Vaporization and sequencing of nucleic acids
First Claim
1. A method for determining, by mass spectrometry, the nucleotide sequence of a polynucleotide comprising the steps of:
- (a) generating from said polynucleotide four populations of nucleic acid molecules having from 65 to 1000 nucleotides, wherein the nucleic acid molecules of each population terminate in one of the nucleotides A, T, G or C;
(b) for each population of nucleic acid molecules forming a mixture of the nucleic acid molecules with a matrix that absorbs laser light in the visible range;
(c) vaporizing said mixture of matrix and nucleic acid molecules by illuminating with visible laser light, wherein said vaporizing occurs without fragmenting the nucleic acid molecules;
(d) ionizing the vaporized nucleic acid molecules;
(e) determining, by mass spectrometry, the molecular weight of the ionized nucleic acid molecules of each population; and
(f) comparing the molecular weights of the nucleic acid molecules generated from the polynucleotide to provide the nucleotide sequence of the polynucleotide.
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Abstract
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.
201 Citations
46 Claims
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1. A method for determining, by mass spectrometry, the nucleotide sequence of a polynucleotide comprising the steps of:
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(a) generating from said polynucleotide four populations of nucleic acid molecules having from 65 to 1000 nucleotides, wherein the nucleic acid molecules of each population terminate in one of the nucleotides A, T, G or C; (b) for each population of nucleic acid molecules forming a mixture of the nucleic acid molecules with a matrix that absorbs laser light in the visible range; (c) vaporizing said mixture of matrix and nucleic acid molecules by illuminating with visible laser light, wherein said vaporizing occurs without fragmenting the nucleic acid molecules; (d) ionizing the vaporized nucleic acid molecules; (e) determining, by mass spectrometry, the molecular weight of the ionized nucleic acid molecules of each population; and (f) comparing the molecular weights of the nucleic acid molecules generated from the polynucleotide to provide the nucleotide sequence of the polynucleotide. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46)
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Specification