DNA sequences by mass spectrometry

• US 6,238,871 B1
• Filed: 05/08/2000
• Issued: 05/29/2001
• 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): Jones, W. Gary
• Assistant Examiner(s): LU, FRANK WEI MIN
• Application Number:
  US09/566,591
• Time in Patent Office: 386 Days
• Field of Search: 435/6, 435/91.1, 435/91.2, 435/183, 435/283.1, 435/287.1, 435/287.2, 436/94, 536/23.1, 536/24.3, 536/24.33, 702/19, 702/20
• US Class Current: 435/5
• CPC Class Codes: C12P 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides
• 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 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: 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

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.

Claims

1. A method for determining the sequence of a nucleic acid comprising the steps of:

a) generating at least two base-specifically terminated fragments from a nucleic acid to be sequenced that comprises nucleotides with a modification at a base, a sugar or a phosphate of a nucleotide, wherein the modification improves the separation or resolution of the fragments when analyzed compared to unmodified fragments;

b) desorbing and ionizing the base-specifically terminated fragments with a single laser source;

c) simultaneously determining the molecular weight value of each of the desorbed and ionized base-specifically terminated fragments by mass spectrometry, and

d) determining the sequence of the nucleic acid by aligning the base-specifically terminated fragments according to molecular weight.

2. The method of claim 1, wherein the modification comprises alteration of mass.

3. The method of claim 1, wherein the modification comprises treating the nucleic acid to be sequenced with cation-exchange.

4. The method of claim 1, wherein the modification comprises removing the negative charge from the phosphodiester backbone.

5. The method of claim 1, wherein the nucleic acid fragments are generated using the following:

an initiator oligonucleotide;

a combination of nucleoside triphosphates selected from the group consisting of adenosine triphosphate ATP, uridine triphosphate UTP, guanosine triphosphate GTP, cytidine triphosphate CTP, inosine triphosphate ITP, a 7-deazanucleoside triphosphates c⁷ATP, a 7-deazanucleoside triphosphates c⁷GTP, and a 7-deazanucleoside triphosphates c⁷ITP;

one or a combination of chain terminating 3′

-deoxynucleoside triphosphates selected from the group consisting of deoxyadenosine triphosphate 3′

-dATP, deoxyuridine triphosphate 3′

-dUTP, deoxyguanosine triphosphate 3′

-dGTP, and deoxycytidine triphosphate 3′

-dCTP; and

an RNA polymerase.

6. The method of claim 1, wherein the base-specifically terminated fragments are purified before a step of determining the molecular weight value by mass spectrometry.

7. The method of claim 6, wherein the base-specifically terminated fragments are purified by the steps comprising:

a) immobilizing the nucleic acid fragments on a solid support;

b) washing out reactants and by-products; and

c) removing the nucleic acid fragments from the solid support.

8. The method of claim 1, wherein the molecular weight value of each fragment is determined by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS).

9. The method of claim 1, wherein the molecular weight value of each fragment is determined by electrospray mass spectrometry (ES-MS).

10. The method of claim 1, wherein the base-specifically terminated nucleic acid fragments are generated using the following:

a nucleotide primer;

a combination of deoxynucleoside triphosphates selected from the group consisting of deoxyadenosine triphosphate dATP, deoxythymidine triphosphate dTTP, deoxyguanosine triphosphate dGTP, deoxycytidine triphosphate dCTP, deoxyinosine triphosphate dITP, a 7-deazadeoxy-nucleoside triphosphates c7dGTP, a 7-deazadeoxynucleoside triphosphates c7dATP, and a 7-deazadeoxynucleoside triphosphates c7dITP;

one or a combination of chain terminating dideoxynucleoside triphosphates selected from the group consisting of dideoxyadenosine triphosphate ddATP, dideoxythymidine triphosphate ddTTP, dideoxyguanosine triphosphate ddGTP, and dideoxycytidine triphosphate ddCTP; and

a DNA polymerase.

11. The method of claim 10, wherein the primer further includes a linking group (L) for reversibly binding the primer to a solid support.

12. The method of claim 11, wherein the base-specifically terminated fragments are coupled by the linking group (L) to a functionality (L′

) on the support creating a cleavable attachment of the complementary nucleic acid to the support.

13. The method of claim 12, wherein the support-bound base-specifically terminated fragments are thoroughly washed to remove all remaining reactants and by-products from the sequencing reaction, and the base-specifically terminated fragments are subsequently cleaved from the solid support.

14. The method of claim 13, wherein the molecular weight value of each of base-specifically terminated fragments is determined by a mass spectrometric technique selected from matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) or electrospray mass spectrometry (ES-MS).

15. The method of claim 5, wherein the initiator oligonucleotide further comprises a linking group (L) for reversibly binding the initiator oligonucleotide to a solid support.

