Mass spectrometric methods for sequencing nucleic acids

• US 6,268,131 B1
• Filed: 12/15/1997
• Issued: 07/31/2001
• Est. Priority Date: 12/15/1997
• Status: Expired due to Term

• Current Assignee: Korea Advanced Institute of Science and Technology, Agena Bioscience Inc. (Mesa Laboratories Inc.)
• Original Assignee: Sequenom Incorporated (Laboratory Corporation Of America Holdings)
• Inventors: Kwon, Young-Soo, Lough, David M., Xiang, Guobing, Kang, Changwon, Little, Maryanne J., Köster, Hubert, Little, Daniel P., Kim, Young Tae, Cantor, Charles
• Primary Examiner(s): Brusca, John S.
• Assistant Examiner(s): Lundgren, Jeffrey S.
• Application Number:
  US08/990,851
• Time in Patent Office: 1,324 Days
• Field of Search: 435/6, 435/91.1, 435/91.2, 435/91.3, 435/518, 435/528, 436/518, 436/528, 536/23.1
• US Class Current: 435/5
• CPC Class Codes: B01J 2219/00317 Microwell devices, i.e. having large numbers of wells
• CPC Class Codes: B01J 2219/00387 Applications using probes
• CPC Class Codes: B01J 2219/00527 Sheets
• CPC Class Codes: B01J 2219/00585 Parallel processes
• CPC Class Codes: B01J 2219/00596 Solid-phase processes
• CPC Class Codes: B01J 2219/00605 the compounds being directly bound or immobilised to solid supports
• CPC Class Codes: B01J 2219/00612 the surface being inorganic
• CPC Class Codes: B01J 2219/00621 by physical means, e.g. trenches, raised areas
• CPC Class Codes: B01J 2219/00626 Covalent
• CPC Class Codes: B01J 2219/00637 by coating it with another layer
• CPC Class Codes: B01J 2219/00648 by the use of solid beads
• CPC Class Codes: B01J 2219/00659 Two-dimensional arrays
• CPC Class Codes: B01J 2219/00689 using computers
• CPC Class Codes: B01J 2219/00691 using robots
• CPC Class Codes: B01J 2219/00702 Processes involving means for analysing and characterising the products
• CPC Class Codes: B01J 2219/00722 Nucleotides
• CPC Class Codes: C12Q 1/6872 involving mass spectrometry
• CPC Class Codes: C12Q 2525/143 incorporating a promoter sequence
• CPC Class Codes: C40B 40/06 Libraries containing nucleotides or polynucleotides, or derivatives thereof
• CPC Class Codes: C40B 60/14 for creating libraries

Abstract

A mass spectrometric method for sequencing nucleic acids using RNA polymerases, including DNA-dependent and RNA-dependent RNA polymerases, is provided. The methods use a modified Sanger sequencing strategy in which RNA polymerase is used to generate a set of nested RNA transcripts obtained by base-specific chain termination. These are analyzed by mass spectrometry. A method of identifying transcriptional terminator sequences or attenuator sequences is also provided.

Claims

1. A method for determining the sequence of a target nucleic acid molecule, comprising:

a) immobilizing a nucleic acid promoter-containing probe on a solid support, wherein;

the nucleic acid promoter-containing probe comprises at least 5 nucleotides at the 3′

-end of the coding strand that is complementary to a single stranded region at the 3′

-end of the target nucleic acid, and a double-stranded portion that comprises the promoter, which is oriented to permit transcription of a hybridized target nucleic acid molecule;

b) hybridizing the target nucleic acid to the single-stranded portion of the immobilized nucleic acid probe;

c) transcribing the target nucleic acid with an RNA polymerase to produce a plurality of base-specifically terminated RNA transcripts, wherein the RNA polymerase recognizes the promoter;

d) determining the molecular weight value of each base-specifically terminated RNA transcript by mass spectrometry; and

e) determining the sequence of the nucleic acid by aligning the base-specifically terminated RNA transcripts according to molecular weight.

2. The method of claim 1, wherein at least two base-specifically terminated RNA transcripts are produced.

3. The method of claim 1, wherein the immobilized nucleic acid promoter-containing probe is produced by immobilizing a single-stranded molecule that comprises a promoter or the complement of a promoter and hybridizing a fragment that comprises the complement thereof, whereby the resulting double-stranded region comprises a promoter.

4. The method of claim 1, further comprising between steps b) and c):

adding a ligase to form a phosphodiester bond between the 3′

hydroxyl group and the 5′

phosphate group of adjacent strands of the nucleic acid probe and the target nucleic acid.

5. The method of claim 1, wherein the promoter is selected from the group consisting of archaebacteria, eubacteria, bacteriophages, DNA viruses, RNA viruses, plants, plant viruses and eukaryotic promoters.

