Generation of Reference Material by the Use of Multiple Displacement Amplification (MDA) for the Detection of Genetically Modified Organisms (GMOs)

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 cost-intensive 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. Real-time 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 suitable for the use as reference material for qualitative GMO analysis.