Process for Labeling and Purification of Nucleic Acids of Interest Present in a Biological Sample to be Treated in a Single Reaction Vessel

• US 20080233632A1
• Filed: 07/21/2005
• Published: 09/25/2008
• Est. Priority Date: 07/23/2004
• Status: Abandoned Application

• Current Assignee: Biomerieux B.V. (Compagnie Merieux Alliance SAS)
• Original Assignee: Biomerieux B.V. (Compagnie Merieux Alliance SAS)
• Inventors: Laayoun, Ali, Ginot, Frederic, Menou, Lionel
• Application Number:
  US11/658,028
• Publication Number:
  US 20080233632A1
• Time in Patent Office: Days
• US Class Current: 435/270
• CPC Class Codes: C07D 233/32 One oxygen atom
• CPC Class Codes: C07D 333/00 Heterocyclic compounds containing five-membered rings having one sulfur atom as the only ring hetero atom
• CPC Class Codes: C07D 495/04 Ortho-condensed systems
• CPC Class Codes: C07H 1/08 from natural products
• 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

Abstract

The present invention relates to a process for labeling and purification of nucleic acids of interest present in a biological sample to be treated, comprising:

• • taking a single reaction vessel,
  • introducing into the reaction vessel:
    • the biological sample,
    • at least one labeling reagent for nucleic acids,
    • at least one solid support enabling the adsorption of said nucleic acids,
    • any ingredient necessary for the labeling of the nucleic acids and/or for the immobilization of said nucleic acids on the support,
  • incubating the contents of the reaction vessel, and
  • isolating the nucleic acids thus labeled.

The invention finds a preferred application in the diagnostics field.

Claims

1. A process for labeling and purification of nucleic acids of interest which are present in a biological sample to be treated, consisting in:

taking a single reaction vessel,

introducing into the reaction vessel;

the biological sample,

at least one labeling reagent for nucleic acids, said labeling reagent being stable to heat and of formula (0);

2. The labeling and purification process as claimed in claim 1, wherein the nucleic acids treated consist of single-strand and/or double-strand, synthetic and/or natural DNA and/or RNA.

3. The labeling and purification process as claimed in claim 1, wherein the introduction of the labeling reagent enables:

the fragmentation of the nucleic acids in a non-specific manner to generate a plurality of nucleic acid fragments, and

the labeling of a plurality of these fragments at the terminal phosphate situated at the 3′ and

/or 5′

end, said terminal phosphate having been liberated during the fragmentation.

4. The labeling and purification process as claimed in claim 3, wherein the labeling of the 3′

or 5′

end of a nucleic acid fragment is performed by attachment of a reactive group borne by a label onto the phosphate in the 2′

position, 3′

position or cyclic 2′

-3′

-mono-phosphate position, with reference to the ribose.

5. The labeling and purification process as claimed in claim 3, wherein the fragmentation and/or labeling of the 3′

or 5′

end of a nucleic acid fragment is performed by attachment of a nucleophilic, electrophilic or halide group borne by a label onto the phosphate in the 2′

position, 3′

position or cyclic 2′

-3′

-monophosphate position, with reference to the ribose.

6. The process as claimed in claim 3, wherein the fragmentation of the nucleic acids is performed by:

enzymatic means (nucleases),

chemical means (metal cations, such as Mg⁺⁺, Mn⁺⁺, Cu⁺⁺, Co⁺⁺ and/or Zn⁺⁺ ions, whether or not in combination with a chemical catalyst, for example N-methyl-imidazole, or any chemical molecule having an affinity for RNA and bearing an imidazole nucleus or a substituted analog) or

physical means (by sonication or by radiation).

7. The process as claimed in claim 3, wherein the labeling of the 3′

or 5′

end of a fragment of RNA is performed by attachment of a molecule R—

X, where R consists of the label and X is the binding agent between the label and the RNA, such as a hydroxyl, amine, hydrazine, alkoxylamine, alkyl halide, phenylmethyl halide, iodoacetamide or maleimide group onto the phosphate linked to the 2′

position, 3′

position or the cyclic 2′

-3′

-monophosphate position of the ribose.