16. The method of claim 15, wherein the base-specifically terminated fragments are coupled by the linking group (L) to a functionality (L′

) on the support creating a cleavable attachment of the complementary nucleic acid to the support.

17. The method of claim 16, wherein the support-bound base-specifically terminated fragments are thoroughly washed to remove all remaining reactants and by-products from the sequencing reaction, and the base-specifically terminated fragments are subsequently cleaved from the support.

18. The method of claim 17, wherein the molecular weight value of each base-specifically terminated fragment is determined by a mass spectrometric technique selected from matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) or electrospray mass spectrometry (ES-MS).

19. A method of determining the sequence of a nucleic acid comprising the steps of:

a) generating four sets of base-specifically terminated nucleic acid fragments, wherein;

the nucleic acid fragments comprise nucleotides with a modification at a base, a sugar or a phosphate of a nucleotide; and

the modification improves the separation or resolution of the fragments when analyzed compared to unmodified fragments;

b) simultaneously determining the molecular weight value of the four sets of the base-specifically terminated fragments by mass spectrometry, and

c) determining the sequence of the nucleic acid by aligning the base-specifically terminated fragments according to molecular weight.

20. The method of claim 19, wherein the base-specifically terminated fragments are purified before a step of determining the molecular weight values by mass spectrometry.

21. The method of claim 20, wherein the base-specifically terminated fragments are purified by the steps comprising

a) immobilizing the nucleic acid fragments on a solid support;

b) washing out reactants and by-products; and

c) removing the nucleic acid fragments from the solid support.

22. The method of claim 20, wherein the molecular weight value of each fragment is determined by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS).

23. The method of claim 20, wherein the molecular weight value of each fragment is determined by electrospray mass spectrometry (ES-MS).

24. The method of claim 20, wherein the base-specifically terminated nucleic acid fragments are generated using the following:

a nucleotide primer;

a combination of deoxynucleoside triphosphates selected from the group consisting of deoxyadenosine triphosphate dATP, deoxythymidine triphosphate dTTP, deoxyguanosine triphosphate dGTP, deoxycytidine triphosphate dCTP, deoxyinosine triphosphate dITP, a 7-deazadeoxynucleoside triphosphates c7dGTP, a 7-deazadeoxynucleoside triphosphates c7dATP, and a 7-deazadeoxynucleoside triphosphates c7dITP;

one or a combination of chain terminating dideoxynucleoside triphosphates selected from the group consisting of dideoxyadenosine triphosphate ddATP, dideoxythymidine triphosphate ddTTP, dideoxyguanosine triphosphate ddGTP, and dideoxycytidine triphosphate ddCTP; and

a DNA polymerase.

25. The method of claim 24, wherein the primer further includes a linking group (L) for reversibly binding the primer to a solid support.

26. The method of claim 25, wherein the base-specifically terminated fragments are coupled by the linking group (L) to a functionality (L′

) on the support creating a cleavable attachment of the complementary nucleic acid to the support.

27. The method of claim 26, wherein the support-bound base-specifically terminated fragments are thoroughly washed to remove all remaining reactants and by-products from the sequencing reaction, and the base-specifically terminated fragments are subsequently cleaved from the solid support.

28. The method of claim 27, wherein the molecular weight value of each of base-specifically terminated fragments is determined by a mass spectrometric technique selected from matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) or electrospray mass spectrometry (ES-MS).

29. The method of claim 19, wherein the nucleic acid fragments are generated using the following:

an initiator oligonucleotide;

a combination of nucleoside triphosphates selected from the group consisting of adenosine triphosphate ATP, uridine triphosphate UTP, guanosine triphosphate GTP, cytidine triphosphate CTP, inosine triphosphate ITP, a 7-deazanucleoside triphosphates c⁷ATP, a 7-deazanucleoside triphosphates c⁷GTP, and a 7-deazanucleoside triphosphates c⁷ITP;

one or a combination of chain terminating 3′

-deoxynucleoside triphosphates selected from the group consisting of deoxyadenosine triphosphate 3′

-dATP, deoxyuridine triphosphate 3′

-dUTP, deoxyguanosine triphosphate 3′

-dGTP, and deoxycytidine triphosphate 3′

-dCTP; and

an RNA polymerase.

30. The method of claim 29, wherein the initiator oligonucleotide further comprises a linking group (L) for reversibly binding the initiator oligonucleotide to a solid support.

31. The method of claim 30, wherein the base-specifically terminated fragments are coupled by the linking group (L) to a functionality (L′

) on the support creating a cleavable attachment of the complementary nucleic acid to the support.

32. The method of claim 31, wherein the support-bound base-specifically terminated fragments are thoroughly washed to remove all remaining reactants and by-products from the sequencing reaction, and the base-specifically terminated fragments are subsequently cleaved from the support.

33. The method of claim 32, wherein the molecular weight value of each base-specifically terminated fragment is determined by a mass spectrometric technique selected from matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) or electrospray mass spectrometry (ES-MS).