Development and application of DNA analytical methods for the detection of GMOs in food

Development and application of DNA analytical methods for the detection of GMOs in food The principle of direct detection of recombinant DNA in food by the polymerase chain reaction (PCR) is discussed following the three main steps: DNA-extraction, amplification by PCR and verification of PCR products. Suitable methods for genomic DNA isolation from homogenous, heterogeneous, low DNA containing matrices (e.g. lecithin), gelatinising material (e.g. starch), derivatives and finished products based on classical protocols and/or a combination with commercially available extraction kits are discussed. Various factors contribute to the degradation of DNA such as hydrolysis due to prolonged heat-treatment, nuclease activity and increased depurination and hydrolysis at low pH. The term “DNA quality” is defined as the degree of degradation of DNA (fragment size less than 400 bp in highly processed food) and by the presence or absence of potent inhibitors of the PCR and is, therefore, a key criterion. In general, no DNA is detectable in highly heat-treated food products, hydrolysed plant proteins (e.g. soya sauce), purified lecithin, starch derivatives (e.g. maltodextrins, glucose syrup) and defined chemical substances such as refined soya oil. If the nucleotide sequence of a target gene or stretch of transgenic DNA is already known specific primers can be synthesised and the segment of rDNA amplified. Detection limits are in the range 20 pg–10 ng target DNA and 0.0001–1% mass fraction of GMO. Amplification products are then separated by agarose gel electrophoresis and the expected fragment size estimated by comparison with a DNA molecular weight marker. Several methods are used to verify PCR results and they vary in reliability, precision and cost. They include specific cleavage of the amplification products by restriction endonucleases or the more time-consuming, but also more specific, transfer of separated PCR-products onto membranes (Southern Blot) followed by hybridisation with a DNA probe specific for the target sequence. Alternatively, PCR products may be verified by direct sequencing. Nested-PCR assays combines high specificity and sensitivity. Methods for the screening of 35S-promoter, NOS-terminator and other marker genes used in a wide range of GMOs, the specific detection of approved products such as FlavrSavr™ tomatoes, Roundup Ready™ Soya, Bt-maize 176 and official validated methods for potatoes and genetically modified micro-organisms, that have a model character, are available. Methods to analyse new GMO products are being validated by interlaboratory tests and new techniques are in development (e.g. EC project: DMIF-GEN). However, these efforts may be hampered by the lack of availability of GMO reference material as well as specific sequence information which so far can only be obtained from the suppliers. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Food Control Elsevier

Development and application of DNA analytical methods for the detection of GMOs in food

Food Control, Volume 10 (6) – Dec 1, 1999

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Publisher
Elsevier
Copyright
Copyright © 1999 Elsevier Science Ltd
ISSN
0956-7135
eISSN
1873-7129
D.O.I.
10.1016/S0956-7135(99)00081-X
Publisher site
See Article on Publisher Site

Abstract

The principle of direct detection of recombinant DNA in food by the polymerase chain reaction (PCR) is discussed following the three main steps: DNA-extraction, amplification by PCR and verification of PCR products. Suitable methods for genomic DNA isolation from homogenous, heterogeneous, low DNA containing matrices (e.g. lecithin), gelatinising material (e.g. starch), derivatives and finished products based on classical protocols and/or a combination with commercially available extraction kits are discussed. Various factors contribute to the degradation of DNA such as hydrolysis due to prolonged heat-treatment, nuclease activity and increased depurination and hydrolysis at low pH. The term “DNA quality” is defined as the degree of degradation of DNA (fragment size less than 400 bp in highly processed food) and by the presence or absence of potent inhibitors of the PCR and is, therefore, a key criterion. In general, no DNA is detectable in highly heat-treated food products, hydrolysed plant proteins (e.g. soya sauce), purified lecithin, starch derivatives (e.g. maltodextrins, glucose syrup) and defined chemical substances such as refined soya oil. If the nucleotide sequence of a target gene or stretch of transgenic DNA is already known specific primers can be synthesised and the segment of rDNA amplified. Detection limits are in the range 20 pg–10 ng target DNA and 0.0001–1% mass fraction of GMO. Amplification products are then separated by agarose gel electrophoresis and the expected fragment size estimated by comparison with a DNA molecular weight marker. Several methods are used to verify PCR results and they vary in reliability, precision and cost. They include specific cleavage of the amplification products by restriction endonucleases or the more time-consuming, but also more specific, transfer of separated PCR-products onto membranes (Southern Blot) followed by hybridisation with a DNA probe specific for the target sequence. Alternatively, PCR products may be verified by direct sequencing. Nested-PCR assays combines high specificity and sensitivity. Methods for the screening of 35S-promoter, NOS-terminator and other marker genes used in a wide range of GMOs, the specific detection of approved products such as FlavrSavr™ tomatoes, Roundup Ready™ Soya, Bt-maize 176 and official validated methods for potatoes and genetically modified micro-organisms, that have a model character, are available. Methods to analyse new GMO products are being validated by interlaboratory tests and new techniques are in development (e.g. EC project: DMIF-GEN). However, these efforts may be hampered by the lack of availability of GMO reference material as well as specific sequence information which so far can only be obtained from the suppliers.

Journal

Food ControlElsevier

Published: Dec 1, 1999

References

  • Polymerase chain reaction in food microbiology
    Candrian, U.
  • Mit Hilfe der Gentechnik erzeugte Lebensmittel: Novel Foods und die Problematik ihres Nachweises
    Hammes, W.P.; Hertel, Ch.
  • Detection of the starch modifying gbss-antisense construct in transgenic potatoes
    Hassan-Hauser, Ch.; Mayer, W.; Hörtner, H.
  • Determination of DNA traces in rapeseed oil
    Hellebrand, M.; Nagy, M.; Mörsel, J.-T.
  • PCR-based analysis for the identification and characterization of food components
    Meyer, R.; Candrian, U.
  • Polymerase chain reaction (PCR) in the quality and safety assurance of food. Detection of soya in processed meat products
    Meyer, R.; Chardonnens, F.; Hübner, P.; Lüthy, J.
  • Detection of DNA in soybean oil
    Pauli, U.; Liniger, M.; Zimmermann, A.

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