DNA SEQUENCING BY ELECTROSPRAY AND ION/ION CHEMISTRY

描述（改编自研究者摘要）：目的 这项提议的一个重要目的是发展高速DNA测序的方法 它可以产生序列数据， 当前最先进的技术，具有可比或上级 精度具体来说，目标是提供完整的序列信息 至少500个碱基的DNA链上。 所提出的方法是基于电喷雾电离/四极离子 使用气相离子/离子化学的阱质谱法。 电喷雾已被证明能够产生气相离子， 延伸远远超过500个碱基的核酸寡聚体。然而，在这方面， 用于高速DNA测序的基于电喷雾的方法还没有 由于频谱拥塞， 电喷雾特有的多重充电现象。的 从单个低聚物形成多个电荷态， 限制了混合物的复杂性， 电喷雾出于这个原因，电喷雾通常遵循分离 方法，如液相色谱法或毛细管电泳，当 适用于混合物。研究人员已经证明质子 由于带相反电荷的 四极离子阱中的离子是一种用于极大地减少 与生物聚合物的混合物相关的光谱拥塞。他们 目前的证据表明，结合电喷雾和离子/离子 化学是非常有前途的一个战略的核心要素， 加速DNA测序。这一战略涉及生成 通过桑格法和直接分析寡核苷酸混合物， 这些混合物与电喷雾结合离子/离子化学。的 该过程的输出类似于通过常规方法产生的输出。 基于测序的测序技术，其中时间尺度是 被一个大规模取代。根据目前的情况 对于这种方法的未知限制因素，读取长度可能达到 高达几千个碱基。

DESCRIPTION (Adapted from the Investigator's Abstract): 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.