Based on the results of the pilot project the proposal suggests to explore and establish the potential of Matrix-Assisted Laser Desorption/Ionisation Mass Spectrometry (MALDI-MS) for the mass spectrometric analysis of polynucleotides and the routine, large scale sequencing of DNA. Even in the most advanced automated instruments for the Sanger technique separation of the polynucleotides on a gel is generally considered the limiting step, which could prohibit routine sequencing of the Human Genome in reasonable time. One of the promising alternative approaches towards this goal is to replace the separation on a gel by mass spectrometry, which in principle has a comparable sensitivity, but is much faster, has a superior absolute accuracy in terms of the exact definition of the polynucleotide (by absolute molecular weight) and is better suited for integration into a fully automated system and data analysis. Before the advent of MALDI-MS mass spectrometry of nucleic acids had been limited to relatively small oligo-nucleotides and sensitivity had been very low. Results obtained during the pilot phase have shown that with MALDI-MS polydeoxyribonucleotides with up to 20 bases give very good results. Between 20 and 30 bases spectra deteriorate significantly, no useful spectra could be recorded for DNA molecules with more than 30 bases. For polyribonucleotides, which generate more stable ions, spectra of molecules with up to 100 bases have been recorded. The results suggest that there is still considerable room for further improvement and that the technique indeed holds a potential for sequencing of polynucleotides, if chemical and physical conditions and parameters are optimized. Accordingly this project therefore has the following goals: (1) extend the mass range for the analysis of deoxyribonucleic acids beyond the currently accessible range of ca. 30 bases, (2) establish the optimum parameters for the analysis of ribonucleic acids with at least 100 bases, (3) analyse without further purification polynucleotide mixtures as obtained routinely from sequencing solutions and (4) demonstrate the potential of the technique for a prototype clinical application by sequencing of (relatively short), well defined DNA-segments in search for point mutations.
Topic(s)Data not available
Call for proposalData not available
Funding SchemeCSC - Cost-sharing contracts
5230 Odense M