Method of nucleic acid analysis

• US 6,268,129 B1
• Filed: 11/24/1997
• Issued: 07/31/2001
• Est. Priority Date: 03/03/1995
• Status: Expired due to Fees

• Current Assignee: Cancer Research Technology Limited (Cancer Research UK)
• Original Assignee: Imperial Cancer Research Technology, Ltd. (Cancer Research UK)
• Inventors: Beck, Stephan A., Gut, Ivo G.
• Primary Examiner(s): Houtteman, Scott W.
• Application Number:
  US08/894,836
• Time in Patent Office: 1,345 Days
• Field of Search: 435/6, 435/91.2, 536/24.3, 536/26.6, 536/25.3
• US Class Current: 435/5
• 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 1/6858 Allele-specific amplification
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 1/6872 involving mass spectrometry
• CPC Class Codes: C12Q 2525/113 incorporating modified backbone
• CPC Class Codes: C12Q 2525/117 incorporating modified base
• CPC Class Codes: C12Q 2525/186 incorporating a non-extendable or blocking moiety
• CPC Class Codes: C12Q 2533/101 Primer extension
• CPC Class Codes: C12Q 2563/167 Mass label
• CPC Class Codes: C12Q 2600/156 Polymorphic or mutational markers

Abstract

A method of analysing a nucleic acid by mass spectrometry comprising the steps of: (1) preparing a nucleic acid molecule comprising a negatively charged non-phosphate sugar-sugar linkage; (2) eliminating the charge from all, or up to all but ten, of the sugar-sugar linkages of the said nucleic acid molecule; (3) introducing the said nucleic acid molecule in which the charge has been wholly or partly eliminated as said into a mass spectrometer; and (4) determining the mass of the said nucleic acid molecule. Preferably, the nucleic acid has no or one charge. A method of preparing a nucleic acid molecule containing no or up to ten negative charges and no or up to ten positive charges comprising the steps of (1) synthesizing a nucleic acid with a phosphorothioate linkage or a phosphoroselenoate linkage between sugar residues, and (2) reacting the said nucleic acid with an alkylating agent so as to eliminate the charge on the said phosphorothioate linkage or said phosphoroselenoate linkage. The methods are useful for DNA sequencing and mutation analysis, and the nucleic acids are useful to suppress gene expression.

Claims

1. A method of analyzing a nucleic acid by mass spectrometry comprising the steps of

(1) providing a positively charged or negatively charged nucleic acid molecule containing one or up to ten negative charges and no or up to ten positive charges, or no negative charges and one or up to ten positive charges, wherein the nucleic acid molecule has a sugar-linkage-sugar backbone and said sugars have a base attached thereto at the 1 position;

and there are fewer negative charges than there are sugar-sugar linkages;

(2) introducing the said nucleic acid molecule into a mass spectrometer; and

(3) determining the mass of the said nucleic acid molecule.

4. A method according to claim 1 wherein the nucleic acid has one charge.

5. A method according to claim 1 wherein the nucleic acid DNA or an analouge or derivative thereof wherein the sugar residue comprises a 2′

-deoxyribose.

6. A method according to claim 1 wherein the said mass spectrometry is matrix-assisted laser desorption ionization time-of-flight mass spectroscopy (MALDI MS).

7. A method according to claim 1 wherein when the nucleic acid is uncharged or positively charged the mass is determined by detection in positive ion mode.

8. A method according to claim 1 wherein when the nucleic acid is negatively charged the mass is determined by detection in negative ion mode.

9. A method according to claim 1 wherein a plurality of nucleic acid molecules with differing molecular mass are introduced into the mass spectrometer and the mass of at least one of the said molecules is determined.

10. A method according to claim 1 wherein an enzymatic chain extension step is used in the preparation of the nucleic acid.

11. A method according to claim 9 wherein a polymerase chain reaction is used in the preparation of the plurality of nucleic acid molecules.

12. A method according to claim 9 wherein a nucleic acid chain terminating reagent is used in the preparation of the plurality of nucleic acids.

13. A method according to claim 12 further comprising the step of determining a nucleotide sequence or detecting a mutation by comparing the mass differences of the said plurality of nucleic acid molecules.

