Real-time nucleic acid detection processes and compositions

• US 9,261,460 B2
• Filed: 03/12/2002
• Issued: 02/16/2016
• Est. Priority Date: 03/12/2002
• Status: Expired due to Term

• Current Assignee: Enzo Life Sciences Incorporated (Enzo Biochem Incorporated)
• Original Assignee: Enzo Life Sciences Incorporated (Enzo Biochem Incorporated)
• Inventors: Rabbani, Elazar, Stavrianopoulos, Jannis G., Donegan, James J., Coleman, Jack, Liu, Dakai
• Primary Examiner(s): HORLICK, KENNETH R
• Application Number:
  US10/096,076
• Publication Number:
  US 20050137388A1
• Time in Patent Office: 5,089 Days
• Field of Search: 435/6, 435/91.2, 536/24.33, 536/22.1, 536/24.31, 536/24.32
• US Class Current: 1/1
• CPC Class Codes: C12Q 1/6818 involving interaction of two or more labels, e.g. resonant energy transfer
• CPC Class Codes: C12Q 1/6834 Enzymatic or biochemical coupling of nucleic acids to a solid phase
• CPC Class Codes: C12Q 1/6844 Nucleic acid amplification reactions
• CPC Class Codes: C12Q 1/6853 using modified primers or templates
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 2533/101 Primer extension
• CPC Class Codes: C12Q 2561/113 Real time assay
• CPC Class Codes: C12Q 2565/101 Interaction between at least two labels
• CPC Class Codes: G01N 21/64 Fluorescence; Phosphorescence
• CPC Class Codes: G01N 21/6486 Measuring fluorescence of biological material, e.g. DNA, RNA, cells G01N21/6428 takes precedence

Abstract

This invention provides compositions for use in real time nucleic acid detection processes. Such real time nucleic acid detection processes are carried out with energy transfer elements attached to nucleic acid primers, nucleotides, nucleic acid probes or nucleic acid binding agents. Real time nucleic acid detection allows for the qualitative or quantitative detection or determination of single-stranded or double-stranded nucleic acids of interest in a sample. Other processes are provided by this invention including processes for removing a portion of a homopolymeric sequence, e.g., poly A sequence or tail, from an analyte or library of analytes. Compositions useful in carrying out such removal processes are also described and provided.

Claims

1. A composition of matter comprising at least two parts:

a first part comprising at least two segments,

wherein said first segment comprises a first nucleic acid primer and at least one first energy transfer element, wherein said first energy transfer element is;

(a) an energy donor and (b) covalently attached directly or via a linker arm to said first nucleic acid primer and said second segment comprises a primer-extended nucleic sequence complementary to at least a portion of a nucleic acid target sequence; and

a second part comprising at least two segments,

wherein said first segment of said second part comprises a second nucleic acid primer and at least one second energy transfer element, wherein said second energy transfer element is;

(a) an energy acceptor and (b) covalently attached directly or via a linker arm to said first nucleic acid primer; and

wherein said second segment of said second part comprises a primer-extended nucleic acid sequence which is identical to at least a portion of said nucleic acid target sequence;

wherein said first nucleic acid primer does not comprise said second energy transfer element, and wherein said second nucleic acid primer does not comprise said first nucleic acid energy element;

wherein said first primer is not complementary to said second primer or nucleic acid in the absence of a target dependent extension step; and

wherein neither of said nucleic acid primers is fixed or immobilized to a solid support.

2. The composition of claim 1, wherein said first nucleic acid primer comprises more than one first energy transfer element.

3. The composition of claim 1, wherein said second nucleic acid primer comprises more than one second energy transfer element.

4. The composition of claim 1, wherein said first part comprises more than one nucleic acid primer, or said second part comprises more than one nucleic acid primer, or both said first part and said second part comprise more than one nucleic acid primer.

5. The composition of claim 4, wherein said more than one nucleic acid primer comprise identical or different. nucleic acid sequences.

