DNA sequencing by mass spectrometry

An intact ionized mass-modified nucleic acid molecule, comprising:

• at least one mass modified nucleotide, wherein the molecule is positively charged, and comprises a member selected from the group consisting of:
• a mass-modified universal primer and a mass-modified initiator oligonucleotide.

An intact positively charged ionized mass-modified nucleic acid molecule, comprising:

• at least one mass-modified nucleotide containing a modified heterocyclic base selected from a group consisting of a cytosine moiety modified at C-5, a thymine moiety modified at C-5, a thymine moiety modified at the methyl group of C-5, a uracil moiety modified at C-5, an adenine moiety modified at C-8, a c7-deazaadenine moiety modified at C-8, a c7-deazaadenine moiety modified at C-7, a guanine moiety modified at C-8, a c7-deazaguanine moiety modified at C-8, a c7-deazaguanine moiety modified at C-7, a hypoxanthine moiety modified at C-8, a c7-deazahypoxanthine moiety modified at C-8, and a c7-deazahypoxanthine moiety modified at C-7.

An intact positively charged ionized mass-modified nucleic acid molecule, comprising:

• at least one mass-modified nucleotide containing a mass-modifying functionality (M) attached to at least one sugar moiety of the nucleotide.

An intact positively charged ionized mass-modified nucleic acid molecule, comprising:

• a mass-modifying functionality (M) attached to at least one sugar moiety of the nucleic acid molecule, wherein
• the sugar is modified at a position selected from the group consisting of an internal C-2′ position, an external C-2′ position, and an external C-5′ position.

An intact ionized mass-modified nucleic acid molecule, comprising at least one mass-modified nucleotide containing a mass-modifying functionality (M) incorporated into the molecule, wherein (M) is selected from the group consisting of F, Cl, Br, I, Si(CH3)3, Si(CH3)2(C2H5), Si(CH3)(C2H5)2, Si(C2H5)3, CH2F, CHF2, and CF3.

An intact positively charged ionized mass-modified nucleic acid molecule, comprising:

• at least two mass-modified nucleotides, wherein a mass-modifying functionality (M) incorporated into the at least one mass-modified nucleotide is generated from a precursor functionality (PF) attached to one or more of a nucleic acid primer, a chain-elongating nucleoside triphosphate or a chain-terminating nucleoside triphosphate, and wherein the precursor functionality (PF) is selected from the group consisting of —N3, —NH2, —SH, —NCS, —OCO(CH2)rCOOH (where r=1-20), —NHCO(CH2)rCOOH (where r=1-20), —OSO2OH, —OCO(CH2)rI (where r=1-20), and —OP(O-Alkyl)N(Alkyl)2.

An intact positively charged ionized mass-modified nucleic acid molecule, comprising:

• two or more mass modified nucleotides selected from the group consisting of a mass-modified 2′-deoxynucleotide, a mass-modified 2′,3′-dideoxynucleotide, a mass-modified nucleotide and a mass-modified 3′-deoxynucleotide, wherein the two or more mass-modified nucleotides are different from each other.

An ionized mass-modified nucleic acid molecule, comprising at least one mass modified nucleotide selected from the group consisting of a mass-modified 2′-deoxynucleotide, a mass-modified 2′,3′-dideoxynucleotide, a mass-modified nucleotide and a mass-modified 3′-deoxynucleotide, wherein the mass modified nucleic acid molecule comprises a modified heterocyclic base selected from a group consisting of modified heterocyclic base is a c7-deazaadenine moiety modified at C-8, a c7-deazaadenine moiety modified at C-7, a c7-deazaguanine moiety modified at C-8, a c7-deazaguanine moiety modified at C-7, a hypoxanthine moiety modified at C-8, a c7-deazahypoxanthine moiety modified at C-8, and a c7-deazahypoxanthine moiety modified at C-7.

An ionized positively charged intact duplex, comprising a mass-modified tag probe bound to a tag sequence present within a base-specifically terminated nucleic acid fragment, wherein the mass-modified tag probe comprises at least one mass-modified nucleotide.

An ionized intact duplex, comprising a mass-modified tag probe bound to a tag sequence present within a base-specifically terminated nucleic acid fragment, wherein the mass-modified tag probe comprises at least one mass-modified nucleotide, wherein at least one of the mass-modified nucleotides comprises a mass-modifying functionality (M) attached to the heterocyclic base.

An ionized intact duplex, comprising a mass-modified tag probe bound to a tag sequence present within a base-specifically terminated nucleic acid fragment, wherein:

• the mass-modified tag probe comprises at least one mass-modified nucleotide;
• and a mass-modifying functionality (M) incorporated into the tag probe is attached to the phosphorus atom forming an internucleotidic linkage of the tag probe.

