GMO Food Project - Emi Leonard

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GMO Food Project




Emi Leonard

School of Chemistry and Biochemistry

Georgia Institute of Technology


November 22
, 2010






Experimentation Dates:
November 3 and November 10
, 2010

Date Due:
November 22
, 2010

Date Submitted:
November 22
, 2010







All
work provided here was the author’s original work, and was done in
compliance with the Honor Code for the Georgia Institute of Technology.



__________________________________




___________


Signature









Date





2


INTRODUCTION

Genetic engineering of food involves the genetic modification of organisms. Many foods
today are genetically modified or contain genetically modified ingredients. Americans have
invested billions of dollars developing bigger, healthier, better
-
tasting, pest
-
resistant
, disease
-
resistant crops, including tomatoes


the food focused on in this experiment

[1]
. However,
despite the agricultural benefits of genetic modification, controversy exists about the possible
adverse effects of genetic modific
ation. The conflict
s

between organic farmers and
environmentalists and research scientist
s and private industries remain

in constant debate

[2]
.
With the push for an organic, natural diet in America more recently, many farmers have resorted
back to growing

crops under controlled breeding. Selection of the most profitable, best
-
looking,
and best
-
tasting foods to breed during the next growing season helps to reduce the need for
genetic modification of plants through unnatural selection. Even with the recent f
ocus on
controlled breeding, a method for detecting genetic modification in food is necessary, as foods
containing genetic modification
should

be labeled accordingly in grocery stores to keep
consumers informed about their purchases.

The goal of this exper
iment was to determine if grape tomatoes contain genetic
modification. This was accomplished by amplifying detectable plant and GMO genes through
polymerase chain reaction (PCR) and running the PCR products on an agarose gel to determine
the fragment sizes

of the bands for each sample.
The specific genes detected by PCR

were the
Photosystem II chloroplast gene, the cauliflower mosaic virus (CaMV) 35S promoter and the
nopaline synthase (NOS) terminator.

The latter two are common vectors for genetic modificat
ion
and are therefore
utilized to test the samples for genetic modification.

3


Tomatoes were the first genetically modified fresh fruits or vegetables commercially
available (
Flavr Savr

tomatoes)

[3]
.

Since then, GM tomatoes have disappeared from the market
.
However, new developments in research on GM tomatoes have revived interest in them. New
techniques would allow researchers to insert foreign DNA into the chloroplast rather than the
nuclear DNA which would reduce concerns of genetic pollution via cross p
ollination. Other
researchers have developed the world’s first salt
-
tolerant tomato

[3]
. While GM tomatoes
haven’t yet returned to the market, the interest in them is increasing.
The grape tomatoes utilized
in this experiment were purchased from Publix and

were from Santa Sweets, Inc. Santa Sweets,
Inc.
produces its grape tomatoes i
n Plant City, FL, Cedarville, NJ
, and Nogales, AZ. Santa
variety tomatoes are a first generation hybrid that
are guaranteed to have not been genetically
altered by the company

[4]
.

Therefore the hypothesis for this experiment was that grape tomatoes
are not genetically modified.


EXPERIMENTAL PROCEDURES


Materials
. The protocol and materials were adapted from the GMO Investigator Kit
produced by Bio
-
Rad. United States Santa Swe
ets
, Inc.

grape tomatoes were purchased from the
Publix supermarket in Atlantic Station in Atlanta, GA and kept refrigerated for one day prior to
use. Bio
-
Rad certified non
-
GMO grain and GMO DNA solution
were used as the negative and
positive control, resp
ectively. The Bio
-
Rad InstaGene™ matrix was used for DNA extraction.
PCR plant and GMO primers detecting for the Photosystem II chloroplast gene and the
cauliflower mosaic virus (CaMV) 35S promoter and nopaline synthase (NOS) terminator genes,
respectively
, were purchased from Bio
-
Rad. PCR was run on the Bio
-
Rad MyCycler™ thermal
cycler. The molecular weight marker and Orange G loading dye obtained from Bio
-
Rad, and the
4


GelStar obtained from Lonza

were provided for electrophoresis
.
The PCR products were run

on
an agarose gel in an Owl EasyCast MiniGel System (Thermo Scientific).
The gel was
photographed using a Fotophoresis™ UV Transilluminator and a digital camera.


