DNA sequencing by mass spectrometry

• US 6,225,450 B1
• Filed: 06/07/1995
• Issued: 05/01/2001
• Est. Priority Date: 01/07/1993
• Status: Expired due to Term

• Current Assignee: Agena Bioscience Inc. (Mesa Laboratories Inc.)
• Original Assignee: Sequenom Incorporated (Laboratory Corporation Of America Holdings)
• Inventors: Koster, Hubert
• Primary Examiner(s): Riley, Jezia
• Application Number:
  US08/481,033
• Time in Patent Office: 2,155 Days
• Field of Search: 435/6, 536/26.13, 536/26.6, 536/28.52, 536/28.54, 536/28.55, 536/54, 536/118, 536/122, 536/24.3, 536/24.33
• US Class Current: 536/22.1
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/6872 involving mass spectrometry
• CPC Class Codes: C12Q 2537/113 Heteroduplex formation
• CPC Class Codes: C12Q 2537/143 Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis
• CPC Class Codes: C12Q 2563/167 Mass label
• CPC Class Codes: C12Q 2565/627 being a mass spectrometer
• CPC Class Codes: G01N 35/1067 for transfer to or from containers having different spacing
• CPC Class Codes: Y10S 435/81 Packaged device or kit
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]
• CPC Class Codes: Y10T 436/24 Nuclear magnetic resonance, electron spin resonance or other spin effects or mass spectrometry
• CPC Class Codes: Y10T 436/25 including sample preparation

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 flirter 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 tag specific probes with mass differentiated molecular weights.

Claims

1. An intact ionized and volatilized mass-modified nucleic acid molecule, comprising at least two mass modified nucleotides, wherein the molecule is positively charged.

3. An intact ionized mass-modified nucleic acid molecule of claim 1, comprising at least one mass-modified nucleotide containing a mass-modifying functionality (M) attached to a heterocyclic base of the nucleotide.

5. An intact ionized mass-modified nucleic acid molecule of claim 1, comprising at least one mass-modified nucleotide containing a mass-modifying functionality (M) attached to at least one phosphorus of the nucleotide.

11. An ionized intact mass-modified nucleic acid molecule of claim 1, 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

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),—

COOH, —

(O(CH₂)_rCO—

)_m—

O—

(CH₂)_r—

COOH, —

Si(Y)₃, —

(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.

2. 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.

8. An ionized mass-modified nucleic acid molecule of claim 2, wherein a mass-modifying functionality (M) is attached to at least one sugar moiety of a 5′

-terminal nucleotide of the primer, and wherein the mass-modifying function (M) is a linking functionality (L).

4. 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.

6. 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.

7. 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.

9. 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.

10. 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.

12. A set of mass-differentiated tag probes wherein,

each tag probe in the set comprises a sequence of nucleotides which is complementary by Watson-Crick base pairing to a tag sequence present within at least one set of base-specifically terminated fragments;

the tag sequences to which each tag probe is complementary are different for each tag probe;

each tag probe in the set comprises at least one mass-modified nucleotide; and

the mass-modified nucleotides are not isotopically labeled and have different mass modifications in each tag probe.

13. The set of mass-differentiated tag probes of claim 12, wherein at least one of the mass-modified nucleotides comprises a mass-modifying functionality (M) attached to the heterocyclic base.

14. The set of mass-differentiated tag probes of claim 13, wherein the mass-modified heterocyclic base is selected from the group consisting of a cytosine moiety modified at C-5, a thymine moiety modified at C-5, a thymine moiety modified at the C-5 methyl group, a uracil moiety modified at C-5, an adenine moiety modified at C-8, a c⁷-deazaadenine moiety modified at C-8, a, 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.

15. The set of mass-differentiated tag probes of claim 12, wherein at least one of the mass-modified nucleotides comprises a mass-modifying functionality (M) attached to the phosphorus atom forming an internucleotidic linkage of the tag probe.

16. The set of mass-differentiated tag probes of claim 12, wherein at least one of the mass-modified nucleotides comprises a mass-modifying functionality (M) attached to the sugar moiety.

17. The set of mass-differentiated tag probes of claim 12, wherein at least one of the tag probes further comprises a cross-linking group (CL) which allows for covalent binding to the corresponding and complementary tag sequences.

18. The set of mass-differentiated tag probes of claim 17, wherein the cross-linking group (CL) is activated photochemically and is derived from at least one photoactivatable group selected from the group consisting of psoralen and an ellipticine.

19. The set of mass-differentiated tag probes of claim 12, wherein at least one of the tag probes 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 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, —

(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.

20. The set of mass-differentiated tag probes of claim 12, wherein one or more mass-modifying functionalities (M) incorporated into the probes are generated from one or more precursor functionalities (PF) attached to the mass-differentiated tag probes, 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.

38. The set of mass-differentiated tag probes of claim 12, wherein one or more mass-modifying functionalities incorporated into the probes are generated from a precursor functionality (PF) attached to the mass-differentiated tag probes, and wherein the precursor functionalities are 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), —

CONH₂, —

NH—

C(S)—

NH₂, OP(O-Alkyl)OH, and O—

CO—

CH₂—

SH.

39. The set of mass-differentiated tag probes of claim 12, wherein the tag probes are 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, N₃, 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.

40. The set of mass-differentiated tag probes of claim 12, wherein the tag probes are 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), —

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—

CH₂—

O—

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—

)_mO—

(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.

21. 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.

22. 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.

23. 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.

24. 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.

25. The ionized duplex of claim 24, wherein the mass-modified heterocyclic base is selected from the group consisting of a cytosine moiety modified at C-5, a thymine moiety modified at C-5, a thymine moiety modified at the C-5 methyl group, a uracil moiety modified at C-5, an adenine moiety modified at C-8, a c⁷-deazaadenine moiety modified at C-8, a, 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.

26. 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.

27. 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.

28. 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.

29. The ionized duplex of claim 28, wherein the cross-linking group (CL) is activated photochemically and is derived from at least one photoactivatable group selected from the group consisting of psoralen and an ellipticine.

30. 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.

31. 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.

32. 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), —

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—

CH₂—

O—

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.

33. 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.

34. 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.

35. 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.

36. 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.

37. 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.

41. 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.

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