DNA sequencing by mass spectrometry via exonuclease degradation

• US 5,851,765 A
• Filed: 05/30/1995
• Issued: 12/22/1998
• Est. Priority Date: 03/19/1993
• 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): Horlick, Kenneth R.
• Assistant Examiner(s): Tung, Joyce
• Application Number:
  US08/454,527
• Time in Patent Office: 1,302 Days
• Field of Search: 250/281, 250/282, 435/6, 435/91.2, 536/22.1, 536/24.3, 935/77
• US Class Current: 435/5
• CPC Class Codes: C07F 9/2408 of hydroxyalkyl compounds
• CPC Class Codes: C07F 9/2429 of arylalkanols
• CPC Class Codes: C07H 19/06 Pyrimidine radicals
• CPC Class Codes: C07H 19/10 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• CPC Class Codes: C07H 19/16 Purine radicals
• CPC Class Codes: C07H 19/20 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• CPC Class Codes: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: C12Q 1/6872 involving mass spectrometry
• CPC Class Codes: C12Q 2521/319 Exonuclease
• CPC Class Codes: C12Q 2521/325 Single stranded exonuclease
• CPC Class Codes: C12Q 2521/543 Immobilised enzyme(s)
• CPC Class Codes: C12Q 2525/117 incorporating modified base
• 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 2537/157 A reaction step characterised by the number of molecules incorporated or released
• CPC Class Codes: C12Q 2565/631 being a biochannel or pore
• CPC Class Codes: G01N 35/1067 for transfer to or from containers having different spacing

Abstract

Methods for determining the sequence of nucleic acids by cleaving the nucleic acid unilaterally from a first end with an exonuclease activity to sequentially release individual nucleotides, identifying each of the sequentially release nucleotides by mass spectrometry, and determining the sequence of the nucleic acid from the identified nucleotides are disclosed. The method is amenable to multiplexing for simulataneously determining more than one nuleic acid sequence.

Claims

1. A multiplex mass spectrometric method for simultaneously determining the sequence of at least two different nucleic acids concurrently, comprising the steps of:

(i) obtaining multiple copies of the at least two different nucleic acids, wherein at least one of the at least two different nucleic acids comprises mass modified nucleotides;

(ii) concurrently cleaving the at least two different nucleic acids unilaterally from a first end to a second end with an exonuclease to generate sequentially released individual nucleotides and optionally, at least two different sets of nucleic acid fragments;

(iii) identifying each of the sequentially released nucleotides by mass spectrometry or determining the molecular weight values of each of the sets of nucleic acid fragments by mass spectrometry; and

(iv) determining the sequence of the at least two different nucleic acids based on the molecular weight values of the sequentially released individual nucleotides or the sets of nucleic acid fragments.

2. The method according to claim 1, wherein the mass-modified nucleotides further serve to modulate activity of the exonuclease.

3. The method according to claim 1, wherein the mass-modified nucleotides comprise a mass-modifying functionality (M) attached to the sequentially released nucleotides.

4. The method according to claim 1, wherein at least one of the mass-modified nucleotides is modified with a mass modifying functionality (M) attached to a 5'"'"' phosphate.

5. The method according to claim 1, wherein at least one of the mass-modified nucleotides is modified with a mass modifying functionality (M) attached to a C-2'"'"' position sugar moiety.

6. The method according to claim 1, wherein at least one of the mass-modified nucleotides is modified with a mass modifying functionality (M) attached to a heterocyclic base.

7. The method according to claim 6, wherein the mass modified nucleotides comprise a modified heterocyclic base selected from the group consisting of a cytosine moiety modified at C-5, an uracil moiety modified at C-5, a thymine moiety modified at the C-5 methyl group, 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.

8. The method according to claim 1, wherein the at least one mass-modified nucleic acid is prepared from a single-stranded template polynucleotide, which is complementary to the nucleic acid sequence by synthesizing the nucleic acid using mass-modified nucleotides.