DNA Extraction
. Non
-
GMO grain, soaked for three hours in deionized water, and a
sliced grape

tomato

(including skin, seeds and fruit) were homogenized into a pipettable slurry
separately in deionized water using a mortar and pestle. The plant control slurry and the grape
tomato slurry were added to separate 500 µL aliquots of InstaGene™ matrix. T
he plant control
and food samples in InstaGene™ matrix were heated in a 95°C water bath for 5 minutes. Both
samples were then centrifuged in a Fisher Scientific Marathon microA centrifuge for 5 minutes
at 5250xg. The resulting supernatant contained the DNA

sample.


Polymerase Chain Reaction
.
Six PCR samples were prepared by adding Plant and
GMO master mixes separately to the plant control, GMO DNA solution, and grape tomato food
sample.
The
Plant and GMO master mix
es

each contained PCR master mix (Taq DNA
P
olymerase,
dNTPs, MgCl
2
, and buffer) and plant primers or GMO primers, respectively. PCR
was run on the 6 samples according to the program described below in Table 1.


Table 1
. PCR Overview

Step

Function

Temperature (°C)

Duration (min)

Number of Cycles

Initial
Denaturation

Denature dsDNA

94

2

1

Amplification

Denature dsDNA

94

1

40

Anneal Primers

59

1

Polymerize

72

2

Final Extension

Polymerize

72

10

1

Hold

Hold Temp

41

Indefinitely

1


Agarose Gel Electrophoresis
. A 2 % agarose gel was prepared using 1.0103 g of
agarose and 50 mL of 1X TAE buffer.
GelStar (1µL) was added to the warm agarose prior to
pouring; the gel was allowed to solidify. Orange G loading dye was added to each PCR product
5


and the PCR molecular w
eight marker in a 1:5 dye to sample ratio. The 6 samples and marker
were loaded into the gel, and the gel was electrophoresed at 120 V for 1 hour. The gel was
photographed using a

Fotophoresis™ UV Transilluminator and a digital camera.


RESULTS


The agaros
e gel (Figure 1) of the PCR products
revealed

the DNA amplification of plant
and GMO genes. The PCR molecular weight marker separated into bands of known sizes: 1000,
700, 500, 200 and 100 base pairs. The migration distances of these bands were measured in

pixels using the Paint function of Microsoft. Estimated fragment sizes for the bands in the
sample lanes were determined (Table 2) from the linear regression of the graph of logMW vs.
migration distances of the molecular weight marker bands (Figure 2).


L
anes 3, 4, and 5 represent the results for the GMO master mix samples. Lane 3 was
loaded with non
-
GMO plant
DNA

and GMO master mix. No significant bands
(GMO DNA
-
containing)
appeared in this lane, as expected. Lane 4, containing GMO
DNA (
positive control
)

and GMO master mix, showed a bright band
at a migration distance corresponding to a fragment
size of 157 base pairs. This fragment size is slightly less than the expected 203 bp or 225 bp for
the GMO DNA, but still suggests a successful positive control.
Lane 5 containing the grape
tomato DNA and GMO master mix displayed no significant bands. Lanes 3, 4, and 5 also
showed bright bands at a fragment size of 74 or 79 base pairs, suggesting the presence of primer
dimers in all three lanes.


Lanes 7, 8, and 9

represent the results for the plant master mix. Lane 7, loaded with non
-
GMO plant DNA (negative control) and plant master mix,
showed a band at 420 base pairs and
at 87 base pairs. Lane 8 was loaded with GMO DNA and plant master mix and displayed a band
6


a
t 372 base pairs. Lane 9, containing grape tomato DNA and plant master mix, displayed bands
at 459 base pairs and 90 base pairs. The known fragment size of the Photosystem II chloroplast
gene amplified in the plant master mix was 455 base pairs; fragment s
izes of 420, 372 and 459
base pairs correspond to this gene. This suggests that the negative control was also successful.
The bands in lanes 7 and 9 at fragment sizes of 87 and 90 base pairs represent primer dimers.
Lane 8, however, lacks primer dimers.


T
he gel
had some smearing in the lanes (lanes 4 and 8) containing GMO DNA; these
lanes also displayed bands with smaller fragment sizes than expected. The bands appeared
broader and less defined than expected in all lanes, suggesting an error in the overall

electrophoresis. However, in general bands appeared where expected and most had minimal
smearing illustrating the success of the DNA extraction and PCR.













7













Figure 1
. Agarose gel (2%) of PCR molecular weight marker and 6 samples.
Lanes 3
-
5
represent samples with GMO master mix and Lanes 7
-
9 represent samples with plant master
mix. P stands for the plant sample (negative control), G stands for the GMO sample (positive
control), and F stands for the food sample (grape tomatoes).

















