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 base-specifically terminated nucleic acid fragments containing deazapurine nucleotides;
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
65 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 base-specifically terminated nucleic acid fragments containing deazapurine nucleotides;
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, 42, 51, 52, 60, 61, 62, 63)
a) reversibly immobilizing the nucleic acid fragments on a solid support; and
b) washing out all remaining reactants and by-products.
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4. The method according to claim 3, further comprising the step of removing the nucleic acid fragments from the solid support.
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5. The method of claim 1, wherein the deazapurine moieties are selected from the group consisting of:
- C7-deazaguanine, C7-deazaadenine, C7-deazainosine triphosphate, C9-deazaadenine, C9-deazaguanine and C9-deazainosine triphosphate.
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6. The method of claim 1, wherein the deazapurine nucleotides comprise at least about 80% of the nucleotide fragment.
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7. A process of claim 1 wherein the mass spectrometer is selected from the group consisting of:
- Matrix-Assisted Laser Desorption/Ionization Time-of-Flight (MALDI-TOF), Electrospray (ES), Ion Cyclotron Resonance (ICR), and Fourier Transform and combinations thereof.
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8. The method according to claim 1, wherein more than one species of nucleic acid are concurrently sequenced by multiplex mass spectrometric nucleic acid sequencing employing nucleic acid primers, chain-elongating nucleotides, and chain-terminating nucleotides, wherein one of the sets of base-specifically terminated fragments is unmodified and the other sets of base-specifically terminated nucleic acid fragments are mass modified, and each of the sets of base-specifically terminated nucleic acid fragments has a sufficient mass difference to be distinguished from the others by mass spectrometry.
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9. The method according to claim 8, wherein at least one of the sets of mass-modified base-specifically terminated fragments is modified with a mass-modifying functionality at a heterocyclic base of at least one nucleotide.
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10. The method according to claim 9, wherein the heterocyclic base-modified nucleotide is selected from the group consisting of a cytosine nucleotide modified at C-5, a thymine nucleotide modified at C-5, a thymine nucleotide modified at the C-5 methyl group, a uracil nucleotide modified at C-5, an adenine nucleotide modified at C-8, an adenine nucleotide modified at C-7, a c7-deazaadenine modified at C-8, a c7-deazaadenine modified at C-7, a guanine nucleotide modified at C-8, a guanine nucleotide modified at C-7, a c7-deazaguanine modified at C-8, a c7-deazaguanine modified at C-7, a hypoxanthine modified at C-8, a c7-deazahypoxanthine modified at C-7, and a c7-deazahypoxanthine modified at C-8.
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11. The method according to claim 8, wherein at least one of the sets of mass-modified base-specifically terminated nucleic acid fragments is modified with a mass-modifying functionality attached to one or more phosphate moieties of the internucleotidic linkages of the fragments.
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12. The method according to claim 8, wherein at least one of the sets of mass-modified base-specifically terminated nucleic acid fragments is modified with a mass-modifying functionality attached to one or more sugar moieties of nucleotides within the set of mass modified base-specifically terminated fragments at at least one sugar position selected from the group consisting of a C-2′
- position, an external C-3′
position, and an external C-5′
position.
- position, an external C-3′
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13. The method according to claim 8, wherein at least one of the sets of mass-modified base-specifically terminated nucleic acid fragments is modified with a mass-modifying functionality (M) attached to the sugar moiety of a 5′
- -terminal nucleotide and wherein the mass-modifying function (M) is the linking functionality (L).
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14. The method according to claim 8, wherein a mass-modifying functionality (M) is attached to a set of base-specifically terminated nucleic acid fragments subsequent to generating the base-specifically terminated nucleic acid fragments and prior to determining the molecular weight values for the nested fragments by mass spectrometry.
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15. The method according to claim 14, wherein the base-specifically terminated nucleic acid fragments are generated using at least one reagent selected from the group consisting of a nucleic acid primer, a chain-elongating nucleotide, a chain-terminating nucleotide and a tag probe which has been modified with a precursor of the mass-modifying functionality, M;
- and a subsequent step comprises modifying the precursor of the mass-modifying functionality to generate the mass-modifying functionality, M, prior to mass spectrometric analysis.
