The objective of this proposal is to develop an innovative technique to increase both the detection efficiency and mass resolution of large DNA ions when matrix-assisted laser desorption/ionization (MALDI) is used for DNA sequencing. MALDI based time-of-flight mass spectrometry (TOFMS) has been proven to be capable of fast sequencing of small DNAs using an ion detector. The time needed for the sequencing is less than 1 ms. However, the sensitivity of ion detection critically depends on the speed of the ions. High-speed ions strike the detector's surface and eject secondary electrons. The electrons are multiplied and the ion signal is amplified. The detection efficiency drops rapidly with the increase of molecular mass since the speed of large DNA is lower than that of the small DNA. Due to the low detection efficiency for the large DNA, a high laser pumping intensity for ablation is used. The high pumping intensity creates complicated collision and energy transfer processes in MALDI and results in a poor mass resolution in sequencing large DNA. The investigators propose to improve the ion detection efficiency by using UV laser-induced photodissociation/fragmentation at the end of the drift tune of a TOFMS. The large parent DNA ions interact with and are fragmented into small ions by a UV laser. The small fragments of DNA ions are then mass-selected and accelerated by pulsed high voltage (HV) electric fields to a speed higher than the critical speed needed for efficient ejection of secondary electron from the surface of an ion detector. With special care being taken, the detection sensitivity of large DNA sequencing is expected to be improved by several orders of magnitude. The improved sensitivity would make possible the application of MALDI-based TOFMS for routine sequencing of very large DNA (m/z-1,000,000-Dalton or higher) and allow the use of lower laser intensity for ablation, which consequently results in a better mass resolution.

Agency
National Institute of Health (NIH)
Institute
National Human Genome Research Institute (NHGRI)
Type
Exploratory/Developmental Grants (R21)
Project #
1R21HG001785-01
Application #
2605475
Study Section
Genome Study Section (GNM)
Program Officer
Graham, Bettie
Project Start
1998-05-01
Project End
2000-04-30
Budget Start
1998-05-01
Budget End
1999-04-30
Support Year
1
Fiscal Year
1998
Total Cost
Indirect Cost
Name
Georgia Southern University
Department
Physics
Type
Schools of Arts and Sciences
DUNS #
063828383
City
Statesboro
State
GA
Country
United States
Zip Code
30460
Zhang, Jing-Yuan; Kong, Yufei; Xu, Zuyan et al. (2002) Optical parametric properties of ultraviolet-pumped cesium lithium borate crystals. Appl Opt 41:475-82
Kong, Y; Zhu, Y; Zhang, J Y (2001) Ionization mechanism of oligonucleotides in matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Rapid Commun Mass Spectrom 15:57-64