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 i s 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.
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