The objective of this proposal is to develop methodology for high speed DNA sequencing that can yield sequence data roughly two orders of magnitude faster than current state-of-the-art technology, with comparable or superior accuracy. Specifically, the goal is to provide full sequence information on DNA stands consisting of at least 500 bases in seconds to minutes. The proposed approach is based on electrospray ionization/quadrupole ion trap mass spectrometry employing gas-phase ion/ion chemistry. Electrospray has been shown to be capable of yielding gas-phase ions of nucleic acid oligomers extending far in excess of 500 bases. However, electrospray-based approaches for high speed DNA sequencing have not been extensively pursued due to spectral congestion associated with multiple charging phenomenon that is characteristic of electrospray. The formation of multiple charge states from a single oligomer severely limits the mixture complexity amenable to direct analysis via electrospray. For this reason, electrospray usually follows a separation method, such as liquid chromatography or capillary electrophoresis, when applied to mixtures. The investigators have demonstrated that proton transfer reactions resulting from the interaction of oppositely-charged ions in a quadrupole ion trap is a robust means for greatly reducing the spectral congestion associated with mixtures of biopolymers. They present evidence that the combination of electrospray and ion/ion chemistry is highly promising as a core element in a strategy for high speed DNA sequencing. This strategy involves the generation of oligonucleotide mixtures via the Sanger method and direct analysis of these mixtures with electrospray combined with ion/ion chemistry. The output of this procedure is analogous to that produced via conventional electrophoresis-based sequencing technologies wherein the time-scale is replaced by a mass scale. Depending upon the nature of the as yet unknown limiting factors for this approach, the read length might reach as high as several thousand bases.