6. The composition of claim 1, wherein said first energy element and said second energy element are independently selected from the group consisting of fluorescein, fluorescein isothiocyanate (FITC), 6-carboxyfluorescein (6-FAM), naphthofluorescein, rhodamine, rhodamine 6G, rhodamine X, rhodal, sulforhodamine 101, tetramethylrhodamine (TAMRA), tetramethylrhodamineisothiocyanate (TRITC), 4,7-dichlororhodamine, eosin, eosinisothiocyanate (EITC), dansyl, hydroxycoumarin, methoxycoumarin or p-(Dimethyl aminophenylazo) benzoic acid (DABCYL), cyanine dyes, and derivatives of any of the foregoing.

7. The composition of claim 6, wherein said cyanine dyes or derivatives comprises Cy3 or Cy5.

8. The composition of claim 1, wherein at least one of said nucleic acid primers is fixed or immobilized to a solid support.

9. The composition of claim 8, wherein said solid support comprises beads, tubes, microtiter plates, glass slides, plastic slides, microchip arrays, wells or depressions.

10. The composition of claim 1, wherein said fixation or immobilization is direct or indirect.

11. The composition of claim 1, wherein said primer extended sequence was added or effected by a DNA polymerase.

12. The composition of claim 1, wherein said primer extended sequence was added or effected by a ligase.

13. A composition of matter that comprises a nucleic acid strand, said strand comprising two segments, the first segment comprising a primer;

and the second segment comprising a sequence derived from a target-dependent extension of said primer,

wherein said primer comprises a first energy transfer element, wherein said first energy transfer element is;

(a) an energy donor and (b) covalently attached directly or via a linker arm to a nucleotide in said first segment,

wherein said primer-extended sequence comprises (i) a second energy transfer element, wherein said second energy transfer element is;

(a) an energy acceptor and (b) covalently attached directly or via a linker arm to a nucleotide in said second segment; and

(ii) sequences derived from enzymatically adding more than one nucleotide to said primer using a nucleic acid target sequence as a template, wherein one of said more than one nucleotide comprises said second energy transfer element.

14. The composition of claim 13, wherein said nucleic acid primer comprises more than one first energy transfer element.

15. The composition of claim 13, wherein said primer-extended sequence comprises more than one second energy transfer element.

16. The composition of claim 13, wherein said first energy element and said second energy element are independently selected from the group consisting of fluorescein, fluorescein isothiocyanate (FITC), 6-carboxyfluorescein (6-FAM), naphthofluorescein, rhodamine, rhodamine 6G, rhodamine X, rhodal, sulforhodamine 101, tetramethyirhodamine (TAMRA), tetramethylrhodamineisothiocyanate (TRITC), 4,7-dichlororhodamine, eosin, eosinisothiocyanate (EITC), dansyl, hydroxycoumarin, methoxycoumarin or p-(Dimethyl aminophenylazo) benzoic acid (DABCYL), cyanine dyes, and derivatives of any of the foregoing.

17. The composition of claim 16, wherein said cyanine dyes or derivatives comprises Cy3 or Cy5.

18. The composition of claim 13, further comprising a second nucleic acid strand, said second strand comprising two segments, the first segment comprising a primer;

and said second segment comprising a primer-extended sequence,

wherein said primer comprises a first energy transfer element, wherein said first energy transfer element is;

(a) an energy transfer donor and (b) covalently attached directly or via a linker arm to a nucleotide in said first segment, and wherein said primer-extended sequence comprises a second energy transfer element, wherein said second energy transfer element is;

(a) an energy transfer acceptor and (b) covalently attached directly or via a linker arm to a nucleotide in said second segment.

19. The composition of claim 13 or 18, wherein at least one of said nucleic acid primers is fixed or immobilized to a solid support.

20. The composition of claim 19, wherein said solid support comprises beads, tubes, microtiter plates, glass slides, plastic slides, microchip arrays, wells or depressions.

21. The composition of claim 20, wherein said fixation or immobilization is direct or indirect.

22. A composition of matter comprising a nucleic acid strand, said strand comprising two segments, the first segment comprising a primer;

and the second segment comprising a primer-extended sequence,

wherein said primer-extended sequence comprises (a) sequences derived from enzymatically adding more than one nucleotide using a nucleic acid target sequence as a template wherein said nucleotides comprise energy transfer elements to said primer and (b) one or more first energy transfer elements, wherein said first energy element is;

(i) an energy donor and (ii) covalently attached directly or via a linker arm to a nucleotide in said second segment and (c) one or more second energy transfer elements wherein said second energy element is;

(i) an energy acceptor and (ii) covalently linked directly or via a linker arm to a nucleotide in said second segment.

