Which further opportunities does this concept have to increase performance and competitiveness in comparative sequence analysis? Improvements in performance can be achieved through changes in the instrumentation, the biochemistry and continuous evolution of the software algorithms applied for mass spectral interpretation. One of the more simple advancements with a direct impact on throughput would be the use of faster lasers. Most of the MALDI-TOF MS instruments currently applied in nucleic acid analysis use nitrogen lasers with a repetition rate of 20 Hz. X/Y movement of sample stage, spectra acquisition (usually 10-20 laser shots are acquired and summed) and real-time interpretation of the spectrum, e.g., the calling of a genotype can currently be achieved in about 1.5 s, with the majority of the time consumption related to data acquisition. The use of much faster lasers (e.g., 200 Hz nitrogen lasers) therefore would increase the sequencing throughput by 2.5-fold (and of course would then also improve depreciation of instrument cost significantly).
Despite increases in throughput, researchers are usually also interested in the opportunity to reduce reagent cost associated with sample processing. The base-specific cleavage assay, as introduced earlier in this chapter, offers considerable cost-saving opportunities. In the long run, these can best be achieved through advances in microfluidics and sample processing in microfluidic devices. A simple calculation reveals the cost saving potential. The PCR is currently performed in 384-well microtiter plate (MTP) format with 5 ml volumes. Some 2 ml volumes of the PCR reaction are used for an individual transcription and cleavage reaction, which usually is performed in 7 ml total volume. The sample is then diluted and the cleavage products are conditioned for MALDI-TOF MS using ion-exchange resin. The final sample volume for each reaction well is usually around 30 ml. Then, only 15 nl of sample are dispensed on a miniaturized chip array for automated MALDI-TOF MS analysis. This means that only about 1/2,000 of the sample is actually required for the final analysis step and therefore cost savings through miniaturization of sample processing are tremendous. The proof-of-principle for analysis is already established in the current format.
Finally, we should also take into account those performance improvements that relate to the applications listed in previous sections of this chapter. The main limitation in an application such as re-sequencing is related to loss of information: some cleavage products may be too small to be detected (their masses fall below the low mass cut-off around 1200 Da), some cleavage products may be too long to be detected (their masses fall above the high mass cutoff, where the sensitivity of axial DE-MALDI-TOF MS is limited) and multiple cleavage products may overlap in mass so that they cannot be resolved and changes in the mass signals remain obscured.
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