Use of base-modified deoxynucleoside triphosphates to improve nucleic acid detection

• US 8,349,556 B2
• Filed: 04/30/2007
• Issued: 01/08/2013
• Est. Priority Date: 04/28/2006
• Status: Active Grant

• Current Assignee: Igor Kutyavin
• Original Assignee: Igor Kutyavin
• Inventors: Kutyavin, Igor
• Primary Examiner(s): Riley, Jezia
• Application Number:
  US12/298,895
• Publication Number:
  US 20100151455A1
• Time in Patent Office: 2,080 Days
• Field of Search: 435/6.1, 435/91.1, 435/91.2, 536/23.1, 536/24.3, 536/26.6, 422/430, 422/68.1
• US Class Current: 435/6.1
• CPC Class Codes: C12Q 1/6832 Enhancement of hybridisation reaction
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 2525/101 incorporating non-naturally occurring nucleotides, e.g. inosine
• CPC Class Codes: C12Q 2525/117 incorporating modified base
• CPC Class Codes: C12Q 2527/107 Temperature of melting, i.e. Tm
• CPC Class Codes: C12Q 2527/125 Specific component of sample, medium or buffer

Abstract

Aspects of the invention provide novel and surprisingly effective methods for the detection of nucleic acids, comprising nucleic acid amplification using base-modified deoxynucleoside 5′-triphosphates (dNTPs). Particular aspects relate to methods for enhancing hybridization properties of oligonucleotide primers and probes in assays detecting nucleic acids, comprise amplifying target DNAs in presence of base-modified duplex-stabilizing deoxyribonucleoside 5′-triphosphates to provide for modified target DNAs, and thereby considerably improving performance of the detection assays. The disclosed methods allow for increasing of the reaction temperature in PCR-based detection systems or, alternatively, reducing the length of the oligonucleotide primers and probes. Certain aspects relates to improvement of real time PCR assays, wherein nucleic acids of interest are detected as the reaction proceeds using fluorescent agents or oligonucleotide FRET probes.

Claims

1. A method for the detection of a target nucleic acid in a sample, comprising:

providing a reaction mixture comprising a target nucleic acid, at least one oligonucleotide primer, a DNA polymerase, and a mixture of deoxynucleoside 5′

-triphosphates containing at least one base-modified duplex-stabilizing dNTP;

amplifying the target nucleic acid, wherein the at least one base-modified duplex-stabilizing dNTP incorporates into amplicons providing copies of a modified DNA with enhanced hybridization properties;

providing at least one oligonucleotide probe and hybridizing the at least one oligonucleotide probe to the modified DNA to form a complex; and

detecting the complex, wherein presence of the complex is indicative of the presence of the target nucleic acid in the sample.

2. The method of claim 1, wherein the target nucleic acid comprises at least one of DNA and RNA.

3. The method of claim 1, wherein the target nucleic acid is RNA and amplifying of the target nucleic acid includes a stage wherein at least one DNA copy of said RNA is synthesized using a reverse transcriptase.

4. The method of claim 1, wherein more than one oligonucleotide primer is used to amplify the target nucleic acid, and wherein the modified DNA serves as a template for at least one of said oligonucleotide primers at any stage of the amplification.

5. The method of claim 1, wherein a plurality of target nucleic acids are amplified and detected.

6. The method of claim 5, wherein at least one oligonucleotide primer and at least one oligonucleotide probe is provided for every target nucleic acid amplified and detected.

7. The method of claim 5, wherein more than one of the oligonucleotide primers is provided for every said target nucleic acid amplified and detected and wherein at least one of the modified DNAs serves as a template for at least one of said oligonucleotide primer at any stage of the amplification.

8. The method of claim 1, wherein detection of the target nucleic acid is performed after the amplification.

9. The method of claim 1, wherein detection of the target nucleic acid is performed in real time.

10. The method of claim 1, wherein the base-modified duplex-stabilizing dNTP represents a fraction of the respective natural dNTP or completely replaces the respective natural dNTP.

11. The methods of claim 1, wherein said reaction mixture comprises more than one of said base-modified duplex-stabilizing dNTP, in each case either replacing or representing a fraction of the respective natural dNTP.

12. The method of claim 1, wherein said base-modified duplex-stabilizing dNTP is of Formula I:

13. The method of claim 1, wherein amplifying and detecting of the target nucleic acid is performed to measure the amount of said target nucleic acid in said sample.

14. The method of claim 1, wherein amplifying of the target nucleic acid comprises use of isothermal amplification.

15. The method of claim 1, wherein amplifying of the target nucleic acid comprises use of detection PCR.

16. The method of claim 1, wherein the at least one oligonucleotide primer contains a label and said label is used in detecting of said modified DNA.

17. The method of claim 16, wherein said label is a fluorescent label.

18. The method of claim 1, wherein the at least one oligonucleotide primer comprises a duplex-stabilizing modification comprising at least one of modified nucleotides and/or a tail conjugated to the 5′

-end of said oligonucleotide primer.

19. The method of claim 18, wherein the tail comprises at least one of an intercalator and a minor groove binder.

20. The method of claim 1, wherein detecting of the modified DNA comprises use of a detecting agent, wherein said detecting agent interacts with the modified DNA providing a detection signal, and wherein detection of the signal is indicative of the presence of the modified DNA in the reaction mixture.

21. The method of claim 20, wherein the detecting agent comprises a fluorescent agent, and wherein the fluorescent agent changes its fluorescence properties upon interaction with said modified DNA thereby providing the detection signal.

22. The method of claim 21, wherein the fluorescent agent comprises a SYBR Green dye.

23. The method of claim 1, wherein the at least one oligonucleotide primer and the at least one oligonucleotide probe are portions or fragments of the same molecule.