An ionized intact duplex, comprising a mass-modified tag probe bound to a tag sequence present within a base-specifically terminated nucleic acid fragment, wherein:

• the mass-modified tag probe comprises at least one mass-modified nucleotide;
• and at least one of the mass-modified nucleotides comprises a mass-modifying functionality (M) attached to the sugar moiety.

An ionized intact duplex, comprising a mass-modified tag probe bound to a tag sequence present within a base-specifically terminated nucleic acid fragment, wherein:

• the mass-modified tag probe comprises at least one mass-modified nucleotide;
• and the tag probe further comprises a cross-linking group (CL) which allows for covalent binding to the tag sequence.

An ionized intact duplex, comprising a mass-modified tag probe bound to a tag sequence present within a base-specifically terminated nucleic acid fragment, wherein:

• the mass-modified tag probe comprises at least one mass-modified nucleotide;
• and the tag probe is mass-modified with a mass-modifying functionality (M) selected from the group consisting of XR, F, Cl, Br, I, Si(CH3)3, Si(CH3)2(C2H5), Si(CH3)(C2H5)2, Si(C2H5)3, CH2F, CHF2, and CF3) wherein X is selected from the group consisting of —O—, —NH—, —S—, —NHC(S)—, —OCO(CH2)rCOO— (where r=1-20), —NHCO(CH2)rCOO— (where r=1-20), —OSO2O— and —OP(O-Alkyl)O— and R is selected from the group consisting of H, methyl, ethyl, propyl, isopropyl, t-butyl, hexyl, benzyl, benzhydryl, halogen, trityl, substituted trityl, aryl, substituted aryl, polyoxymethylene, monoalkylated polyoxymethylene, a polyethylene imine, —(NH(CH2)rNHCO(CH2)rCO—)m—NH—(CH2)r—NH—CO—(CH2)r—COOH, —(NH(CH2)rCO—)m—NH—(CH2)r—COOH, —O(CH2)rCO—)m—O—(CH2)r—COOH, —Si(Y)3, —(NHCHaaCO)m—HCHaaCOOH, —(CH2CH2O)m—CH2CH2OH, and —(CH2CH2O)m—CH2CH2O—Y, where m is in the range of 0 to 200, Y is a lower alkyl group selected from a group consisting of methyl, ethyl, propyl, isopropyl, t-butyl and hexyl, r is in the range of 1 to 20, and aa represents the amino acid side chain of a naturally occurring amino acid.

An ionized intact duplex, comprising a mass-modified tag probe bound to a tag sequence present within a base-specifically terminated nucleic acid fragment, wherein:

• the mass-modified tag probe comprises at least one mass-modified nucleotide;
• and the tag probe is mass-modified with a mass-modifying functionality (M) selected from the group consisting of XR, F, Cl, Br, I, Si(CH3)3, Si(CH3)2(C2H5), Si(CH3)(C2H5)2, Si(C2H5)3, CH2F, CHF2, and CF3, wherein X is selected from the group consisting of —O—, —NH—, —S—, —NHC(S)—, —OCO(CH2)rCOO— (where r=1-20), —NHCO(CH2)rCOO— (where r=1-20), —OSO2O— and —OP(O-Alkyl)O— and R is selected from the group consisting of H, methyl, ethyl, propyl, isopropyl, t-butyl, hexyl, benzyl, benzhydryl, halogen, trityl, substituted trityl, aryl, substituted aryl, (—NH(CH2)rNHCO(CH2)rCO—)m—NH—(CH2)r—NH—CO—(CH2)r—COOH, (—NHCH2)rCO—)m—NH—(CH2)r—COOH, (—O(CH2)rCO—)m—O—(CH2)r—COOH, —Si(Y)3, —(NHCHaaCO—)m—NHCHaaCOOH, —(CH2CH2O)m—CH2CH2OH, and —(CH2CH2O)m—CH2CH2O—Y, where m is in the range of 0 to 200, Y is a lower alkyl group selected from a group consisting of methyl, ethyl, propyl, isopropyl, t-butyl and hexyl, r is in the range of 1 to 20, and aa represents the amino acid side chain of a naturally occurring amino acid.

An ionized intact duplex, comprising a mass-modified tag probe bound to a tag sequence present within a base-specifically terminated nucleic acid fragment, wherein:

• the mass-modified tag probe comprises at least one mass-modified nucleotide;
• and one or more mass-modifying functionalities (M) incorporated into the tag probe are generated from one or more precursor functionalities (PF) attached to the tag probe, and wherein the precursor functionalities (PF) are selected from a group consisting of —N3, —NH2, —SH, —NCS, —OCO(CH2)rCOOH (where r=1-20), —NHCO(CH2)rCOOH (where r=1-20), —OSO2OH, —OCO(CH2)rI (where r=1-20), and —OP(O-Alkyl)N(Alkyl)2.