9. The method according to claim 8, wherein the mass-modified nucleic acid is synthesized using a primer having a sequence which allows the nucleic acid to be anchored to the solid support.

10. The method according to claim 8, wherein the mass-modified nucleic acid is synthesized using a DNA polymerase and mass-modified deoxyribonucleoside triphosphates (dNTPs).

11. The method according to claim 8, wherein the mass-modified nucleic acid is synthesized using an RNA polymerase and mass modified ribonucleoside triphosphates (NTPs).

12. The method according to claim 8, wherein synthesis of the nucleic acid is initiated in the presence of an initiator oligonucleotide which can be reversibly immobilized on a solid support.

13. The method according to claim 1, wherein mass-modified nucleotides are introduced at phosphodiester bonds.

14. The method of claim 13, wherein the mass modified nucleotides are alpha-thio nucleoside triphosphates.

15. A kit for exonuclease sequencing at least two different species of nucleic acids by mass spectrometry, comprising

(i) an exonuclease for cleaving the nucleic acids unilaterally from a first end to sequentially release individual nucleotides;

(ii) a set of nucleotides for synthesizing the different species of nucleic acids, at least a portion of the nucleotides being mass-modified such that sequentially released nucleotides of each of the different species of nucleic acids are distinguishable,

(iii) a polymerase for synthesizing the nucleic acids from complementary templates and the set of nucleotides, and

(iv) a solid support for immobilizing one of the nucleic acids or the exonuclease.

16. The kit according to claim 15, wherein the nucleic acids are 2'"'"'deoxyribonucleic acids (DNA).

17. The kit according to claim 15, wherein the nucleic acids are ribonucleic acids (RNA).

18. The kit according to claim 15, wherein the exonuclease is selected from the group consisting of snake venom phosphodiesterase, spleen phosphodiesterase, Bal-31 nuclease, E. coli exonuclease I, E. coli exonuclease III, E. coli exonuclease VII, Mung Bean Nuclease, S1 Nuclease, an exonuclease activity of E. coli DNA polymerase I, an exonuclease activity of a Klenow fragment of DNA polymerase I, an exonuclease activity of T4 DNA polymerase, an exonuclease activity of T7 DNA polymerase, an exonuclease activity of Taq DNA polymerase, an exonuclease activity of DEEP VENT DNA polymerase, and an exonuclease activity of VENT, DNA polymerase.

19. The kit according to claim 15, wherein the solid support is selected from a group consisting of a capillary, a flat membrane, glass beads, cellulose beads, polystyrene beads, Sephadex beads, Sepharose beads, polyacrylamide beads and agarose beads.

20. The kit according to claim 15, wherein the solid support is functionalized to facilitate covalent attachment of the nucleic acids.

21. The kit according to claim 15, wherein the exonuclease is immobilized by covalent attachment to the solid support.

22. The kit according to claim 15, wherein the mass-modified nucleotide comprises a mass-modifying functionality (M) attached to a nucleotide moiety.

24. The kit according to claim 15, further comprising an instruction manual providing the exonuclease sequencing protocol.

25. The kit according to claim 15, wherein the mass modified nucleotides are alpha-thio nucleoside triphosphates.

26. The kit according to claim 15, wherein at least one of the mass-modified nucleotides is modified with a mass modifying functionality (M) attached to a C-2'"'"' position of a sugar moiety.

27. The kit according to claim 15, wherein at least one of the mass-modified nucleotides is modified with a mass modifying functionality (M) attached to a heterocyclic base.

28. The kit according to claim 27, wherein the mass-modified nucleotide comprises a modified heterocyclic base selected from the group consisting of a cytosine moiety modified at C-5, an uracil moiety modified at C-5, a thymine moiety modified at the C-5 methyl group, 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.

23. The kit according to claim 23, wherein the mass-modifying functionality (M) is attached to a nucleotide moiety at a position selected from a group consisting of a 5'"'"' phosphate, a C-2'"'"' position of a sugar moiety, and a heterocyclic base.