This research project aims to develop a new method to detect and quantify specific DNA sequences without PCR amplification.
<p>Current methods of GM detection and quantification are predominately ELISA and PCR based. Whilst PCR is an excellent tool for the qualitative detection of specific DNA fragments it is a less than ideal method of quantification due to its logarithmic nature of reaction.
<p>The reaction frequencies are never 100% efficient over the number of cycles required to effect detection and the enzymes are prone to inhibition by certain substances that commonly occur in food. However, it is difficult to devise a system that is comparable in sensitivity and selectivity without amplification.
<p>Quantitative 'real time' PCR is efficient in utilising small fragments of specific DNA sequences to facilitate detection and quantification. However, its results are not as accurate or reproducible as desired. It also requires amplification, which is not ideal for investigating the initial concentration of an analyte.
<p>This project therefore aims to couple the traditional strengths of both molecular biology and mass spectrometry to detect and quantify specific DNA sequences without PCR amplification by investigating and developing non-amplification methods.
DNA sequences to be used in the project will be defined. At the start of the project it is envisaged that the analysis of a particular genetically modified organism (GMO) will involve two assays: one for a DNA sequence that is specific to a particular GM event and allows the analysis of a number of coinciding events.
<p>The second assay will be for an endogenous gene that is species specific and to which all signals can be normalised.
<p>The DNA sequences will then be examined for the presence of appropriate endonuclease restriction sites. Several endonucleases may be required to produce size-paired fragments (one from the GM specific DNA and the other from the endogenous gene).
<p>These fragments will be concentrated from the digestion by their hybridisation with biotintylated capture probes. The biotinylated double stranded DNA will then be isolated from the digest mixture using superparamagnetic beads coupled to the biotin binding protein steptavadin.
<p>The isolated captured fragments will then be analysed using mass spectrometry. A full consideration of the results will be carried out to define the ions of interest and their suitability for quantification. When the basic method of detection has been established, the project will widen to examine different matrices that contain lower levels of GM events. The effects of this change will then be analysed.
<p>The project will then move to commercial samples and to validating the method. The limit of detection and the limit of quantification will be determined by testing a series of baked goods containing different levels of Monsanto's RoundUp Ready soya within a range of background unmodified soya levels.
<p>This will also enable the limits of the method to be established. Once these parameters have been established, further validation will be achieved. The analytical parameters such as reproducibility will be established and compared with that of real-time PCR.
<p>Find more about this project and other FSA food safety-related projects at the <a href="http://www.food.gov.uk/science/research/" target="_blank">Food Standards Agency Research webpage</a>.