44. A method according to claim 1, wherein said nucleic acid molecule comprises a non-phosphate sugar-sugar linkage.

2. A method of analyzing a nucleic acid by mass spectrometry wherein the nucleic acid molecule has a sugar-linkage-sugar backbone and said sugars have a base attached thereto at the 1 position;

said method comprising the steps of

(1) preparing a nucleic acid molecule comprising a negatively charged non-phosphate sugar-sugar linkage;

(2) eliminating charge from the sugar-sugar linkages of the said nucleic acid molecule by covalent modification of the negatively charged non-phosphate sugar-sugar linkage so that the resulting nucleic acid molecule is positively charged or negatively charged and said molecule contains one or up to ten negative charges, being fewer charges than there are sugar-sugar linkages;

(3) introducing the said nucleic acid molecule in which the charge has been partly eliminated as said into a mass spectrometer; and

(4) determining the mass of the said nucleic acid molecule.

3. A method according to claim 2 wherein the said nucleic acid molecule has one phosphate sugar-sugar linkage.

14. A method of preparing a nucleic acid molecule containing no or up to ten negative charges or a nucleic acid molecule containing no or up to ten positive charges wherein the nucleic acid molecule has a sugar-linkage-sugar backbone and said sugars have a base attached thereto at the 1 position;

said method comprising the steps of

(1) synthesizing a nucleic acid with a phosphorothioate linkage or a phosphoroselenoate linkage between sugar residues, and

(2) reacting the said nucleic acid with an alkylating agent so as to eliminate the charge on the said phosphorothioate linkage or said phosphoroselenoate linkage wherein step (1) further comprises (a) synthesizing said nucleic acid with a taggable group, said taggable group being capable of accepting a positive charge either directly or indirectly and either before or after step (2), or (b) synthesizing said nucleic acid with a precursor that comprises a positively charged moiety.

17. A method according to claim 14 wherein the nucleic acid with a phosphorothioate linkage or a phosphoroselenoate linkage is synthesized chemically.

18. A method according to claim 14 wherein the nucleic acid is synthesized by copying a nucleic acid template using a primer, a polymerase and dNTP-α

-S or dNTP-α

-Se.

19. A method according to claim 18 wherein the primer is an oligonucleotide wherein each of the sugar-sugar linkages is either a phosphorothioate or a phosphoroselenoate linkage.

20. A method according to claim 18 wherein the primer is positively charged.

21. A method according to claim 20 wherein the primer comprises an amino residue at its 5′

end.

22. A method according to claim 18 wherein the primer is negatively charged which negative charge is not eliminated upon reaction with the said alkylating agent.

23. A method according to claim 22 wherein the primer comprises a phosphate as a sugar-sugar linkage.

24. A method according to claim 14 wherein each of the sugar-sugar linkages is either a phosphorothioate or a phosphoroselenoate linkage.

26. A method according to claim 14 wherein the alkylating agent reacts selectively, but substantially completely, with the phosphorothioate S atom or phosphoroselenoate Se atom.

27. A method according to claim 14 wherein the alkylating agent reacts substantially completely with the phosphorothioate S atom or phosphoroselenoate Se atom and substantially completely with other reactive groups in the said nucleic acid.

28. A method according to claim 14 wherein the alkylating agent is any of R-Hal wherein R is any of a primary, secondary or tertiary alkyl, alkenyl, —

CO-alkyl, —

CO-aryl, —

CH(Hal/Cl/F) alkyl, —

CH(Hal/Cl/F)-aryl, —

C(Hal/Cl/F)(Hal/Cl/F)alkyl and —

C(Hal/Cl/F)aryl and wherein Hal is independently Br or I and (Hal/Cl/F) is independently any of Hal or Cl or F;

or an alkene or vinyl such as H₂C═

CH-alkyl and H₂C═

CH-aryl.

29. A method according to claim 28 wherein the alkylating agent is any of methyliodide, methylbromide, ethyliodide, ethylbromide, propyliodide, propylbromide, butyliodide, butylbromide, 1-iodopropane2-one, 1-iodopropane-3-one, 1-bromopropane-2-one, 1-bromopropane-3-one, 1-iodo-2-fluoropropane, 1-iodo-2-chloro-propane, 1-bromo-2-fluoropropane and 1-bromo-2-chloropropane.