8. The process as claimed in claim 3, wherein the labeling on the phosphate group is effected by means of 5-(bromomethyl)-fluorescein.

9. The labeling and purification process as claimed in claim 1, wherein the labeling reagent is contacted with the nucleic acids in homogenous solution, in an essentially aqueous buffer, said labeling reagent being stable to heat and of formula (0).

10. The process as claimed in claim 9, wherein the labeling reagent used is of formula (1):

11. The process as claimed in claim 9, wherein the reagent is of formula (2):

12. The process as claimed in claim 9, wherein the reagent is of formula (3):

13. The process as claimed in claim 9, wherein the reagent is of formula (4):

14. The process as claimed in claim 9, wherein the reagent is of formula (21):

15. The process as claimed in claim 9, wherein the reagent is of formula (22):

16. The process as claimed in claim 9, wherein the reagent is of formula (23):

17. The process as claimed in claim 9, wherein the labeling reagent used contains R³ and R⁴ which independently of one another represent:

H, NO₂, OCH₃, —

CO—

NH—

(CH₂)₃—

(O—

CH₂—

CH₂)₃—

CH₂—

NH—

R² or —

CO—

NH—

(CH₂)₃—

(O—

CH₂—

CH₂)₄—

CH₂—

NH—

R².

18. The process as claimed in claim 9, wherein the reagent is of formula (7):

19. The process as claimed in claim 9, wherein the reagent is of formula (24):

20. The process as claimed in claim 9, wherein the structure R²-(L)_n- in the reagent is of formula (5):

23. The process as claimed in claim 9, wherein the reagent is of formula (14):

24. The process as claimed in claim 9, wherein the reagent is of formula (26):

25. The process as claimed in claim 9, wherein the reagent is of formula (15):

26. The process as claimed in claim 9, wherein the reagent is of formula (27):

27. The process as claimed in claim 9 wherein the constituent L of the reagent contains a —

(O—

CH₂—

CH₂)—

moiety, repeated from 1 to 20 times.

46. The process as claimed in claim 1, wherein the solid support consists of particles of silica.

47. The process as claimed in claim 1, wherein the solid support consists of magnetic particles covered with silica.

48. The process as claimed in claim 46 wherein the particles of silica comprising the solid support have particle sizes lying between 0.1 and 500 μ

m.

49. The process as claimed in claim 1, wherein one of the supplementary ingredients enabling the labeling consists of an alcohol preferably Isopropanol.

50. The process as claimed in claim 49, wherein Isopropanol comprises 70% v/v of the final mixture.

51. The process as claimed in claim 1, wherein one of the supplementary ingredients enabling the cell liberation and thus the adsorption of the nucleic acids onto the solid support consists of a chaotropic agent.

52. The process as claimed in claim 51, wherein the chaotropic agent used is a guanidium salt, sodium iodide, potassium iodide, sodium (iso)thiocyanate, urea or mixtures of these derivatives.

53. The process as claimed in claim 52, wherein the guanidium salt used is guanidium (iso)thiocyanate.

54. The process as claimed in claim 1, wherein the solid phase-nucleic acid complexes are separated from the liquids by sedimentation and rejection of the supernatant, then washing of the complexes with a washing buffer containing a chaotropic substance.

55. The process as claimed in claim 54, wherein the solid phase-nucleic acid complexes, after washing with the washing buffer, are then washed again with one or several organic solvents, and then subjected to a drying process.

56. The process as claimed in claim 55, wherein the nucleic acid present in the solid phase-nucleic acid complexes, after washing and drying of the complexes, is eluted by means of an elution buffer.

57. The process as claimed in claim 1, wherein the solid phase-nucleic acid complexes thus obtained are contacted with a mixture wherein the components are present for the purpose of amplifying the nucleic acid, whether attached to said solid phase, or eluted therefrom.

58. The process as claimed in claim 1, wherein the incubation step comprises maintaining the treated sample for 5 to 45 minutes, at a temperature lying between 45 and 85°

C.

59. The process as claimed in claim 58, wherein after the incubation step, the sample is brought back to ambient temperature for approximately 5 minutes.

