DEVELOPMENT OF MALDI-TOF FOR DNA SEQUENCING TECHNOLOGIES

描述：（申请人的摘要）一个广泛的，长期的目标 申请人的研究计划之一是开发/利用先进的激光 用于生物分析的光谱和质谱技术。 该第一 该项目是专门设计的，以进一步发展矩阵辅助激光 解吸电离（MALDI）飞行时间（TOF）质谱法 用于低成本、高精度的DNA测序。 MALDI-TOF测序 方法是一种新兴的方法，可以提供大量的 提高了DNA测序的速度，降低了测序的成本。 由于人类基因组计划的资助， 用MALDI-TOF进行DNA测序。 但是大量的额外工作 以实现基因组测序的高通量和低成本。 在这一应用中遇到的两个基本问题一直很低 检测灵敏度和强金属盐效应。 过程中 在我们最近的研究中，我们发现使用Parafilm代替 金属作为MALDI样品基底可以显着提高 MALDI-TOF检测灵敏度高，减少金属盐影响。 因此，在第一个项目中，我们将开发这种新方法， MALDI-TOF测序DNA。 我们的具体目标是实现低fmol检测 以最小的样品纯化对大DNA进行测序的灵敏度。 到 为实现这一目标，我们将：（1）优化这种新型MALDI样品制备方法 通过使用3-羟基吡啶甲酸（3-HPA）作为DNA分析的方案 主要基质材料;（2）研究这种新的样品制备方法， 使用其他有效的矩阵系统来搜索DNA的最佳矩阵， 测序：（3）调查使用其他材料作为样品 基质，以寻找最佳的基质材料;（4）调查 使用前三部分中开发的优化样本协议 用于DNA混合物样品的MALDI-TOF分析的研究， 测序产物;（5）研究优化方案的使用 沿着分子束， 基于MALDI-TOF，用于测序数百个碱基的大DNA。

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.