23. The composition of claim 22, wherein said first energy element and said second energy element are independently selected from the group consisting of fluorescein, fluorescein isothiocyanate (FITC), 6-carboxyfluorescein (6-FAM), naphthofluorescein, rhodamine, rhodamine 6G, rhodamine X, rhodal, sulforhodamine 101, tetramethylrhodamine (TAMRA), tetramethylrhodamineisothiocyanate (TRITC), 4,7-dichlororhodamine, eosin, eosinisothiocyanate (EITC), dansyl, hydroxycoumarin, methoxycoumarin or p-(Dimethyl aminophenylazo) benzoic acid (DABCYL), cyanine dyes, and derivatives of any of the foregoing.

24. The composition of claim 23, wherein said cyanine dyes or derivatives comprises Cy3 or Cy5.

25. The composition of claim 22, further comprising a second nucleic acid strand, said second strand comprising two segments, wherein the first segment of said second strand comprises a primer, wherein the second segment of said second strand comprises a primer-extended sequence,

wherein said primer-extended sequence of said second segment comprises one or more first energy transfer elements, wherein said first energy transfer element is:

(a) an energy donor and (b) covalently attached directly or via a linker arm to a nucleotide in said primer extended sequence and one or more second energy transfer elements, wherein said second energy transfer element is;

(a) an energy acceptor and (b) covalently attached directly or via a linker arm to a nucleotide in said primer-extended sequence, and

wherein said nucleic acid strand is hybridized to said second nucleic acid strand.

26. The composition of claim 22 or 25, wherein at least one of said nucleic acid primers is fixed or immobilized to a solid support.

27. The composition of claim 26, wherein said solid support comprises beads, tubes, microtiter plates, glass slides, plastic slides, microchip arrays, wells or depressions.

28. The composition of claim 26, wherein said fixation or immobilization is direct or indirect.

29. The composition of claim 22 or 25, wherein at least one of said nucleic acid primers further comprises a quencher.

30. A composition of matter that comprises a nucleic acid strand, said strand comprising two or more ribonucleotides, wherein at least one ribonucleotide is covalently attached directly or via a linker arm to a first energy transfer element, wherein said first energy transfer element is an energy donor and wherein at least one other ribonucleotide is covalently attached directly or via a linker arm to a second energy transfer element, wherein said second energy transfer element is an energy acceptor, and wherein said one ribonucleotide and said one other ribonucleotide are different ribonucleotides in the nucleic acid strand.

31. The composition of claim 30, wherein said first energy element and said second energy element are independently selected from the group consisting of fluorescein, fluorescein isothiocyanate (FITC), 6-carboxyfluorescein (6-FAM), naphthofluorescein, rhodamine, rhodamine 6G, rhodamine X, rhodal, sulforhodamine 101, tetramethylrhodamine (TAMRA), tetramethylrhodamineisothiocyanate (TRITC), 4,7-dichlororhodamine, eosin, eosinisothiocyanate (EITC), dansyl, hydroxycoumarin, methoxycoumarin or p-(Dimethyl aminophenylazo) benzoic acid (DABCYL), cyanine dyes, and derivatives of any of the foregoing.

32. The composition of claim 31, wherein said cyanine dyes or derivatives comprises Cy3 or Cy5.

33. The composition of claim 30, wherein said nucleic acid strand is fixed or immobilized indirectly to a solid support.

34. The composition of claim 33, wherein said solid support comprises beads, tubes, microtiter plates, glass slides, plastic slides, microchip arrays, wells or depressions.

35. A composition of matter that comprises two parts, wherein the first part is a nucleic acid strand comprising two segments, the first segment comprising a nucleic acid primer, wherein said nucleic acid primer comprises one or more fluorescent first energy transfer elements, wherein said first energy transfer element is (i) covalent attached directly or via a linker arm to said nucleic acid primer in said first part and (ii) is an energy transfer donor, and the second segment comprises a primer-extended sequence,

wherein the second part comprises a nucleic acid binding agent non-covalently bound to said nucleic acid strand,

wherein said nucleic acid binding agent is not a nucleic acid, and

wherein said nucleic acid binding agent comprises one or more second energy transfer elements, wherein said second energy transfer element is an energy transfer acceptor.

