Generation of Reference Material by the Use of Multiple Displacement Amplification (MDA) for the Detection of Geneticall
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Generation of Reference Material by the Use of Multiple Displacement Amplification (MDA) for the Detection of Genetically Modified Organisms (GMOs) Lillian Roth & Jutta Zagon & Ines Laube & Arne Holst-Jensen & Hermann Broll
Received: 16 January 2008 / Accepted: 18 February 2008 / Published online: 21 May 2008 # Springer Science + Business Media, LLC 2008
Abstract The identification of genetically modified (GM) organisms (GMOs) in unknown samples by polymerase chain reaction (PCR) requires the use of a positive control sample, containing the target sequence derived from the respective GMO. For this purpose, either DNA extracted from suitable reference material or plasmids bearing the sequence are used. In the case of isolated genomic DNA, the preparation is cost-intensive and time-consuming, and material availability may be limited. Once the sequence is cloned into a plasmid, it may be simpler and less costintensive to purify the DNA, but contamination risk is substantially higher. The potential of multiple displacement amplification (MDA) as a new tool to generate reference material for GMO detection was studied taking the GM maize MON810 as a case. MDA yield of maize-specific DNA and amplification efficiency in dependence of the amount of starting material were estimated using real-time PCR. Applicability of the amplified DNA for the use as reference material was tested with regard to real-time PCR performance and MDA bias. Depending on the amount of genomic DNA (gDNA) input into MDA, amplification rates in the range of 30-fold (100 ng input of gDNA) to 23,000-fold (0.1 ng input of gDNA) were achieved. Realtime PCR performance and gene representation of amplified DNA (mdaDNA) are comparable to those of gDNA. Our results demonstrate that the DNA amplified by MDA is L. Roth (*) : J. Zagon : I. Laube : H. Broll Bundesinstitut für Risikobewertung (BfR), Thielallee 88-92, 14195 Berlin, Germany e-mail: [email protected] A. Holst-Jensen National Veterinary Institute, P.O. Box 8156 Dep., 0033 Oslo, Norway
suitable for the use as reference material for qualitative GMO analysis. Keywords Whole Genome Amplification . Multiple Displacement Amplification . Phi29 DNA Polymerase . Reference Material . GMO . Detection
Introduction According to the Regulation (EC) No. 1829/2003 and the Regulation (EC) No. 1830/2003, products which are, consist of, or contain GMOs need to go through an authorization procedure in which safety assessment is a core element. After authorization, products have to be labeled and need to be traceable in each stage of production, processing, and distribution. Only in cases where the GMO level of each individual ingredient is below 0.9% and presence is technically unavoidable or resulting from adventitious contamination is labeling not required. This rule applies to food as well as feed products. Moreover, according to current legislation, it is necessary for the GMO-producing companies to provide the Community Reference Laboratory for GM Food and Feed (CRL-GMFF) with control samples of the GMO and
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