Figure 2
. Plot of Standard Curve of the PCR Molecular Weight Marker. By measuring the
migration distance of the bands in the MW marker lane, a linear regression function of the
logMW versus the migration distance (in pixels) graph was calc
ulated. This function was used to
determine the fragment sizes corresponding to the bands in each of the sample lanes.



y =
-
0.0012x + 3.7946

R² = 0.9962

1.5
1.7
1.9
2.1
2.3
2.5
2.7
2.9
3.1
500
700
900
1100
1300
1500
logMW

Migration Distance (pixels)

MW Marker Standard Curve

8


Table
2
.
Fragment Sizes of the Bands in the Agarose Gel Calculated from the Standard Curve



Migration Distance
(pixels)

Molecular Weight

(base pairs)

GMO Master Mix

Plant

1604

74

GMO

1332

157

1580

79

Grape Tomatoes

1580

79

Plant Master Mix

Plant

976

420

1544

87

GMO

1020

372

Grape Tomatoes

944

459

1532

90



DISCUSSION


The goals of this experiment were to extract DNA
from plant samples and to run an
agarose gel of PCR products to test for genetic modification of a grape tomato. Polymerase chain
reaction was used to amplify DNA fragments coding for detectable plant and GMO genes. The
detectable plant gene was the Photos
ystem II chloroplast gene, and the detectable GMO genes
were cauliflower mosaic virus 35S promoter and the nopaline synthase terminator genes. Since
the plant and GMO primers only amplify DNA coding for the detected genes, only sample DNA
containing the co
ding DNA sequence was amplified. Agarose gel electrophoresis, used to
separate DNA samples by fragment size, was run on the PCR products to check for the presence
of bands corresponding to fragment sizes of the amplified DNA sequences of the plant and GMO
genes.


Overall Conclusions
. The results of the agarose gel revealed that Santa Sweets, Inc.
grape tomatoes are not genetically modified. The extracted DNA from the grape tomato test
sample did not contain the DNA
sequence encoding for the cauliflower mosaic virus 35S
promoter or the nopaline synthase terminator genes, demonstrating that the grape tomato tested
9


had not been genetically modified. Bands appeared at 420, 372 and 459 base pairs for the plant,
GMO and gr
ape tomato samples, respectively, when combined with plant master mix. Since the
expected size of the Photosystem II chloroplast gene is 455 base pairs, these bands verify that
these samples are all plants, or more precisely, photosynthetic organisms.
The
grape tomato
sample contains a band at 459 base pairs confirming that it is a plant (and that DNA extraction
was successful).

For the samples prepared with GMO master mix, only the GMO positive control showed
a band at 157 base pairs. The expected fragment

size for the GMO detectable genes is 203 or 225
base pairs; therefore this band confirms the presence of genetically modified DNA in the GMO
positive control DNA.

The presence of a band at 420 bp for the plant sample and
a band at 157
bp for the GMO sampl
e, as well as the lack of a band around 200 bp in the non
-
GMO plant
sample
demonstrate the success of the negative
and positive
control
s
, validating the conclusions
made.

The grape tomato sample lacks a band near 200 base pairs suggesting that it is not
genetically modified. The success of both controls paired with the results support the conclusion
that the grape tomato tested was not genetically modified by the detectabl
e genes.

Anomalies
.
The gel had some smearing in lanes 4 and 8 containing GMO DNA; these
lanes also displayed bands with smaller fragment sizes than expected. This was most likely due
to an excess of DNA in these samples. The bands in these lanes were extr
emely wide and intense
suggesting a great deal of DNA was present; an excess of DNA would result in smearing and
would force the sample to spread out, skewing the calculated fragment size as seen.

The bands
also appeared broader and less defined than expec
ted in all lanes, suggesting an error in the
overall electrophoresis. This was probably due to an excess of DNA in all samples (too much
was loaded into each well) and to stopping the electrophoresis too early.
As discussed earlier,
10


excess DNA results in w
ider bands. Also, t
he longer a gel runs, the further the fragments are
allowed to migrate creating the separation desired in a gel. Had the gel been run for as little as
ten more minutes, it is possible that the fragments would have separated further creat
ing tighter,
more defined bands.


Five of the six samples displayed bands between 74 and 90 base pairs, corresponding to
primer dimers. Primer dimers occur when two primers anneal due to complementarity in their 3’
ends. During polymerization they are exte
nded resulting in primer dimers that are approximately
twice the size of the initial primer. During the following the cycles, these primer dimers continue
to compete for binding to the primer resulting in amplification and therefore visibility on the gel

[
5]
.

