G01024: An investigation into the use of electrospray-TOF to quantify specific DNA fragments generated from GMOs
Tuesday 23 March 2004
This research project aims to develop a new method to detect and quantify specific DNA sequences without PCR amplification.
Background
Real-time polymerase chain reaction (PCR) amplification is regarded as the ‘method of choice’ for quantification of genetically modified (GM) DNA, as there are currently no reliable alternatives. The project attempted to explore alternatives to PCR that could be used for the quantification of GM DNA in food matrices.
Research Approach
The aim was to couple molecular biology and mass spectrometric techniques to produce a robust and sensitive method for the detection and quantification of specific GM DNA sequences.
Results and findings
A high degree of sensitivity was demonstrated with synthetic DNA oligonucleotides and a specific GM fragment was identified by mass spectrometry after genomic digest and capture. Further work would be required before this approach could be successfully adapted for identification of genetically modified organisms (GMOs) in food.
Real-time PCR amplification is regarded as the ‘method of choice’ for quantification of GM DNA as there are currently no reliable alternatives. This project was an ambitious attempt to combine techniques from molecular biology and mass spectrometry (MS) to achieve reproducible and sensitive quantification of specific DNA sequences without amplification. This involved the use of biotin labelled synthetic capture oligonucleotides (oligo’s), complimentary to the target sequence that can be added to the DNA and allowed to hybridise. Biotin labelled double stranded DNA can then be recovered using superparamagnetic beads covalently coupled with streptavidin. Steptavidin is a protein that has a high binding affinity for biotin and allows a rapid and efficient isolation of biotin-labelled target molecules. This serves as a concentration step during the purification process and compensates for the lack of sensitivity of MS when compared to PCR.
GM event-specific DNA sequences from Roundup® Ready(RR) soya were used in this study as these sequences are in the public domain, however, it was envisaged that the methods devised could be adapted for any DNA sequence. Initially, an In silico approach was used to identify suitably sized oligo’s that span the RR soya DNA sequence of interest. These oligo’s were then synthesised commercially and used to optimise the biotin-streptavidin capture process described above. Once this process was completed, genomic DNA was isolated and purified from RR soya using an established procedure. Aliquots of this material were then cleaved using appropriate restriction enzymes to produce fragments of the required length. After hybridisation the biotin labelled target molecules were recovered and analysed by MS. Two methods were compared during this study: electrospray ionisation (ESI) MS and matrix assisted laser desorption ionisation (MALDI) MS.
Once finally optimised the biotin-streptavidin capture process delivered a very effective means of concentrating the low levels of specific oligo’s present in genomic DNA digests. The procedure was effective at presenting the captured oligo to the MS to allow characterisation. However, the captured oligo was not the expected size. Therefore, although the analytical method described has gone some way to proving that, in principal, a technique that does not involve PCR can be used to detect and characterise small DNA fragments. More investigation is required to address the problems encountered.