24. The method of claim 1, wherein a plurality of target nucleic acids are amplified and detected, and wherein at least one said oligonucleotide primer is provided for every target nucleic acid amplified and/or detected.

25. The method of claim 1, wherein at least one of a plurality of oligonucleotide primers and a plurality of oligonucleotide probes are used in amplifying and/or detecting the target nucleic acid.

26. The method of claim 1, wherein at least one of the oligonucleotide primers, oligonucleotide probes, and modified DNAs is immobilized on a solid support at the amplifying or detecting stages, or at both stages.

27. The method of claim 1, wherein the base-modified duplex-stabilizing dNTP represents a fraction of the respective natural dNTP or completely replaces the respective natural dNTP.

28. The method of claim 1, wherein at least one of the oligonucleotide primer, oligonucleotide probe, and modified DNA contains a label and wherein this label affords detecting of the modified DNA.

29. The method of claim 28, wherein the at least one oligonucleotide probe contains the label.

30. The method of claim 29, wherein the label comprises at least one of a fluorescent label and a fluorescence-polarization label.

31. The method of claim 1, wherein the oligonucleotide probe is a FRET probe, wherein the FRET probe changes its fluorescent properties upon forming the complex with the modified DNA, and wherein the changes are indicative of the presence of the complex.

32. The method of claim 31, wherein the FRET probe comprises a hybridization-triggered FRET probe.

33. The method of claim 32, wherein the hybridization-triggered FRET probe comprises at least one of a Scorpion primer and a Beacon probe.

34. The method of claim 31, wherein the FRET probe comprises at least one of a cleavable FRET probe and a cleavable FRET probe comprising a TaqMan probe.

35. The method of claim 15, wherein the detection PCR is performed using at least two oligonucleotide primers, and wherein the oligonucleotide primers provide for exponential amplification of the target nucleic acid.

36. The method of claim 35, wherein the detection PCR comprises quantitative PCR.

37. A method for the detection of a target nucleic acid in a sample, comprising:

amplifying the target nucleic acid using detection PCR in a reaction mixture comprising;

a target nucleic acid, at least two oligonucleotide primers to provide for exponential amplification of the target nucleic acid, a DNA polymerase, and a mixture of deoxynucleoside 5′

-triphosphates containing at least one base-modified duplex-stabilizing dNTP, wherein the at least one base-modified duplex-stabilizing dNTP incorporates into amplicons providing copies of a modified DNA with enhanced hybridization properties; and

detecting said modified DNA using at least one oligonucleotide probe that hybridizes to said modified DNA to form a complex, and detecting the complex, wherein presence of the complex is indicative of the presence of the target nucleic acid in the sample.

38. The method of claim 37, wherein the target nucleic acid comprises DNA.

39. The method of claim 37, wherein the target nucleic acid is RNA and amplifying the target nucleic acid includes a stage wherein at least one DNA copy of said RNA is synthesized using a reverse transcriptase.

40. The method of claim 37, wherein one of the oligonucleotide primers and the oligonucleotide probe are portions of fragments of the same molecule.

41. The method of claim 37, wherein a plurality of target nucleic acids are amplified and detected, and wherein at least two of the oligonucleotide primers and at least one of the oligonucleotide probes are provided for every target nucleic acid.

42. The method of claim 37, wherein detecting the modified DNA is performed after the amplification.

43. The method of claim 37, wherein detection of said target nucleic acid is performed in real time.

44. The method of claim 37, wherein at least one of the oligonucleotide primer, oligonucleotide probe and modified DNA is immobilized on a solid support at the amplifying or detecting stages, or at both stages.

45. The method of claim 37, wherein the base-modified duplex-stabilizing dNTP represents a fraction of the respective natural dNTP or completely replaces the respective natural dNTP.

46. The method of claims 37, wherein amplifying the target nucleic acid comprises use of more than one base-modified duplex-stabilizing dNTP, in each case either replacing or representing a fraction of the respective natural dNTP.

47. The method of claim 37, wherein the base-modified duplex-stabilizing dNTP is of Formula I:

48. The method of claim 37, wherein amplifying and detecting of the target nucleic acid is performed to measure the amount of said target nucleic acid in the sample.

49. The method of claim 37, wherein the oligonucleotide probe contains a label.

50. The method of claim 49, wherein the label comprises at least one of a fluorescent label and a fluorescence-polarization label.

51. The method of claim 37, wherein either one of the oligonucleotide primer or both primers, or the oligonucleotide probe, or both the primer or primers and the probe contain one or more duplex-stabilizing modification comprising at least one of modified nucleotides and/or a tail conjugated to the 5′

-end of said oligonucleotide primer.

52. The method of claim 51, wherein the at least one oligonucleotide primer comprises a duplex-stabilizing modification comprising at least one of modified nucleotides and/or a tail conjugated to the 5′

-end of said oligonucleotide primer, and wherein the tail comprises at least one of an intercalator and a minor groove binder.

53. The method of claim 37, wherein the oligonucleotide probe comprises a FRET probe, wherein the FRET probe changes its fluorescent properties upon forming the complex with the modified DNA, and wherein the changes are indicative of the presence of the complex.

54. The method of claim 53, wherein the FRET probe comprises a hybridization-triggered FRET probe.

55. The method of claim 54, wherein the hybridization-triggered FRET probe comprises at least one of a Scorpion primer and a Beacon probe.

56. The method of claim 53, wherein the FRET probe comprises at least one of a cleavable FRET probe and a cleavable FRET probe comprising a TaqMan probe.

57. The method of claim 37, wherein the detection PCR comprises quantitative PCR.