36. The composition of claim 35, wherein said first energy element and said second energy element are independently selected from the group consisting of fluorescein, fluorescein isothiocyanate (FITC), 6-carboxyfluorescein (6-FAM), naphthofluorescein, rhodamine, rhodamine 6G, rhodamine X, rhodal, sulforhodamine 101, tetramethylrhodamine (TAMRA), tetramethylrhodamineisothiocyanate (TRITC), 4,7-dichlororhodamine, eosin, eosinisothiocyanate (EITC), dansyl, hydroxycoumarin, methoxycoumarin or p-(Dimethyl aminophenylazo) benzoic acid (DABCYL), cyanine dyes, and derivatives of any of the foregoing.

37. The composition of claim 36, wherein said cyanine dyes or derivatives comprises Cy3 or Cy5.

38. The composition of claim 35, wherein said nucleic acid binding agent comprises a protein or a polypeptide.

39. The composition of claim 38, wherein said protein or polypeptide comprises T4 gene 32 protein, SSB protein or antibodies.

40. The composition of claim 35, wherein said nucleic acid binding agent comprises a single strand nucleic acid binding dye.

41. The composition of claim 35, wherein said nucleic acid strand is fixed or immobilized to a solid support.

42. The composition of claim 41, wherein said solid support comprises beads, tubes, microtiter plates, glass slides, plastic slides, microchip arrays, wells or depressions.

43. The composition of claim 41, wherein said fixation or immobilization is direct or indirect.

44. A composition of matter that comprises

(a) two nucleic acid strands,

wherein at least one of said strands comprises two segments, the first segment comprising a nucleic acid primer, and the second segment comprising a primer-extended sequence, wherein said nucleic acid primer comprises one or more fluorescent first energy transfer elements, wherein said first energy transfer element is (i) covalently attached to said nucleic acid primer and (ii) is an energy transfer donor, and

(b) a nucleic acid binding agent non-covalently bound to said two nucleic acid strands,

wherein said nucleic acid binding agent comprises one or more second energy transfer elements, wherein said second energy transfer element is an energy transfer acceptor,

wherein said nucleic acid binding agent is not a nucleic acid, and

wherein said two nucleic acid strands are hybridized to each other.

45. The composition of claim 44, wherein said first energy element and said second energy element are independently selected from the group consisting of fluorescein, fluorescein isothiocyanate (FITC), 6-carboxyfluorescein (6-FAM), naphthofluorescein, rhodamine, rhodamine 6G, rhodamine X, rhodal, sulforhodamine 101, tetramethylrhodamine (TAMRA), tetramethylrhodamineisothiocyanate (TRITC), 4,7-dichlororhodamine, eosin, eosinisothiocyanate (EITC), dansyl, hydroxycoumarin, methoxycoumarin or p-(Dimethyl aminophenylazo) benzoic acid (DABCYL,), cyanine dyes, and derivatives of any of the foregoing.

46. The composition of claim 45, wherein said cyanine dyes or derivatives comprises Cy3 or Cy5.

47. The composition of claim 44, wherein said second strand comprises two segments, the first segment comprising a second nucleic acid primer, and the second segment comprising a primer-extended sequence.

48. The composition of claim 47, wherein said second primer comprises one or more first energy transfer elements.

49. The composition of claim 44, wherein said nucleic acid binding agent comprises a protein or a polypeptide.

50. The composition of claim 49, wherein said protein or polypeptide comprises histones or antibodies.

51. The composition of claim 44, wherein said nucleic acid binding agent comprises an intercalator.

52. The composition of claim 51, wherein said intercalator comprises SYBR green I, ethidium bromide, a homodimer or a heterodimer that comprises ethidium bromide or derivatives.

53. The composition of claim 44, wherein at least one of said nucleic acid strands is fixed or immobilized to a solid support.

54. The composition of claim 53, wherein said solid support comprises beads, tubes, microtiter plates, glass slides, plastic slides, microchip arrays, wells or depressions.