6. The method of claim 1, wherein the RNA polymerase is a DNA-dependent RNA polymerase.

7. The method of claim 1, wherein the RNA polymerase is an RNA-dependent RNA polymerase.

8. The method of claim 1, wherein the RNA polymerase is selected from the group consisting of archaebacteria, eubacteria, bacteriophages, DNA viruses, RNA viruses, plants and eukaryotic RNA polymerases.

9. The method of claim 1, wherein the RNA polymerase is selected from the group consisting of Escherichia coli RNA polymerase, T7 RNA polymerase, SP6 RNA polymerase, T3 RNA polymerase and Qβ

replicase.

10. The method of claim 1, wherein prior to immobilization of the nucleic acid, the surface of the support is derivatized by reacting the surface with an aminosilane to produce primary amines on the surface of the support.

11. The method of claim 10, wherein the aminosilane is 3-amino-propyltriethoxysilane.

12. The method of claim 10, further comprising reacting the primary amines on the surface of the support with a thiol-reactive cross-linking reagent to form a thiol-reactive solid support.

13. The method of claim 12, wherein the thiol-reactive cross-linking reagent is N-succinimidyl (4-iodoacetyl) aminobenzoate (SIAB).

14. The method of claim 12, wherein the immobilization of the nucleic acid probe to a solid support is effected by reacting the thiol-reactive solid support with a nucleic acid probe having a free 5′

- or 3′

-thiol group, whereby a covalent bond between the thiol group and the thiol-reactive solid support is formed.

15. The method of claim 1, wherein the nucleic acid probe is covalently bound to a surface the solid support at a density of at least 20 fmol/mm².

16. The method of claim 1, wherein the nucleic acids are immobilized on the surface of the solid support in the form of an array.

17. The method of claim 1, wherein the solid support is silicon.

18. The method of claim 1, wherein the surface comprises a plurality of wells comprising the immobilized nucleic acid molecule.

19. The method of claim 18, wherein the wells have a rough interior surface.

20. The method of claim 18, wherein the solid support has a rough surface.

21. The method of claim 17, wherein the surface of the wells is etched.

22. The method of claim 1, wherein the mass spectrometry analysis is selected from the group consisting of Matrix Assisted Laser Desorption/lonization, Time-of-Flight (MALDI-TOF) analysis, Electronspray (ES), Ion Cyclotron Resonance (ICR) and Fourier transform.

23. The method of claim 1, wherein transcription is performed in the presence of one or more 3′

-deoxyribonucleotides.

24. The method of claim 1, further comprising:

adding a matrix material to the surface of the support, and

determining the molecular weight of the synthesized single-stranded ribonucleic acid using mass spectrometry analysis.

25. The method of claim 1, wherein the hybridization of the nucleic acid to be sequence to the solid support results in the formation of a nick in the coding strand corresponding to positions beyond +6 relative to the start of transcription from the promoter.

26. The method of claim 25, wherein the nick is at position +7, +8, +9or +19.

27. The method of claim 1, wherein transcription is performed in the presence of at least one modified ribonucleoside triphosphate analog, whereby the resulting RNA molecule has decreased secondary structure compared to an RNA molecule produced from unmodified ribonucleotide triphosphates.

31. The method of claim 1, wherein in step c), the transcription is carried out in the presence of a modified ribonucleotide, whereby RNA polymerase turnover rate is increased.

32. The method of claim 31, wherein the modified ribonucleotide is selected from the group consisting of 4-thio UTP, 5-bromo UTP and 5-iodo CTP.

28. A method of identifying transcriptional terminator sequences or attenuator sequences in a target nucleic acid molecule, comprising:

a) immobilizing a nucleic acid promoter-containing probe on a solid support, wherein the nucleic acid promoter-containing probe comprises at least 5 nucleotides at the 3′

-end of the coding strand that is complementary to a single stranded region at the 3′

-end of the target nucleic acid, and a double-stranded portion that comprises the promoter, which is oriented to permit transcription of a hybridized target nucleic acid molecule;

b) hybridizing the target nucleic acid molecule to the immobilized nucleic acid probe;

c) transcribing the target nucleic acid with an RNA polymerase to produce a sequence-terminated RNA transcript, wherein the RNA polymerase recognizes the promoter; and

d) determining the molecular weight value of the RNA transcript by mass spectrometry, wherein the observed mass of the RNA is indicative of the presence of a the terminator sequence or attenuator in the target nucleic acid molecule.

29. The method of claim 28 that is a method for identifying an attenuator, wherein the termination occurs prior to a coding sequence.

30. The method of claim 28 that is a method for identifying a terminator, wherein the termination occurs after or at the end of a coding sequence.

33. The method of claim 28, wherein in step c), the transcription is carried out in the presence of a modified ribonucleotide, whereby the RNA polymerase turnover rate is increased.

34. The method of claim 33, wherein the modified ribonucleotide is selected from the group consisting of 4-thio UTP, 5-bromo UTP and 5-iodo CTP.