As primers tend to be 30
-
50 base pairs, the bands seen between 74 and 90 base pairs are
about twice this

and most likely correspond to primer dimers.
This anomaly occurs in both the
plant and GMO primers; however, the GMO primer dimer bands are much mo
re intense than
those of the plant primer dimers. This is possibly due to the sequence of the primers. Primer
dimers
form more readily if the initial annealing of the primers is stable. Constructs that contain
more G
-
C pairs or overlap longer are more stab
le

[5]
. It is possible that the DNA sequences of
the GMO primer dimers either overlap further or contain more G
-
C pairs than those of the plant
primer dimers. It should also be noted that the GMO DNA in lanes 4 and 8 seems to contain
fewer primer dimers th
an the surrounding lanes. Lane 8 actually contains no primer dimers. A
possible explanation of this phenomenon is that the large amount of
complementary
DNA out
-
competes the primer for annealing. In lanes 3 and 5 the primers had no complementary DNA to
bin
d to, so they seem to have simply bound to themselves resulting in the large quantity of
primer dimers. In lane 4, however,
the primers had a higher affinity for the GMO DNA than for
themselves, resulting in the lower intensity of the primer dimer band. In

lanes 7 and 9, the
11


primers had a higher affinity for the plant DNA so the primer dimer bands are less intense than
the plant bands. However, in lane 8 the huge quantity of GMO plant DNA resulted in complete
primer
-
plant DNA annealing and no primer dimer f
ormation.


Broader Impact
. The controversy with genetically modified organisms, primarily crops,
raises health and environmental concerns. Researchers and scientists are constantly searching for
a safe and effective way to enhance crops without creating un
pleasant effects. The results of this
experiment

illustrate the effectiveness of controlled breeding of plants as a possible
compromise.
The grape tomato tested was ripe, fresh, plump, and yet not genetically modified despite the fact
that it is currently
not in season. It was instead grown under controlled breeding and progressive
growing practices.

As the genetic modification of crops becomes more and more popular, reliable methods
for detecting genetic modification are necessary for traceability of these

plants. Due to low
concentrations of DNA and difficulties in extracting and isolating DNA from plants, detecting
genetic modification in plants has proven more difficult than expected. This experiment
proposed just one method for detecting genetic modific
ation, but there are genes other than the
CaMV 35S promoter and NOS terminator used to genetically modify plants that the method
failed to detect. Also, with increasing technology in the field, detection of the genetic
modification must remain cost
-
efficie
nt. Development of microarrays, mass spectrometry,
biosensors, and near infrared spectroscopy techniques are the main focus of possible new
detection methods

[6]
.

Overall, DNA extraction and PCR were successful techniques applied to amplify
DNA

sequences c
oding for plant
and

GMO detectable genes. The agarose gel run on the PCR products
revealed that the Santa Sweets, Inc. grape tomatoes were not genetically modified to contain
12


either the CaMV 35S promoter or the NOS terminator genes. Therefore, the hypothes
is, stating
that the grape tomatoes were not genetically modified, was accepted by the results of this
experiment
.



REFERENCES

[1] Ambra R
;

Azzini E
;

Durazzo A
;

Foddai MS
;

Maiani G. (2008) Assessment of the nutritional
values of genetically modified wheat
, corn and tomato crops
,
J Agric Food Chem 56
,
9206
-
9214.


[2] Dona, Artemis; Arvantioyannis, Ioannis S. (2009) Health Risks of Genetically Modified
Foods,
Critical Reviews in Food Science and Nutrition 49
, 164
-
175.


[3]
Bukenya, J; Wright, N. (2007)
Determinants of Consumer Attitudes and Purchase Intentions
With Regard to Genetically Modified Tomatoes
,
Agribusiness 23
, 117
-
130.


[4]
www.santasweets.com

(used to obtain product information)


[5] Chou, Q; Russell, M; Birch, D; Raymond, J; Bloch, W. (1992)
Prevention of pre
-
PCR mis
-
priming and primer dimerization

improves low
-
copy
-
number amplifications
,
Nucleic
Acids Research 20
, 1717
-
1723.


[6] Lu, J; Shi, X; Mo, Q; Li, X. (2008) Safety problem
s and detection technology of genetically
-
modified foods
, Xiandai Yufang Yixue 35
, 3951
-
3953.