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BIOL1500 – Lab 10

Using DNA to Identify GMO foods


PCR/DNA fingerprinting

Using genetic information to identify GMO foods

In this lab you will be doing the following:


DNA isolation on food products (corn or soy / organic and nonorganic)
DNA amplification by polymerase chain reaction (PCR) on food DNA to
detect the presence of genetic modification.
Using genetically modified controls and samples will be analyzed using
agarose gel electrophoresis.

Objectives
At the end of this lab, you will be able to perform:
• a method to detect genetic modification in food.
• a method for isolating DNA.
• a method called PCR for amplifying DNA .
• a method for separating DNA by size using agarose gel electrophoresis.
Genetically Modified Foods

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.
Scientific America GMO
A Typical Genetic Modification Procedure

Figure 9.1
BIOL1500 – Lab 10

• Goal: Compare the DNA from non GMO foods and GMO foods

• Problem: The amount of DNA present usually is pretty limited


- Need a way to amplify the small amount of DNA so that
analysis/comparison can be done

• In 1983, Kary Mullis developed a technique called polymerase chain


reaction (PCR) that functions to amplify specific regions of DNA
- PCR = Targeted DNA replication in a test tube

PCR

Solution: Use PCR to amplify parts of the genome that contain target sequences
BIOL1500 – Lab 10
GMO – PCR

• Basic steps of PCR (each repeated 30-35 times)_thermal cycler


1. Denaturation – Separating the double-stranded DNA into 2 single strands
- Heat the DNA to 95°C
2. Annealing - Sequence specific primers are
added to the DNA
- Primers = Short pieces of DNA that are
complimentary to a specific part of the
genome (e.g. region of tandem repeats)
- One primer sits at the start of the gene
and the other sits at the end.
- Switch to 45- 55°C to allow the primers to bind
to the template DNA
- These primers are so specific that they
will not bind to any of the other
2.9 billion nucleotides
3. Extension – DNA polymerase makes a new
strand from each template (using each primer
as a start. 75°C optimum temp
- Only replicates the part of the genome that is recognized by the primers
BIOL1500 – Lab 10

• What are you going to be doing today?


- You will be extracting DNA from GM and non-GM foods
- Set up the PCR tubes –adding the extracted DNA with a Master Mix (MM)
The GMM master mix anneal to target DNA of GM plants
The PMM master mix anneal to target chloroplast gene in plants
•Using PCR/specific primers, you will amplify a part of the genome of plants

- You will work in 5 groups


- Following the instructions on handout
- NOTE: The lab staff will set up the PCR reactions in a thermocycler and run them
overnight. You will analyze your results next week.
- Next lab we will go over how to analyze PCR products using electrophoresis

• What is due and when?


- Answer questions on handout after we view our gels next lab
Last Lab
DNA Fingerprinting

Part II
DNA Fingerprinting
TEAM
PART 2
ADD THE LOADING DYE TO THE DNA SAMPLES
Use micropipettors to add 10 µl of Loading Dye
(LD) to each DNA sample tube – make sure the
dye and sample mix and are at the bottom of
the tube
Use a new tip for each sample
LANE SAMPLE LOAD VOL
1 Non GMO PMM 20 ul
2 Non GMO GMM 20 ul
3 Test GMO PMM 20 ul
4 Test GMO GMM 20 ul
5 + control PMM 20 ul
6 + control GMM 20 ul
DNA Fingerprinting
LOAD DNA SAMPLES
INTO THE GEL
Use the micropippete
to add 20 µl of the
DNA samples into
the wells in the gel
Use new tip each time
DNA Fingerprinting
TEAM
POUR TBE BUFFER INTO THE ELECTROPHORESIS CHAMBER
START THE ELECTROPHORESIS
RUN THE DNA GEL for 30 minutes at 100 volts

Run the gel until the


yellow bands from the
loading dye have gone
all the way across the
gel or as long as time
permits.
DNA FINGERPRINTING

• DNA CUT BY RESTRICTION ENZYMES


• FRAGMENTS SEPERATED BY
ELECTROPHORESIS
• “FINGERPRINT” IS THE PATTERN OF
FRAGMENTS (BANDS)
DNA Fingerprinting

OBSERVE THE DNA GEL


LARGER
What are those bands?
Each is a fragment of DNA DNA FRAGMENTS
/ BANDS
of a particular size.
The smallest pieces move SMALLER
the fastest or furthest
through the gel material.
The pattern shows the
number of cuts that were
made and the size of the
pieces that were made.
Results of Electrophoresis

Compare your
test food
With the non
GMO band
and the +
control to
determine If
your sample
food source
Contains GM
genes
BIOL1500 – Lab 10
Using DNA for Forensic Science
PCR/DNA fingerprinting

• DNA is routinely used as a "molecular fingerprint" in paternity cases, criminal


investigations, infectious diseases, and palaeontology

• Why use DNA?


- Most (99.5%) of our genome doesn’t differ too much from individual to individual
- However, certain parts of the genome are unique in each person (like a
fingerprint) (~.5% vary from person to person)
- Which parts?
- The garbage part of our genome (regions of chromosomes that don’t
contain any genes, non coding regions) contains stretches of sequence that is
repeated over and over. These are called tandem repeats or STR’s

CCC TCAT TCAT TCAT TCAT TTTCAAA

- Different people contain different numbers of these tandem repeats


- I may have 3 copies of the above repeat in my DNA and my neighbor might
have 8 copies of same repeat
 We can use these differences to identify specific individuals in the population