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DNA SEQUENCE LADDER READOUT BY MASS SPECTROMETRY

通过质谱读取 DNA 序列梯

基本信息

批准号：
6138896
负责人：
PETER WILLIAMS
金额：
$ 16.13万
依托单位：
ARIZONA STATE UNIVERSITY-TEMPE CAMPUS
依托单位国家：
美国
项目类别：
财政年份：
1999
资助国家：
美国
起止时间：
1999-01-01 至 2001-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/6138896
关键词：
biomedical equipment development genetic mapping mass spectrometry matrix assisted laser desorption ionization nucleic acid sequence

项目摘要

A new mass spectrometric approach is proposed for rapid readout of Sanger dideoxy DNA sequence ladders. Ions will be generated using the relatively new technique known as Massive Cluster Impact (MCI) desorption. MCI generates vapor-phase ions from solid or liquid (solution) targets by the hypervelocity impact of massive, highly-charged glycerol or water dusters produced in vacuum by an electrospray process. The hypervelocity cluster impact has been shown to shock-heat and desorb massive biomolecular ions with a low degree of internal excitation and consequently long lifetimes, allowing mass spectrometric detection with good mass resolution. Ions arc generated in vacuum, allowing efficient transport into the mass spectrometer and minimizing sample size requirements. The initial extent of multiple charging in the desorbed ions is relatively low. To accomplish these studies, a cluster ion source and sample introduction system will be interfaced to a time-of-flight mass spectrometer with pulsed orthogonal ion extraction and an ion reflector for high mass resolution. Methods to control the degree of charging will be investigated: these include varying the cluster charge and energy, and doping the target and/or the cluster feed liquid with proton donors or acceptors to perturb the competition of the DNA analyte for charged solution species or the excess protons carried into the surface by the impacting cluster. Facile charge control which minimizes multiple charging should allow optimized analyses of multicomponent DNA sequence ladder mixtures. To investigate the competing roles of cluster charge and ionization equilibria in determining analyte ion charge multiplicity, experiments will also be performed in which analyte-containing clusters are electrosprayed and desolvated by impact on clean solid and liquid targets. MCI desorption of DNA from affinity capture surfaces will be investigated, in order to facilitate sample cleanup and minimize sample size requirements.

提出了一种新的质谱快速读出方法桑格双脱氧DNA序列梯。会产生离子使用相对较新的大规模集群技术冲击（MCI）解吸。 MCI产生气相离子，固体或液体（溶液）目标的超高速撞击大量的，高电荷的甘油或水尘产生于通过电喷雾过程真空。超高速星团撞击已经显示出冲击加热和解吸大量生物分子离子具有较低程度的内部激励，寿命，允许质谱检测，分辨率离子在真空中产生，允许有效的传输并使样品尺寸要求最小化。的解吸离子中多重充电的初始程度相对低为了完成这些研究，引入系统将与飞行时间质量接口具有脉冲正交离子提取和离子高质量分辨率的反射器。方法控制将调查收费的程度：这些包括不同的簇电荷和能量，以及掺杂靶和/或具有质子供体或受体的团簇进料液体， DNA分析物对带电溶液物质的竞争或者说是撞击过程中携带到表面的多余质子集群易于充电控制，最大限度地减少多次充电应该允许多组分DNA序列优化分析阶梯混合物研究集群的竞争角色测定分析物离子的电荷和电离平衡电荷多重性，也将进行实验，其中含有分析物的团簇被电喷雾并通过对清洁固体和液体目标的影响。 DNA的MCI解吸从亲和捕获表面将进行研究，以便于样品清理并最大限度地减少样品量要求。