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42. The method of claim 1, wherein the deazapurine is C7-deazaguanine.
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51. The method of claim 5, wherein the deazapurine is selected from the group consisting of C9-deazaadenine, C9-deazaguanine and C9-deazainosine.
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52. The method of claim 5, wherein the deazapurine is selected from the group consisting of C7-deazaguanine, C7-deazaadenine and C7-deazainosine.
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60. The method of claim 1, wherein four sets of base-specifically terminated nucleic acid fragments from a nucleic acid to be sequenced are generated.
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61. The method of claim 1, wherein the base-specifically terminated fragments are linked to a solid support.
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62. The method of claim 1, wherein prior to determining the molecular weight of the base-specifically terminated fragments by mass spectrometry the fragments are exposed to a single laser source.
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63. The method of claim 1, wherein the deazapurine is C7-deazainosine.
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16. A method of sequencing a target nucleic acid, comprising the steps of:
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a) reversibly linking oligonucleotide primers to a solid support;
b) hybridizing to the primers, at least a portion of the target nucleic acid and generating via chain elongation of the primer and subsequent termination, at least two base-specifically terminated nucleic acid fragments containing deazapurine nucleotides;
c) determining the molecular weight value of each based-specifically terminated fragment by mass spectrometry, wherein;
the molecular weight values of at least two base-specifically terminated fragments are determined concurrently; and
the nested fragments are cleaved from the solid support during mass spectrometry; and
d) determining the nucleotide sequence by aligning the base specifically terminated fragments according to molecular weight. - View Dependent Claims (17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 53, 54, 64, 65)
a) reversibly immobilizing the nucleic acid fragments on a solid support; and
b) washing out all remaining reactants and by-products.
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19. The method according to claim 18, further comprising the step of removing the nucleic acid fragments from the solid support.
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20. The method of claim 16, wherein the deazapurine moieties are selected from the group consisting of:
- C7-deazaguanine, C7-deazaadenine, C7-deazainosine triphosphate, C9-deazaadenine, C9-deazaguanine and C9-deazainosine triphosphate.
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21. The method of claim 16, wherein deazapurine nucleotides comprise at least about 80% of the nucleotide fragment.
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22. A process of claim 16 wherein the mass spectrometer is selected from the group consisting of:
- Matrix-Assisted Laser Desorption/Ionization Time-of-Flight (MALDI-TOF), Electrospray (ES), Ion Cyclotron Resonance (ICR), and Fourier Transform and combinations thereof.
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23. The method according to claim 16, wherein more than one species of nucleic acid are concurrently sequenced by multiplex mass spectrometric nucleic acid sequencing employing nucleic acid primers, chain-elongating nucleotides, and chain-terminating nucleotides, wherein one of the sets of base-specifically terminated fragments is unmodified and the other sets of base-specifically terminated nucleic acid fragments are mass modified, and each of the sets of base-specifically terminated nucleic acid fragments has a sufficient mass difference to be distinguished from the others by mass spectrometry.
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24. The method according to claim 23, wherein at least one of the sets of mass-modified base-specifically terminated fragments is modified with a mass-modifying functionality (M) at a heterocyclic base of at least one nucleotide.
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25. The method according to claim 24, wherein the heterocyclic base-modified nucleotide is selected from the group consisting of a cytosine nucleotide modified at C-5, a thymine nucleotide modified at C-5, a thymine nucleotide modified at the C-5 methyl group, a uracil nucleotide modified at C-5, an adenine nucleotide modified at C-8, an adenine nucleotide modified at C-7, a c7-deazaadenine modified at C-8, a c7-deazaadenine modified at C-7, a guanine nucleotide modified at C-8, a guanine nucleotide modified at C-7, a c7-deazaguanine modified at C-8, a c7-deazaguanine modified at C-7, a hypoxanthine modified at C-8, a c7-deazahypoxanthine modified at C-7, and a c7-deazahypoxanthine modified at C-8.
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26. The method according to claim 23, wherein at least one of the sets of mass-modified base-specifically terminated nucleic acid fragments is modified with a mass-modifying functionality (M) attached to one or more phosphate moieties of the internucleotidic linkages of the fragments.
