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

http://www.vu.edu.au/

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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


RESUL & RESUL & DISCUSSIONDISCUSSION

RESUL & RESUL & DISCUSSIONDISCUSSION CONCLUSIONCONCLUSIONCONCLUSIONCONCLUSIONINTRODUCTIONINTRODUCTIONINTRODUCTIONINTRODUCTION

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• The herbicide-tolerant Roundup ReadyTM (RR)

Soybean (Monsanto) in Brazil

Herbicide tolerant soybean





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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???





Polymerase chain reaction (PCR)

Kary Mullis accepting the Nobel Prize PCR amplification

in 1993

Nested PCR increases the confident level

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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





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

12:00m0:30s

95o

62o

72o

1:00m

0:30s∞

4o

Temperature

Time

50 cycles1. 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 447GMO05 CCACTGACGTAAGGGATGACG

GMO08 TGGGGTTTATATGGAAATTGGAA 169GMO07 ATCCCACTATCCTTCGCAAGA

Primers GMO5 and GMO7 are complementary to the CaMV 35S promoterPrimers GMO9 hybridizes to the CP4 EPSPS sequence and GMO8to the CTP sequence

GMO8 GMO7 GMO5

GMO9

CaMV35S promoter CTPCP4 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





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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.





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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).





All soybean mixed samples containing 0.01–10% GM soybean.This fragment was absent for 0% GM soybean andalso 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.





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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





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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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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

Education

rr lane

pcr product

gm soybean

pcr conditions3

rr soybean detection

pcr protocol2

pcr primers3

negative control soybean