A MALDI-TOF instrument with much higher resolution (Mr 10,000 instead of the Mr 800 currently achieved) and higher sensitivity in the high mass range
(8000+ Da) will be helpful to improve identification and characterization of sequence changes. These performance characteristics can, for example, be achieved with recently introduced orthogonal-extraction MALDI-TOF mass spectrometers (also called O-TOF) (Berkenkamp et al, 2003; Loboda et al, 2003). The higher mass resolution reduces spectral overlap of mass signals (with the restriction of course that the isotopic envelope inherent to ''natural'' nucleic acid building blocks is still limiting) and enables processing of spectra with much higher peak densities. This is particularly interesting, if instrumental improvement in mass resolving power is coupled with the use of isotopically depleted nucleotides during the transcription and cleavage process. In simulations using the same data set as employed for the results displayed in Figure 2, the combination of O-TOF and isotopically depleted nucleotides for base-specific cleavage increased the discovery rates of heterozygous sequence changes (all possible single base sequence changes simulated) from an average 95% (at 500 bp amplicon length) to an average 98% (Sebastian Bocker, University of Bielefeld, personal communication). Early experiments to improve performance of MALDI-TOF MS analysis of nucleic acids with isotop-ically depleted nucleotides have already been published (Abdi et al., 2001, 2002; Tang et al, 2002). An additional benefit of the use of orthogonal TOF mass spectrometers may be the achievable throughput. They can be equipped with 1 kHz lasers, which will allow further improvements and data acquisition speed.
While the reduction of overlapping mass signals (by improved mass resolution and use of isotopically depleted nucleotides) helps significantly in avoiding ambiguities in the identification and characterization of sequence changes, it cannot resolve issues related to the reconstruction of regions where significant information is lost in the low mass range. This issue, however, can be tackled by modification of the biochemistry. The base-specific cleavage process described above uses complete cleavage. Due to the complete cleavage, we isolate the cleavage products from their original position within the amplification product. Hence, we lose the information about how the cleavage products were ordered. Furthermore, the size distribution of cleavage products is dependent on the sequence context and distribution of cleavage sites. In some cases, many of the cleavage products will fall under our low-mass cut-off.
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