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27. The method according to claim 23, wherein at least one of the sets of mass-modified base-specifically terminated nucleic acid fragments is modified with a mass-modifying functionality (M) attached to one or more sugar moieties of nucleotides within the set of mass modified base-specifically terminated fragments at at least one sugar position selected from the group consisting of a C-2′
- position, an external C-3′
position, and an external C-5′
position.
- position, an external C-3′
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28. The method according to claim 23, wherein at least one of the sets of mass-modified base-specifically terminated nucleic acid fragments is modified with a mass-modifying functionality (M) attached to the sugar moiety of a 5′
- -terminal nucleotide and wherein the mass-modifying function (M) is the linking functionality (L).
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29. The method according to claim 23, wherein a mass-modifying functionality (M) is attached to a set of base-specifically terminated nucleic acid fragments subsequent to generating the base-specifically terminated nucleic acid fragments and prior to determining the molecular weight values for the nested fragments by mass spectrometry.
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30. The method according to claim 29, wherein the base-specifically terminated nucleic acid fragments are generated using at least one reagent selected from the group consisting of a nucleic acid primer, a chain-elongating nucleotide, a chain-terminating nucleotide and a tag probe which has been modified with a precursor of the mass-modifying functionality, M;
- and a subsequent step comprises modifying the precursor of the mass-modifying functionality, M, to generate the mass-modifying functionality, M, prior to mass spectrometric analysis.
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53. The method of claim 20, wherein the deazapurine is selected from the group consisting of C9-deazaadenine, C9-deazaguanine and C9-deazainosine.
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54. The method of claim 20, wherein the deazapurine is selected from the group consisting of C7-deazaguanine, C7-deazaadenine and C7-deazainosine.
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64. The method of claim 20, wherein the deazapurine is C7-deazaguanine.
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65. The method of claim 20, wherein the deazapurine is C7-deazainosine.
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31. 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) hybridizing to the primers, at least a portion of the target nucleic acid and generating via chain elongation of the primer with chain-elongating nucleoside triphosphates and subsequent termination with chain-terminating nucleoside triphosphates, at least two, base-specifically terminated nucleic acid fragments containing deazapurine nucleotides;
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 during mass spectrometry; and
d) determining the nucleotide sequence of the nucleic acid by aligning the fragments according to molecular weight. - View Dependent Claims (32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 55, 56, 57)
one or more of the nucleotides in the primer, chain-terminating nucleoside triphosphates and/or chain-elongating nucleoside triphosphates is mass modified.
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43. A method for determining the sequence of a nucleic acid comprising the steps of:
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a) generating at least two base-specifically terminated nucleic acid fragments from a nucleic acid to be sequenced, wherein;
the fragments are generated under conditions which comprise cation exchange; and
the conditions permit sequencing of oligomers that are 50-mers;
b) determining the molecular weight value of each of the base-specifically terminated fragments 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 (48, 50, 58)
a plurality of nucleic acid fragments are concurrently sequenced;
one of the sets of base-specifically terminated fragments is unmodified and the other sets of base-specifically terminated nucleic acid fragments are mass modified; and
each of the sets of base-specifically terminated nucleic acid fragments has a sufficient mass difference from the others to be distinguished from the others by mass spectrometry.
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58. The method of claim 43, wherein four sets of base-specifically terminated nucleic acid fragments from a nucleic acid to be sequenced are generated.
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44. A method for determining the sequence of a nucleic acid comprising the steps of:
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a) generating at least two base-specifically terminated nucleic acid fragments from a nucleic acid to be sequenced, wherein;
the fragments are generated under conditions wherein the fragments contain deazapurines; and
the conditions result in enhanced separation or resolution of the fragments when analyzed by mass spectrometry compared to the absence of the conditions;
b) determining the molecular weight value of each of the base-specifically terminated fragments 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 (45, 47, 49, 59)
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46. A method for determining the sequence of a nucleic acid, comprising the steps of:
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a) generating at least two base-specifically terminated nucleic acid fragments wherein;
at least one of the nucleic acid fragments comprises two different mass modifications b) determining the molecular weight value of each base-specifically terminated fragment by mass spectrometry; and
c) determining the sequence of the nucleic acid by aligning the one or more sets of base-specifically terminated nucleic acid fragments according to molecular weight.
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Specification