55. The composition of claim 53, wherein said fixation or immobilization is direct or indirect.

56. A composition of matter that comprises

(a) a nucleic acid strand comprising two segments, said first segment comprising a primer;

and said second segment comprising a primer-extended sequence, wherein said primer-extended sequence comprises one or more first energy transfer elements, wherein said first energy transfer element is;

(i) an energy transfer donor and (ii) covatently attached directly or via a linker arm to a nucleotide in said second segment; and

(b) a nucleic acid binding agent, non-covalently bound to said nucleic acid strand,

wherein said nucleic acid binding agent is not a nucleic acid, and

wherein said nucleic acid binding agent comprises one or more second energy transfer elements,

wherein said second energy transfer element is an energy transfer acceptor.

57. The composition of claim 56, wherein said first energy element and said second energy element are independently selected from the group consisting of fluorescein, fluorescein isothiocyanate (FITC), 6-carboxyfluorescein (6-FAM), naphthofluorescein, rhodamine, rhodamine 6G, rhodamine X, rhodal, sulforhodamine 101, tetramethylrhodamine (TAMRA), tetramethylrhodamineisothiocyanate (TRITC), 4,7-dichlororhodamine, eosin, eosinisothiocyanate (EITC), dansyl, hydroxycoumarin, methoxycournarin or p-(Dimethyl aminophenylazo) benzoic acid (DABCYL), cyanine dyes, and derivatives of any of the foregoing.

58. The composition of claim 57, wherein said cyanine dyes or derivatives comprises Cy3 or Cy5.

59. The composition of claim 56, wherein said nucleic acid binding agent (b) comprises a protein or a polypeptide.

60. The composition of claim 59, wherein said protein or polypeptide comprises T4 gene 32 protein, SSB protein or antibodies.

61. The composition of claim 56, wherein said nucleic acid binding agent (b) comprises a single strand nucleic acid binding dye.

62. The composition of claim 56, wherein said nucleic acid strand (a) is fixed or immobilized to a solid support.

63. The composition of claim 62, wherein said solid support comprises beads, tubes, microtiter plates, glass slides, plastic slides, microchip arrays, wells or depressions.

64. The composition of claim 62, wherein said fixation or immobilization is direct or indirect.

65. A composition of matter that comprises

(a) two nucleic acid strands hybridized to each other,

wherein at least one of said strands comprises two segments, the first segment comprising a nucleic acid primer, and the second segment comprising a primer-extended sequence, wherein said primer-extended sequence comprises one or more first energy transfer elements, wherein said first energy transfer element is:

(i) an energy transfer donor and (ii) covalently attached directly or via a linker arm to a nucleotide in said second segment, and

(b) a nucleic acid binding agent non-covalently bound to said two nucleic acid strands,

wherein said nucleic acid binding agent is not a nucleic acid, and

wherein said nucleic acid binding agent comprises one or more second energy transfer elements, wherein said second energy transfer element is an energy transfer acceptor.

66. The composition of claim 65, wherein said first energy element and said second energy element are independently selected from the group consisting of fluorescein, fluorescein isothiocyanate (FITC), 6-carboxyfluorescein (6-FAM), naphthofluorescein, rhodamine, rhodamine 6G, rhodamine X, rhodal, sulforhodamine 101, tetramethylrhodamine (TAMRA), tetramethylrhodamineisothiocyanate (TRITC), 4,7-dichlororhodamine, eosin, eosinisothiocyanate (EITC), dansyl, hydroxycoumarin, methoxycoumarin or p-(Dimethyl aminophenylazo) benzoic acid (DABCYL), cyanine dyes, and derivatives of any of the foregoing.

67. The composition of claim 66, wherein said cyanine dyes or derivatives comprises Cy3 or Cy5.

68. The composition of claim 65, wherein said nucleic acid binding agent (b) comprises a protein or a polypeptide.

69. The composition of claim 68, wherein said protein or polypeptide comprises histones or antibodies.

70. The composition of claim 65, wherein said nucleic acid binding agent (b) comprises an intercalator.

71. The composition of claim 70, wherein said intercalator comprises SYBR green I, ethidium bromide, a homodimer or a heterodimer that comprises ethidium bromide or derivatives.

