown into complete plants. The process is inefficient, costly, and time consuming! Sugar Beet Debate • Read the article individually and then share what you have learned with your group members. As a group you will be asked to address the advantages and disadvantages of three options from the viewpoint of your assigned stakeholder. 1. Issue a partial deregulation for the interim to allow planting in certain geographic areas and/or under certain conditions 2. Wholly deregulate the HT sugar beet, allowing it to be grown anywhere anytime 3. Refuse to deregulate HT sugar beet, allow for farmers to harvest existing beets, but no more planting. Sugar Beet Debate 1) Introduce yourself as your character 2) Which option would your assigned stakeholder advocate? If you pick (1) the partial deregulation option, describe the terms of the partial deregulation (geographic and conditions). 3) Support your option in terms of scientific, economic, and cultural justifications. 4) What are the potential adverse effects of the proposed options? Benefits and Risks Genetic modification of crops can produce four general benefits: 1) agricultural-increased yield, 2) environmental-reduced use of pesticides, herbicides, and fuel, 3) nutritional-improved quality of foods, and 4) disease prevention- foods that work like edible vaccines. Such risks may include: Exposure to possible allergens and toxins, harm to the environment, antibiotic resistance, and the spread of introduced genes to non-target plants by out- crossing and pollen drift Other Debates GMO corn and Monarchs Possible reading assignments: 1) Anthony M. Shelton and Mark K. Sears. (2001) The monarch butterfly controversy: scientific interpretations of a phenomenon. The Plant Journal 27(6), 483-488. 2) Losey, J., Raynor, L. and Carter, M.E. (1999) Transgenic pollen harms Monarch larvae. Nature, 399, 214. Regulation of Golden Rice Possible reading assignment: Potrykus, I. 2010. Regulation must be revolutionized. Nature 466: 561. How can we check whether the PCR generated the anticipated DNA fragment? Gel Electrophoresis Gel Electrophoresis • Gel electrophoresis separates DNA molecules based on charge and size. • DNA is negatively charged and is repelled by the negative electrode (cathode) and attracted by the positive electrode (anode) when an electric current applied across the gel. • Longer DNA fragments move more slowly than shorter fragments. Gel Electrophoresis • What results do you expect in each line? Lane Sample Sample 1 Non- GMO food control with plant primers Sample 2 Non-GMO food control with GMO primers Sample 3 Test food with plant primers Sample 4 Test food with GMO primers Sample 5 GMO positive control DNA with plant primers Sample 6 GMO positive control DNA with GMO primers Plant primers: 455 bp GMO primers: 200bp Expect Band? Analyzing Results • Step1: Check Plant PCR (Lane 5) • Step 2: Check GMO PCR (Lane 6)* • Step 3: Check DNA was extracted from non-GMO food (Lane 1) • Step 4: Check PCR reactions are not contaminated (Lane 2)* • Step 5: Check DNA was extracted from test food (Lane 3) • Step 6: Is test food GMO positive (Lane 4)? Analyzing Results Analyzing Results Analyzing Results • What was your test food? • Is your test food GMO positive?