DNA sequencing by mass spectrometry
First Claim
1. A method for determining the sequence of a nucleic acid, comprising the steps of:
- a) generating at least two conditioned, base-specifically terminated nucleic acid fragments from a nucleic acid to be sequenced;
b) determining the molecular weight value of each base-specifically terminated fragment by mass spectrometry, wherein the molecular weight values of at least two base-specifically terminated fragments are determined concurrently; and
c) determining the sequence of the nucleic acid by aligning the base-specifically terminated nucleic acid fragments according to molecular weight.
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Abstract
The invention describes a new method to sequence DNA. The improvements over the existing DNA sequencing technologies are high speed, high throughput, no electrophoresis and gel reading artifacts due to the complete absence of an electrophoretic step, and no costly reagents involving various substitutions with stable isotopes. The invention utilizes the Sanger sequencing strategy and assembles the sequence information by analysis of the nested fragments obtained by base-specific chain termination via their different molecular masses using mass spectrometry, as for example, MALDI or ES mass spectrometry. A further increase in throughput can be obtained by introducing mass-modifications in the oligonucleotide primer, chain-terminating nucleoside triphosphates and/or in the chain-elongating nucleoside triphosphates, as well as using integrated tag sequences which allow multiplexing by hybridization of tag specific probes with mass differentiated molecular weights.
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Citations
42 Claims
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1. A method for determining the sequence of a nucleic acid, comprising the steps of:
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a) generating at least two conditioned, base-specifically terminated nucleic acid fragments from a nucleic acid to be sequenced; b) determining the molecular weight value of each base-specifically terminated fragment by mass spectrometry, wherein the molecular weight values of at least two base-specifically terminated fragments are determined concurrently; and c) determining the sequence of the nucleic acid by aligning the base-specifically terminated nucleic acid fragments according to molecular weight. - 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)
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31. A method of sequencing a nucleic acid, comprising the steps of:
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a) reversibly linking an oligonucleotide primer to a solid support; b) generating at least two conditioned, base-specifically terminated nucleic acid fragments; c) determining the molecular weight value of each nested fragment in each of the four sets of base-specifically terminated fragments by matrix assisted laser desorption/ionization mass spectrometry wherein the molecular weight values of at least two base-specifically terminated fragments are determined concurrently and wherein the nested fragments are cleaved from the solid support by a laser during mass spectrometry; and d) determining the nucleotide sequence by aligning the base specifically terminated fragments according to molecular weight. - View Dependent Claims (32, 33, 34, 35, 36, 37, 38)
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39. A method of multiplex analysis of nucleic acid sequences, comprising the steps of:
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a) reversibly linking a nucleic acid primer to a solid support; b) generating at least two conditioned, base-specifically terminated nucleic acid fragments; c) determining the molecular weight value of each fragment by matrix assisted laser desorption/ionization mass spectrometry wherein the molecular weight values of at least two base-specifically terminated fragments are determined concurrently and wherein the fragments are cleaved from the solid support by a laser during mass spectrometry; and d) determining the nucleotide sequence by aligning the fragments according to molecular weight;
wherein at least one reagent selected from a group consisting of, a nucleic acid primer, a chain-elongating nucleotide, and a chain-terminating nucleotide which has been mass-modified;
wherein each set of base-specifically terminated fragments has a sufficient mass difference from the other sets of base-specifically terminated fragments so as to be unique; and
wherein the molecular weight values of the nested fragments of two or more sets of unseparated base-specifically terminated fragments are determined concurrently. - View Dependent Claims (40, 41, 42)
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Specification