30. A method according to claim 29 wherein the alkylating agent is any of methyl iodide, ethyl iodide or propyl iodide.

31. A method according to claim 30 wherein the alkylating agent is ethyl iodide.

42. A method of analyzing a nucleic acid by mass spectrometry comprising the steps of

(1) preparing a nucleic acid according to the method of claim 14;

(2) introducing the said nucleic acid molecule into a mass spectrometer; and

(3) determining the mass of the said nucleic acid.

43. A method according to claim 42 wherein the sugar-sugar linkages of the nucleic acid are substantially all phosphate sugar-sugar linkages.

15. A method of preparing a positively charged or negatively charged nucleic acid molecule containing one or up to ten negative charges and no or up to ten positive charges or no negative charges and one or up to ten positive charges, said nucleic acid molecule comprising a sugar-linkage-sugar backbone wherein said sugars have a base attached thereto at the 1 position;

said method comprising the steps of

(1) enzymatically synthesizing a nucleic acid with a phosphorothioate linkage or a phosphoroselenoate linkage between sugar residues, and

(2) reacting the said nucleic acid with an alkylating agent so as to eliminate the charge on the said phosphorothioate linkage or said phosphoroselenoate linkage, wherein said nucleic acid molecule consists of at least 4 nucleoside moieties.

16. A method of analyzing a nucleic acid by mass spectrometry comprising the steps of

(1) providing a positively or negatively charged nucleic acid molecule obtainable by the method of claim 15, containing one or up to ten negative charges and no or up to ten positive charges, or no negative charges and one or up to ten positive charges, wherein there are fewer negative charges than there are sugar-sugar linkages;

(2) introducing the said nucleic acid molecule into a mass spectrometer; and

(3) determining the mass of the said nucleic acid molecule.

25. A method of preparing a nucleic acid molecule containing no or up to ten negative charges or a nucleic acid molecule containing no or up to ten positive charges wherein the nucleic acid molecule has a sugar-linkage-sugar backbone and said sugars have a base attached thereto at the 1 position;

said method comprising the steps of

(1) synthesizing a nucleic acid with a phosphorothioate linkage or a phosphoroselenoate linkage between sugar residues, and

(2) reacting the said nucleic acid with an alkylating agent so as to eliminate the charge on the said phosphorothioate linkage or said phosphoroselenoate linkage, wherein the nucleic acid chain is synthesized enzymatically and is terminated using a ddNTP, a ddNTP-α

-S or a ddNTP-α

-Se.

32. A method of preparing a nucleic acid molecule containing one or up to ten negative charges wherein the nucleic acid molecule has a sugar-linkage-sugar backbone and said sugars have a base attached thereto at the 1 position;

said method comprising the steps of synthesizing enzymatically the said nucleic acid using a dNTP uncharged at least at the α

-phosphorus position, wherein the nucleic acid is synthesized additionally using a primer and a polymerase and wherein the said dNTP uncharged at least at the α

-phosphorus position is enzymatically incorporated into the said nucleic acid molecule.

33. A method according to claim 32 wherein the uncharged dNTP is the dNTP shown in FIG. 12 wherein R₃ is H, R₁ is any of BH₂, CH₃, F, Cl or H, and R₂ is as given in FIG. 12.

34. A method according to claim 32 wherein the primer is an oligonucleotide wherein each of the sugar-sugar linkages is either uncharged or is phosphorothioate or a phosphoroselenoate linkage capable of having its charge eliminated.

35. A method according to claim 32 wherein the primer is positively charged.

36. A method according to claim 35 wherein the primer comprises an amino residue at its 5′

end.

37. A method according to claim 34 wherein the primer comprises a phosphate as a sugar-sugar linkage.

38. A nucleic acid molecule comprising a sugar-linkage-sugar backbone wherein said sugars have a base attached thereto at the 1 position;

said molecule further containing one to ten phosphate sugar-sugar linkages wherein all other sugar-sugar linkages are uncharged comprising at least 20 nucleoside residues.

39. A nucleic acid according to claim 38 comprising at least 50 nucleoside residues.

40. A nucleic acid with one or up to ten positive charges wherein the sugar-sugar linkages are uncharged.

41. A nucleic acid according to claim 40 wherein each sugar-sugar linkage is either an alkylated phosphorothioate moiety or an alkylated phosphoroselenoate moiety.