DNA amplification and analysis: miniPCRTM GMO Lab Heart-Shaped Bananas Science for everyone, everywhere DNA amplification and analysis: miniPCRTM GMO Lab Heart-Shaped Bananas Release date: June 2015 © Copyright by Amplyus LLC, all rights reserved

Goals for the lab Understand how desired traits can be introduced in plants through recombinant DNA technologies Assess processed foods and plants for the presence of transgenic elements Foster evidence-based discussion about genetic modification

Putting DNA technology in your hands Powerful Portable Engaging Affordable Voiceover: why this

miniPCR is not a black box Heated lid 1 Self-heating block 2 Efficient cooling 3 4 On-board microprocessor

Today’s experiments will help us better understand GMOs Transgenic bananas produce ß-carotene A precursor to Vitamin A Vitamin A deficiency a global nutritional challenge Leading cause of preventable blindness in children 250M child. deficient in Vit A. 0.5M blind Genetically engineered foods Source of agricultural and economic growth Active ongoing debate in society We will use biotechnology to detect genetically engineered foods PCR for transgenic sequences DNA electrophoresis and visualization

Common challenges in agriculture Insects Draught/Compaction Fungus/disease Weeds

Agricultural crops have diverged from their wild ancestors Over last 4,000 years all major crop species have been domesticated: e.g., rice, wheat, and maize Classical genetics: selective breeding for plants with desired traits Early biotechnology: cross-breeding and hybrid plants Genetic engineering: ability to confer very specific traits rapidly by introducing particular genes directly into plants Teosinte: ancestor of modern corn http://nrm101-summer2010.community.uaf.edu/2010/07/12/a-history-of-corn/

Molecular diagnostics DNA technology has transformed food production And many other aspects of society Molecular diagnostics Text Consumer genomics Text Text Personalized medicine PCR Food and agriculture Text Text Human evolution Text Forensics

Typical components of a transgenic cassette CaMV35S Promoter NOS Terminator Transgene Elements of a transgenic cassette The CaMV35S promoter from the cauliflower mosaic virus (a virus that infects cauliflower plants) enables strong and generalized transcription of the transgene across all tissues of the host plant The transgene is the DNA sequence that we wish to express in the host plant to confer it a specific new trait (e.g. production of vitamin A precursor) The NOS terminator from the Ti plasmid in Agrobacterium tumefaciens is the most commonly used transcription terminator because of its ability to be recognized in most plant species Source: McBride and Summerfelt, 1990

We produce (and consume) many genetically engineered crops Examples of genetically engineered crops commonly grown in the US Crop Traits % modified (US) Apples Delayed browning Approved 2015 Canola Tolerance of glyphosate herbicides 87% (2005) Corn Tolerance of glyphosate herbicides. Insect resistance. Higher ethanol production 85% (2013) Cotton Insect resistance 82% (2013) Papaya (Hawaiian) Resistance to ringspot virus 80% Rice Enriched with beta-carotene (a source of vitamin A) Not yet on the market Soybeans Tolerance of glyphosate. Reduced saturated fats. Insect resistance. 93% (2013) Sugar beet Tolerance of glyphosate, glufosinate. 95% (2010) Tomatoes Suppression of polygalacturonase (PG), retarding fruit softening Taken off the market (commercial failure) Source: Wikipedia

Lab plan overview 1 2 3 DNA Extraction PCR Amplification Gel Electrophoresis Endogenous plant gene Tubulin ~187 bp product Transgene (GMO) CMV35s 125 bp product

Setting up DNA extraction from foods Place a small piece of test food or plant tissue on a clean surface. Crush the food into small crumbs (1-2mm) Move 1-2 mm food crumb into PCR tube and add 50 µL of Lysis buffer Incubate 5 min. at 95C in miniPCR using miniPCR in Heat Block Mode After removal from heat, add 5 µl of neutralization buffer. Mix.

Incubate 5 minutes at 95°C

Endogenous plant gene (internal control) Detecting plant transgenes by PCR Duplex PCR: internal control and transgene Endogenous plant gene (internal control) Tubulin ~187 bp product CaMV35S Promoter NOS Terminator Transgene GMO transgene 125 bp product Source: McBride and Summerfelt, 1990

Setting up a PCR experiment Template DNA GMO Lab primers DNA Polymerase Mix Buffer containing Mg2+ dNTPs Taq FWD primer REV primer A T G C

Prepare 4 new tubes for PCR (200µL tubes) EZ PCR Master mix 5X (Taq DNA Polymerase + PCR Buffer + Mg2+ + dNTPs) 5 µL 20 µL GMO Lab Primer mix (Forward and Reverse primers) DNA sample from food A DNA sample from food B GMO control Non-GMO control 2 µL A B G NG Also add your initials to side of tube

Programming PCR parameters Initial denaturation: 94°C 60 seconds Denaturation: 94°C 10 seconds Annealing 55°C 10 seconds Extension 72°C 15 seconds x35 cycles Final extension 72°C 30 seconds

Monitoring PCR amplification What is happening to DNA molecules at each step? Denaturation Annealing Extension Why do we need to add an enzyme (Taq polymerase)? What temperature is optimal for most enzymes? What makes Taq unique? How many more molecules of DNA will we have with each PCR cycle? And at the end of the entire PCR reaction? We call this exponential amplification How many target genes are we aiming to amplify in each tube? Why is this a duplex PCR?

The Polymerase Chain Reaction (PCR) Complex DNA sample The world us full of DNA Region of interest Amplified DNA (~1B copies) Find and replicate a specific DNA target

How PCR works: 3 steps to copy DNA 1 94°C Denaturation 50-60°C 2 Primer 1 Primer 2 Annealing 72°C 3 Taq DNA polymerase dNTPs Extension

Agarose gel electrophoresis

Load 15 µL per PCR sample Don’t pierce bottom! Load order: 1. non-GMO control 2. GMO control 3. Unknown A 4. Unknown B 5. DNA ladder (10 µL)

Detecting GMO sequences in foodstuffs miniPCR GMO Lab Detecting GMO sequences in foodstuffs Cheetos Doritos Papaya Taco shells GMO CTRL non-GMO CTRL Tubulin: ~187 bp product CaMV promoter: 125 bp product

Extension activity: Optimizing GMO detection for various foods

Optimizing GMO detection for various foods Extension activity: Optimizing GMO detection for various foods Vary number of PCR cycles Cheetos #1 Cheetos #2 GMO control 30 35 40 30 35 40 30 35 40 Vary incubation time for DNA extraction 1 min 5 min 10 min 15 min #1 #2 #1 #2 #1 #2 #1 #2 Other variables: annealing temperature, time

Appendix: Copy cycles amplify DNA exponentially