21. A process for labeling and purification of nucleic acids of interest present in a biological sample to be treated, comprising:

taking a single reaction vessel,

introducing into the reaction vessel;

the biological sample,

at least one labeling reagent for nucleic acids, the labeling reagent being contacted with the nucleic acids in homogenous solution, in an essentially aqueous buffer, said labeling reagent being stable to heat, and of formula (6);

60. The labeling and purification process as claimed in claim 21, wherein the nucleic acids treated consist of single-strand and/or double-strand, synthetic and/or natural DNA and/or RNA.

22. A process for labeling and purification of nucleic acids of interest present in a biological sample to be treated, comprising:

taking a single reaction vessel,

introducing into the reaction vessel;

the biological sample,

at least one labeling reagent for nucleic acids, the labeling reagent being contacted with the nucleic acids in homogenous solution, in an essentially aqueous buffer, said labeling reagent being stable to heat, and of formula (25);

28. A process for labeling and purification of nucleic acids of interest present in a biological sample to be treated, comprising:

taking a single reaction vessel,

introducing into the reaction vessel;

the biological sample,

at least one labeling reagent for nucleic acids, the labeling reagent enabling the labeling and the fragmentation a single or double-strand nucleic acid according to the following steps;

fragmenting the nucleic acid,

attaching a label onto at least one of the fragments by means of a labeling reagent selected from the compounds of formula (19);

29. The process as claimed in claim 28, wherein the labeling reagent is selected from the compounds of formula (20):

30. The process as claimed in claim 28, wherein Z is:

31. The process as claimed in claim 28, wherein the fragmentation and the labeling are performed in two steps.

32. The process as claimed in claim 28, wherein the fragmentation and the labeling are performed in one step.

33. The process as claimed in claim 28, wherein the labeling is performed in essentially aqueous homogeneous solution.

34. The process as claimed in claim 28, wherein the fragmentation is performed by enzymatic, physical or chemical means.

35. A process for labeling and purification of nucleic acids of interest present in a biological sample to be treated, comprising:

taking a single reaction vessel,

introducing into the reaction vessel;

the biological sample,

at least one labeling reagent for nucleic acids, the labeling reagent being contacted in homogeneous solution, in an essentially aqueous buffer, said labeling reagent being stable to heat and of formula (8);

36. The process as claimed in claim 35, wherein the reagent is of formula (9):

37. The process as claimed in claim 35, wherein, in the formula of the reagent, p is less than or equal to m.

38. The process as claimed in claim 35, wherein the reagent is of formula (10):

39. The process as claimed in claim 35, wherein the reagent is of formula (11):

40. The process as claimed in claim 35, wherein in the reagent, R² comprises a D-Biotin residue of formula (12):

41. The process as claimed in any claim 35, wherein in the reagent, R¹ comprises:

CH₃, and R³ and R⁴ each represent;

H.

42. The process as claimed in claim 35, wherein the structure -(L)_n- of the reagent comprises:

spermine or N,N′

-bis(3-aminopropyl)-1,4-diaminobutane;

NH₂—

(CH₂)₃—

NH—

(CH₂)₄—

NH—

(CH₂)₃—

NH₂, or

spermidine or N-(3-aminopropyl)-1,4-butanediamine;

H₂N—

(CH₂)₄—

NH—

(CH₂)₃—

NH₂, or

a derivative containing an alanine moiety;

NH₂—

CH₂—

CH₂—

COOH.

45. The process as claimed in claim 35 wherein the constituent L of the reagent contains an —

(O—

CH₂—

CH₂)—

moiety, repeated from 1 to 20 times, -Z- then being represented by —

NH—

, —

NHCO—

or —

CONH—

.

43. A process for labeling and purification of nucleic acids of interest present in a biological sample to be treated, comprising:

taking a single reaction vessel,

introducing into the reaction vessel;

the biological sample,

at least one labeling reagent for nucleic acids, the heat-stable labeling reagent is contacted in homogenous solution, in an aqueous buffer, and is of formula (13);

44. The process as claimed in claim 43, wherein the reagent is of formula (16):