nested pcr detecting gmo

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• Authors: Fa´ bio Cristiano Angonesi Brod, Cibele dos Santos Ferrari,
Luciana Lehmkuhl Valente, Ana Carolina Maisonnave Aris
(Universidade Federal de Santa Catarina, Brazil)
By Khang DT

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I. INTRODUCTIONI. INTRODUCTION
II. MATERIAL AND METHODII. MATERIAL AND METHOD
III. RESULTS AND DISCUSSIONIII. RESULTS AND DISCUSSION
IV. CONCLUSIONIV. CONCLUSION

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• Genetically modified orgamism (GMO)
Multifunction Productivity
MATERIAL &MATERIAL &
METHODMETHOD
METHODMETHOD
RESUL &RESUL &
DISCUSSIONDISCUSSION
RESUL &RESUL &
DISCUSSIONDISCUSSION CONCLUSIONCONCLUSIONCONCLUSIONCONCLUSIONINTRODUCTIONINTRODUCTIONINTRODUCTIONINTRODUCTION

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• The herbicide-tolerant Roundup ReadyTM (RR)
Soybean (Monsanto) in Brazil
Herbicide tolerant soybean
METHODMETHOD
METHODMETHOD
RESUL &RESUL &
RESUL &RESUL &

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• Article 21 For commerce of foodstuffs and food
ingredients, destined for human or animal
consumption, containing GMO above the 1%
threshold based on product level, the consumer
must be informed of the transgenic origin of this
product.’
• Labeling requirement
HOW TO DETECT GMO???
METHODMETHOD
METHODMETHOD
RESUL &RESUL &
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Polymerase chain reaction (PCR)
Kary Mullis accepting the Nobel Prize PCR amplification
in 1993
Nested PCR increases the confident level
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METHODMETHOD
METHODMETHOD
RESUL &RESUL &
RESUL &RESUL &
DNA
Nested PCR
region
PCR
region

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1. Samples1. Samples
The soy products (six defatted soy flours, six infant
formulas containing 14% soy protein isolate and
25 powdered soymilks) were purchased from
local supermarkets and pharmacies in Floriano´
polis,Brazil.
2. DNA extraction2. DNA extraction
CTAB protocol
3. PCR conditions3. PCR conditions
4. Agarose gel electrophoresis4. Agarose gel electrophoresis
METHODMETHOD
METHODMETHOD
RESUL &RESUL &
RESUL &RESUL &

2. PCR protocol2. PCR protocol
• 1XPCR buffer (20 mmol/l Tris-HCl, pH 8.4, 50 mmol/l KCl),
• 2.5 mmol/l MgCl2,
• 0.2 mmol/l of each dNTP,
• 0.5 mM of each primer,
• 1 unit of Taq DNA polymerase (Invitrogen)
• 2 ml of DNA (maximum 50 ng)
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PCR CONDITIONSPCR CONDITIONS
95o
12:00m
0:30s
95o
62o
72o
1:00m
0:30s
∞
4o
Temperature
Time
50 cycles
1. PCR thermal cycle1. PCR thermal cycle

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3. PCR primers3. PCR primers
Primer Sequence (50-30) Amplicon size (bp)
LEC1 GTGCTACTGACCAAGGCAAACTCAGCA 164
LEC2 GAGGGTTTTGGGGTGCCGTTTTCGTCAAC
GMO09 CATGAAGGACCGGTGGGAGAT 447
GMO05 CCACTGACGTAAGGGATGACG
GMO08 TGGGGTTTATATGGAAATTGGAA 169
GMO07 ATCCCACTATCCTTCGCAAGA
Primers GMO5 and GMO7 are complementary to the CaMV 35S promoter
Primers GMO9 hybridizes to the CP4 EPSPS sequence and GMO8
to the CTP sequence
GMO8 GMO7
GMO5
GMO9
CaMV35S promoterCTPCP4 EPSPS
DNA

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1. Samples1. Samples
The soy products (six defatted soy flours, six infant
formulas containing 14% soy protein isolate and
25 powdered soymilks) were purchased from
local supermarkets and pharmacies in Floriano´
polis,Brazil.
2. DNA extraction2. DNA extraction
CTAB protocol
3. PCR conditions3. PCR conditions
4. Agarose gel electrophoresis4. Agarose gel electrophoresis
METHODMETHOD
METHODMETHOD
RESUL &RESUL &
RESUL &RESUL &

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Fig. 1. Amplification of soybean lectin gene.
Lane 1: 50 bp ladder (Promega); lane 2: negative control (water); lane 3:
positive control (soybean DNA); lanes 4–14: powder soymilks.
METHODMETHOD
METHODMETHOD
RESUL &RESUL &
RESUL &RESUL &

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Fig. 2. RR soybean detection by nested PCR.
Lane 1: 50 bp ladder (Promega); lane 2: positive control (soybean 0.1% RR); lane 3:
negative control (water); lane 4: soybean 0% RR; lane 5: soybean 0.001% RR;
lane 6: soybean 0.01% RR; lane 7: soybean 0.1% RR; lane 8: soybean 1% RR;
lane 9: soybean 10% RR (8 ml PCR product+2ml loading buffer per lane).
METHODMETHOD
METHODMETHOD
RESUL &RESUL &
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All soybean mixed samples
containing 0.01–10% GM
soybean.
This fragment was absent
for 0% GM soybean and
also for 0.001% GM
soybean

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Lane 1: 50 bp ladder (Invitrogen); lane 2: negative control (water); lane 3: negative
control (soybean 0% RR); lane 4: positive control (soybean 10% RR); lanes 5–10:
soy flours, first DNA extraction; lanes 11–16: soy flours, second DNA extraction.
METHODMETHOD
METHODMETHOD
RESUL &RESUL &
RESUL &RESUL &

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Lane 1: 50 bp ladder (Invitrogen); lane 2: negative control (water); lane 3: positive
control (soybean 0.1% RR); lane 4: negative control (soybean 0% RR); lanes 5–15:
powder soymilks
METHODMETHOD
METHODMETHOD
RESUL &RESUL &
RESUL &RESUL &

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METHODMETHOD
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• In six analysed soy flour samples, four
samples were positive for the presence of
RR soybean
and in 25 analysed soymilk powder
samples, 15 showed a positive signal of
169 bp length for the nested PCR
detection of RR soybean.

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METHODMETHOD
METHODMETHOD
RESUL &RESUL &
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This study shows that the nested PCR method developed
by Meyer and Jaccaud (1997) is adequate to determine
the presence of GM soybean in samples of soybean
flour, soymilk powder and infant formula containing
protein isolate.
This nested PCR method could be employed to distinguish
GM from nonGM products, because the legal
requirement for the labeling of foods containing GMO.

nested pcr detecting gmo

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