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The electronic form of this document may be cited in the following style:
Human Genome Program, U.S. Department of Energy, DOE Human Genome Program Contractor-Grantee Workshop IV, 1994.

Abstracts scanned from text submitted for November 1994 DOE Human Genome Program Contractor-Grantee Workshop. Inaccuracies have not been corrected.

Electrophore-Labeled DNA for Enhanced Sensitivity in Matrix-Assisted Laser Desorption Mass Spectrometry

Phillip F. Britt, Gregory B. Hurst, and Michelle V. Buchanan
Chemical and Analytical Sciences Division, Oak Ridge National Laboratory, Oak Ridge, TN 37831

In order to speed up sequencing and other DNA analyses that yield information encoded as a series of DNA fragments of different molecular weights, strategies based on matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) are being developed as alternatives to the lengthy gel electrophoresis separation and detection method that is currently used. One disadvantage of MALDI-MS, particularly for larger DNA fragments, is the relatively poor sensitivity that results from the low apparent efficiency for production of ions from the neutral DNA molecules desorbed by the laser [1,2]. We are therefore investigating methods to improve MALDI-MS sensitivity by increasing this ionization efficiency. Our first approach has been to derivatize DNA with an electrophore in order to exploit the presence of electrons in the MALDI plume. The electrophore-tagged DNA should efficiently capture these electrons, potentially resulting in increased ionization efficiency and enhanced sensitivity.

We have attached a series of electrophore tags to the 5' terminus of the M13-40 sequencing primer 5'-GTTTTCCCAGTCACGAC-3'. Electrophore groups tested to date include pentafluorophenyl-, p-nitrophenyl-, m-nitrophenyl-, and 9-fluorenone. Using a methodology similar to that used to attach tags for fluorescence detection of sequencing ladders on gels [3,4], the aminohexyl derivative of the primer is reacted with either the N-hydroxysuccinimide or the isothiocyanate derivative of the electrophore. MALDI-MS analysis of the products shows that the electrophore-labeled primer can be successfully synthesized, and that the label remains attached to the primer throughout the MALDI-MS measurement process. The next phase of the work will be to examine an expanded series of electrophore tags to identify a candidate that is less susceptible to processes leading to loss of signal, such as rapid autodetachment (loss of the captured electron) or dissociative attachment. In addition, modifications to the mass spectrometer that will provide more favorable conditions for electron capture by the labeled oligonucleotides are in the design phase.

Research sponsored by the National Institutes of Health, National Center for Human Genome Research, Grant No. 1 R55 HG/OD00819-01A1, under Interagency Agreement 1884-F026-A1 with the U.S. Department of Energy under Contract DE-AC05-840R21400 with Martin Marietta Energy Systems, Inc.

[1.] Nelson, R.; Rainbow, D.; Lohr, P.; Williams, P. Science 1989, 246, 1585.
[2.] Romano, L.; Levis, R. J. Am. Chem. Soc. 1991, 113, 9665.
[3.] Telser, J.; Cruickshank, K.A.; Morrison, L.E.; Netzel, R.L.; Chan, C.-K. J. Am. Chem. Soc. 1989, 111, 7226-7232.
[4.] Smith, L.M.; Fung, S.; Hunkapiller, M.W.; Hunkapiller, T.J.; Hood, L.E. Nucleic Acids Res. 1985, 13, 2399-2412.