DEVELOPMENT OF MALDI-TOF FOR DNA SEQUENCING TECHNOLOGIES

描述：(申请人摘要)一个广泛的、长期的目标 申请人的研究计划之一是开发/利用先进激光 用于生物分析的光谱和质谱学技术。这是第一次 为进一步发展矩阵辅助激光器而专门设计的项目 解吸电离飞行时间质谱法 用于低成本、高精度的DNA测序。MALDI-TOF测序 方法是新兴的方法之一，可以提供实质性的 提高DNA测序速度，降低测序成本。 由于人类基因组计划的资助，已经取得了巨大的进展 使用MALDI-TOF进行DNA测序。但大量的额外工作 是实现基因组测序的高通量和低成本所必需的。 在这一应用中遇到的两个基本问题很少 检测灵敏度和较强的金属盐效应。在课程期间 在我们最近的研究中，我们发现使用Parafilm来取代 金属作为MALDI样品衬底可以显著提高 MALDI-TOF检测灵敏度高，降低了金属盐的影响。 因此，在第一个项目中，我们将为 MALDI-TOF DNA测序。我们的具体目标是实现低fmol值的检测 大样本DNA测序的灵敏度和最小的样品纯化。至 为了实现这一目标，我们将：(1)优化这种新的MALDI样品制备 以3-羟基烟酸(3-HPA)为模板的DNA分析方法 主要基质材料；(2)研究这种新的样品制备方法 利用其他有效的基质系统寻找DNA的最佳基质 测序：(3)调查其他材料作为样本的使用情况 以寻找最佳的衬底材料；(4)考察 使用前三部分中开发的优化样本方案 DNA混合物样品的MALDI-TOF分析研究 测序产物；(5)考察优化后的方案的使用情况 在前四个部分的研究中发展起来的，以及分子束 基于MALDI-TOF，用于对数百个碱基的大DNA进行测序。

DESCRIPTION: (Applicant's abstract) One of the broad, long term objective of the applicant's research program is to develop/utilize advanced laser spectroscopic and mass spectrometric techniques for bioanalysis. This FIRST project is specially designed to further develop the Matrix Assisted Laser Desorption ionization (MALDI) Time-Of-Flight (TOF) mass spectrometric method for low-cost, high accuracy DNA sequencing. The MALDI-TOF sequencing approach is one of the emerging methods that could offer substantial increases in the rate of DNA sequencing and reduce the cost of sequencing. As a result of the Human Genome Project funding, great advances have been made in using MALDI-TOF for DNA sequencing. But substantial additional work is required to achieve high throughput and low-cost for genomic sequencing. Two of the essential problems encountered in this application have been low detection sensitivity and the strong metal salt effect. During the course of our recent study, we discovered that the use of Parafilm to replace metals as the MALDI sample substrates can significantly improve the MALDI-TOF detection sensitivity and reduce the metal salt effect. Therefore, in this FIRST project, we will develop this new approach for MALDI-TOF sequencing DNA. Our specific aim is to attain low fmol detection sensitivity in sequencing large DNA with minimal sample purification. To achieve this goal, we will: (1) optimize this new MALDI sample preparation protocol for DNA analysis by using 3-hydroxypicolinic (3-HPA) acid as the main matrix material; (2) investigate this new sample preparation method by using other effective matrix systems to search for the best matrix for DNA sequencing: (3) investigate the use of other materials as the sample substrates to search for the best substrate materials; (4) investigate the use of the optimized sample protocol developed in the first three-part investigations for the MALDI-TOF analysis of DNA mixture samples and sequencing products; (5) investigate the use of the optimized protocol developed in the first four-part investigations, along with molecular beam based MALDI-TOF, for sequencing large DNA of several hundred bases.