DNA sequencing by mass spectrometry
First Claim
1. A kit for sequencing one or more species of nucleic acids by multiplex mass spectrometric nucleic acid sequencing, comprising:
- a) a solid support having a linking functionality (L'"'"');
b) a set of nucleic acid primers suitable for initiating synthesis of a set of nucleic acids which are complementary to the different species of nucleic acids, the primers each including a linking group (L) able to interact with the linking functionality (L'"'"') and reversibly link the primers to the solid support and optionally, a tag probe;
c) a set of chain-elongating nucleotides for synthesizing the complementary nucleic acids;
d) a set of chain-terminating nucleotides for terminating synthesis of the complementary nucleic acids and generating sets of base-specific terminated complementary nucleic acid fragments; and
e) a polymerase for synthesizing the complementary nucleic acids from the nucleic acid primers, chain-elongating nucleotides and terminating nucleotides,wherein in the absence of a tag probe, at least one reagent selected from the group consisting of the primers, the chain-elongating nucleotides, and the chain-terminating nucleotides is mass modified to provide distinction between each set of base-specifically terminated nucleotides of each species of nucleic acid by mass spectrometry.
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Accused Products
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.
364 Citations
26 Claims
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1. A kit for sequencing one or more species of nucleic acids by multiplex mass spectrometric nucleic acid sequencing, comprising:
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a) a solid support having a linking functionality (L'"'"'); b) a set of nucleic acid primers suitable for initiating synthesis of a set of nucleic acids which are complementary to the different species of nucleic acids, the primers each including a linking group (L) able to interact with the linking functionality (L'"'"') and reversibly link the primers to the solid support and optionally, a tag probe; c) a set of chain-elongating nucleotides for synthesizing the complementary nucleic acids; d) a set of chain-terminating nucleotides for terminating synthesis of the complementary nucleic acids and generating sets of base-specific terminated complementary nucleic acid fragments; and e) a polymerase for synthesizing the complementary nucleic acids from the nucleic acid primers, chain-elongating nucleotides and terminating nucleotides, wherein in the absence of a tag probe, at least one reagent selected from the group consisting of the primers, the chain-elongating nucleotides, and the chain-terminating nucleotides is mass modified to provide distinction between each set of base-specifically terminated nucleotides of each species of nucleic acid by mass spectrometry. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14)
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15. A kit for sequencing nucleic acids by mass spectrometry, comprising:
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a) a solid support having a linking functionality (L'"'"'); b) a set of nucleic acid primers suitable for initiating synthesis of a set of complementary nucleic acids which are complementary to the different species of nucleic acids, the primers each including a linking group (L) able to interact with the linking functionality (L'"'"') and reversibly immobilize the primers on the solid support; c) a set of chain-elongating nucleotides for synthesizing the complementary nucleic acids; d) a set of chain-terminating nucleotides for terminating synthesis of the complementary nucleic acids and generating sets of base-specific terminated complementary nucleic acid fragments; and e) a polymerase for synthesizing the complementary nucleic acids from the primers, chain-elongating nucleotides and chain-terminating nucleotides, wherein the chain-terminating nucleotides are mass-modified so that addition of one species of the chain-terminating nucleotides to the complementary nucleic acid can be distinguished by mass spectrometry from addition of all other species of chain-terminating nucleotides concurrently analyzed. - View Dependent Claims (16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26)
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Specification