DNA SEQUENCING BY ELECTROSPRAY AND ION/ION CHEMISTRY

通过电喷雾和离子/离子化学进行 DNA 测序

• 批准号: 2761362
• 负责人: SCOTT A MCLUCKEY
• 金额: $ 18.18万
• 依托单位: LOCKHEED MARTIN ENERGY RESEARCH CORP
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-02-01 至 2001-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2761362
• 关键词: DNA; chemical transfer reaction; electrospray ionization mass spectrometry; ion transport; nucleic acid sequence; technology /technique development

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.

描述(改编自《调查者摘要》)：目标 这项提议的目的是开发高速DNA测序的方法学 这可以产生序列数据，其速度大约比 当前最先进的技术，具有可比或卓越的 精确度。具体来说，目标是提供完整的序列信息 在DNA站上，至少有500个碱基，在几秒钟到几分钟内。 提出的方法是基于电喷雾电离/四极离子。 采用气相离子/离子化学的陷阱质谱仪。 已证明电喷雾能够产生气相离子 延伸超过500个碱基的核酸低聚物。然而， 基于电喷雾的高速DNA测序方法还没有 由于与以下技术相关的频谱拥塞 多次充电现象是电喷雾的特征。这个 单一齐聚物严重地形成多个电荷态 限制了适用于直接分析的混合物的复杂性 电喷雾。出于这个原因，电喷雾通常在分离之后进行 在下列情况下，可采用高效液相色谱或毛细管电泳法 用于混合物的。研究人员已经证明了质子 相反电荷相互作用引起的转移反应 四极离子陷阱中的离子是一种强有力的方法，可以极大地减少 与生物聚合物混合物相关的光谱拥堵。他们 目前的证据表明，电喷雾和离子/离子的组合 化学作为高技术战略的核心元素前景看好 快速DNA测序。这一战略涉及到产生 通过桑格法和直接分析的寡核苷酸混合物 这些混合物结合了电喷雾和离子/离子化学。这个 此程序的输出类似于通过传统方法产生的输出 基于电泳法的测序技术，其中时间尺度是 被质量秤取代。取决于目前为止的性质 此方法的未知限制因素，读取长度可能达到 高达几千个垒。