Wayne State University

Robert J Levis

Louis J Romano

Method for analyzing a nucleic acid molecule, without fragmenting the molecule, by vaporizing a mixture of the molecule and a matrix by illuminating the mixture with visible laser light absorbed by the matrix and not by the nucleic acid molecule. The method is useful for determining the nucleotide sequence of a polynucleotide by using mass spectrometry to determine the molecular weights of individual single-stranded nucleic acid molecules in a population including a plurality of single-stranded nucleic acid molecules generated from the polynucleotide, each molecule having a different molecular weight, and one defined terminus and one variable terminus which terminates at a specific nucleotide.

Vaporization and sequencing of nucleic acids

Described is a method and apparatus for analyzing an organic sample. In the preferred embodiment, this method and apparatus allows the determination of the base sequence of a nucleic acid by determining the molecular weights of the components of a biological sample. The method uses either a pre-existing chromophore or the covalent attachment of an ionizable chromophore to a biological sample followed by the vaporization of these molecules by exposure to an intense pulse of electromagnetic radiation in the presence of a matrix which strongly absorbs the radiation. The gaseous molecules are subsequently extracted into an evacuated ionization chamber and then exposed to electromagnetic radiation at a wavelength which specifically excites the chromophore covalently attached to the biological sample. The molecular weights of these ionized species are then determined by mass spectroscopic analysis. This method of molecular weight determination allows for a DNA sequencing method. Four samples of DNA molecules are prepared such that each is covalently linked to an ionizable chromophore and each is fragmented by a means which results in fragments within each of the four samples that terminate at a different one of the nucleotides A, C, G or T. Each of these four fragmented samples is subsequently introduced into the said apparatus in order to determine their molecular weights hence providing the strand length of the fragments. The data generated may be analyzed by high-speed computer, the four data sets correlated, and the sequence deduced.

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Grosse Pointe Park, MI, United States of America

Robert J Levis