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
•
•
•
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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?