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.

Agency
National Institute of Health (NIH)
Institute
National Human Genome Research Institute (NHGRI)
Type
First Independent Research Support & Transition (FIRST) Awards (R29)
Project #
1R29HG001815-01
Application #
2677536
Study Section
Special Emphasis Panel (ZHG1-HGR-P (M1))
Program Officer
Graham, Bettie
Project Start
1998-07-01
Project End
2003-06-30
Budget Start
1998-07-01
Budget End
1999-06-30
Support Year
1
Fiscal Year
1998
Total Cost
Indirect Cost
Name
Cleveland State University
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
City
Cleveland
State
OH
Country
United States
Zip Code
44115
Wang, Liming; Zhang, Jian; Banerjee, Sipra et al. (2010) Sumoylation of vimentin354 is associated with PIAS3 inhibition of glioma cell migration. Oncotarget 1:620-7
Sun, Xiyuan; Liu, Yiding; Lutterbaugh, Jim et al. (2006) Detection of mononucleotide repeat sequence alterations in a large background of normal DNA for screening high-frequency microsatellite instability cancers. Clin Cancer Res 12:454-9
Liu, Yiding; Sun, Xiyuan; Guo, Baochuan (2003) Matrix-assisted laser desorption/ionization time-of-flight analysis of low-concentration oligonucleotides and mini-sequencing products. Rapid Commun Mass Spectrom 17:2354-60
Sun, Xiyuan; Hung, K; Wu, L et al. (2002) Detection of tumor mutations in the presence of excess amounts of normal DNA. Nat Biotechnol 20:186-9
Sun, X; Ding, H; Hung, K et al. (2000) A new MALDI-TOF based mini-sequencing assay for genotyping of SNPS. Nucleic Acids Res 28:E68
Guo, B; Wang, S; Fan, Y (2000) Improving the performance of MALDI-TOF in oligonucleotide analysis using a new SDIFA technology. Anal Chem 72:5792-7
Hung, K C; Ding, H; Guo, B (1999) Use of poly(tetrafluoroethylene)s as a sample support for the MALDI-TOF analysis of DNA and proteins. Anal Chem 71:518-21