CCQM BAWG – P113, Relative Quantification of Genomic DNA Fragments Extracted from a Biological Tissue

The US agricultural system is a major exporter of raw materials such as grain and finished foods, a large portion of which consists of commodity crops. Many of these crops are genetically modified (GM) primarily with traits beneficial to their production. Many importing countries regulate which type of GM crops are allowed and often require labeling when the concentration of these materials in the food or grain shipment reaches a specified level. Thus testing is used both prior to export and at the point of import of US products. These tests often involve testing for specific DNA sequences that identify the genetically modified event or for the protein product of introduced genes. Because of the proliferation of testing protocols and reference materials, trade in grain and foods can be blocked due to inconsistencies in the testing and leading to financial loss.

The provision of a traceable standard to the biological community is an area of active research for many National Measurement Institutes (NMIs). The quantification of the relative amount of DNA sequences extracted from a biological tissue remains a complex analytical procedure and relies on the availability of such standards. Real-time PCR is currently the measurement method most commonly applied to identify and quantify DNA sequences. The goal of this study was to demonstrate the ability to quantify DNA sequences present in a biological tissue using an independent calibrant system, in this case, using a plasmid calibrant for genomic DNA. This study will help determine if the calibrant system is fit for the intended use of quantifying the amount of biotech material in a batch of maize. The study was organized by the Institute for Reference Materials and Measurement (IRMM), Geel, BE. Fourteen NMIs participated and results were presented at the Consultative Committee for Amount of Substance (CCQM), Bioanalytical Working Group (BAWG) meeting in November 2008.

Participants in the study were provided with a material to serve as a calibrant for quantitative PCR consisting of a plasmid containing the DNA sequences of segments of an endogenous gene target and a transgenic gene target. The ratios of transgenic and endogenous genes was certified to be 1:1 and quantified in units of copy number of plasmid. The endogenous gene target came from the high mobility group gene, which is a maize specific target. The transgenic target came from a genetically engineered maize event named 1507. The unknown samples to be analyzed were maize powders that contained a defined mass fraction of genetically engineered maize 1507 mixed with wild type maize. The unknown samples were blinded certified reference materials (CRMs) which had already been tested for homogeneity and stability.

The unknown samples first had to undergo DNA extraction and purification to produce DNA suitable for real-time PCR. Then the samples were quantified for both endogenous gene and transgenic gene in units of copy numbers of each gene target. The ratios of transgenic and endogenous genes were expressed as a percent along with uncertainties.

• As published in the Draft Report of the CCQM-P113 (Oct 31, 2008), NIST’s measurements fell well within the values calculated by the NMI hosting the pilot study.
• Based on this preliminary data, use of a plasmid calibrant for relative quantification of DNA sequences extracted from a biological tissue may be fit for the intended use of quantifying the amount of genetically engineered material in a batch of maize. Use of this calibrant may permit correct labeling of food products.