An ionized intact duplex, comprising a mass-modified tag probe bound to a tag sequence present within a base-specifically terminated nucleic acid fragment, wherein:

• the mass-modified tag probe comprises at least one mass-modified nucleotide;
• and one or more mass-modifying functionalities (M) incorporated into the tag probe are generated from one or more precursor functionalities (PF) attached to the tag probe, and wherein the precursor functionalities (PF) are selected from a group consisting of —N3, —NH2, —SH, —NCS, —OCO(CH2)rCOOH (where r=1-20), —NHCO(CH2)rCOOH (where r=1-20), —OSO2OH, —OCO(CH2)rI (where r=1-20), —CONH2, —NH—C(S)—NH2, OP(O-Alkyl)OH, and O—CO—CH2—SH.

An ionized intact mass-modified nucleic acid molecule, comprising at least one mass modified nucleotide, wherein a mass-modifying functionality (M) incorporated into the molecule is generated from a precursor functionality (PF) attached to one or more of a nucleic acid primer, a chain-elongating nucleoside triphosphate or a chain-terminating nucleoside triphosphate, and wherein the precursor functionality (PF) is selected from a group consisting of —N3, —NH2, —SH, —NCS, —OCO(CH2)rCOOH (where r=1-20), —NHCO(CH2)rCOOH (where r=1-20), —OSO2OH, —OCO(CH2)rI (where r=1-20), —CONH2, —NH—C(S)—NH2, OP(O-Alkyl)OH, and O—CO—CH2—SH.

An ionized intact mass-modified nucleic acid molecule, comprising at least two mass modified nucleotides, wherein a mass-modifying functionality (M) incorporated into the molecule is XR, wherein X is selected from the group consisting of —O—, —NH—, —S—, —NHC(S)—, —OCO(CH2)rCOO— (where r=1-20), —NHCO(CH2)rCOO— (where r=1-20), —OSO2O— and —OP(O-Alkyl)O— and R is selected from the group consisting of H, methyl, ethyl, propyl, isopropyl, t-butyl, hexyl, benzyl, benzhydryl, halogen, trityl, substituted trityl, aryl, substituted aryl, (—NH(CH2)rNHCO(CH2)rCO—)m—NH—(CH2)r—NH—CO—(CH2)r—COOH, (—NH(CH2)rCO—)m—NH—(CH2)r—COOH, (—O(CH2)rCO—)m—O—(CH2)r—COOH, —Si(Y)3, —(NHCHaaCO—)m—NHCHaaCOOH, —(CH2CH2O)m—CH2CH2OH, and —(CH2CH2O)m—CH2CH2O—Y, where m is in the range of 0 to 200, Y is a lower alkyl group selected from a group consisting of methyl, ethyl, propyl, isopropyl, t-butyl and hexyl, r is in the range of 1 to 20, and aa represents the amino acid side chain of a naturally occurring amino acid.

An ionized intact mass-modified nucleic acid molecule, comprising at least two mass modified nucleotides, wherein a mass-modifying functionality (M) incorporated into the molecule is XR, wherein X is selected from the group consisting of —O—, —NH—, —S—, —NHC(S)—, OCO(CH2)rCOO— (where r=1-20), —NHC(O), —CONH—, —NH—C(S)—NH—, —NHCO(CH2)rCOO— (where r==1-20), —OSO2O—, —O—CO—CH2—S— and —OP(O-Alkyl)O— and R is selected from the group consisting of H, N3, methyl, ethyl, propyl, isopropyl, t-butyl, hexyl, benzyl, benzhydryl, halogen, trityl, substituted trityl, aryl, substituted aryl, (CH2)m—CH2—OH, (CH2)m—CH2O—Y, (CH2CH2NH)m—CH2—CH2—NH2, —(NH(CH2)rNHCO(CH2)rCO—)m—NH—(CH2)r—NH—CO—(CH2)r—COOH, —(NH(CH2)rCO—)m—NH—(CH2)r—COOH, —(NH—CHY—CO)m—NH—CHY—COOH, (—O(CH2)rCO—)m—O—(CH2)r—COOH, —Si(Y)3, —(NHCHaaCO—)m—NHCHaaCOOH, CH2F, CHF2, CF3, —(CH2CH2O)m—CH2CH2OH, and —(CH2CH2O)m—CH2CH2O—Y, where m is in the range of 0 to 200, Y is a lower alkyl group selected from a group consisting of methyl, ethyl, propyl, isopropyl, t-butyl and hexyl, r is in the range of 1 to 20, and aa represents the amino acid side chain of a naturally occurring amino acid.

An intact positively charged ionized and volatilized mass-modified nucleic acid molecule, comprising at least one mass-modified nucleotide selected from the group consisting of a mass-modified 2′-deoxynucleotide, a mass-modified 2′,3′-dideoxynucleotide and a mass-modified 3′-deoxynucleotide.

An intact ionized and volatilized mass-modified nucleic acid molecule, comprising at least two mass modified nucleotides.