72. The composition of claim 65, wherein both nucleic acid strands (a) comprise two segments, the first segment comprising a nucleic acid primer, and the second segment comprising a primer-extended sequence.

73. The composition of claim 72, wherein said primer-extended sequences comprise first energy transfer elements.

74. The composition of claim 65, wherein at least one of said nucleic acid strands (a) is fixed or immobilized to a solid support.

75. The composition of claim 74, wherein said solid support comprises beads, tubes, microtiter plates, glass slides, plastic slides, microchip arrays, wells or depressions.

76. The composition of claim 74, wherein said fixation or immobilization is direct or indirect.

77. The composition of claim 65, wherein said nucleic acid primer further comprises a quencher.

78. A composition of matter that comprises:

(i) a nucleic acid strand, said strand comprising one or more ribonucleotides, wherein at least one ribonucleotide comprises a first energy transfer element, wherein said first energy transfer element is;

(a) an energy transfer donor and (b) covalently attached directly or via a linker arm to said ribonucleotide; and

(ii) a nucleic acid binding agent, non-covalently bound to said nucleic acid strand comprising one or more second energy transfer elements, wherein said nucleic acid binding agent is not a nucleic acid and wherein said second energy transfer element is an energy transfer acceptor from said donor.

79. The composition of claim 78, wherein said first energy element and said second energy element are independently selected from the group consisting of fluorescein, fluorescein isothiocyanate (FITC), 6-carboxyfluorescein (6-FAM), naphthofluorescein, rhodamine, rhodamine 6G, rhodamine X, rhodal, sulforhodamine 101, tetramethylrhodamine (TAMRA), tetramethylrhodamineisothiocyanate (TRITC), 4,7-dichlororhodamine, eosin, eosinisothiocyanate (EITC), dansyl, hydroxycoumarin, methoxycoumarin or p-(Dimethyl aminophenylazo) benzoic acid (DABCYL), cyanine dyes, and derivatives of any of the foregoing.

80. The composition of claim 79, wherein said cyanine dyes or derivatives comprises Cy3 or Cy5.

81. The composition of claim 78, wherein said nucleic acid binding agent (ii) comprises a protein or a polypeptide.

82. The composition of claim 81, wherein said protein or polypeptide comprises T4 gene 32 protein, SSB protein or antibodies.

83. The composition of claim 78, wherein said nucleic acid binding agent (ii) comprises a single strand nucleic acid binding dye.

84. The composition of claim 78, wherein said nucleic acid strand is fixed or immobilized indirectly to a solid support.

85. The composition of claim 84, wherein said solid support comprises beads, tubes, microtiter plates, glass slides, plastic slides, microchip arrays, wells or depressions.

86. A composition of matter that comprises a hybridized first and second nucleic acid strand, said first strand comprising an RNA transcript, wherein at least one ribonucleotide of said transcript comprises a first energy transfer element, wherein said first energy transfer element is:

(a) an energy transfer donor and (b) covalently attached directly or via a linker arm to said ribonucleotide;

said second strand comprising;

a nucleic acid probe hybridized to said first strand or a portion thereof, wherein said nucleic acid probe comprises one or more second energy transfer elements, wherein said second energy transfer element is;

(a) an energy transfer acceptor and (b) covalently attached directly or via a linker arm to said nucleic acid probe,

wherein at least one of said nucleic acid strands is fixed or immobilized to a solid support.

87. The composition of claim 86, wherein said nucleic acid probe comprises modified nucleotides or nucleotide analogs.

88. The composition of claim 86, wherein said nucleic acid probe comprises ribonucleotides or deoxyribonucleotides, or both.

89. The composition of claim 86, wherein said first energy element and said second energy element are independently selected from the group consisting of fluorescein, fluorescein isothiocyanate (FITC), 6-carboxyfluorescein (6-FAM), naphthofluorescein, rhodamine, rhodamine 6G, rhodamine X, rhodal, sulforhodamine 101, tetramethylrhodamine (TAMRA), tetramethylrhodamineisothiocyanate (TRITC), 4,7-dichlororhodamine, eosin, eosinisothiocyanate (EITC), dansyl, hydroxycoumarin, methoxycoumarin or p-(Dimethyl aminophenylazo) benzoic acid (DABCYL), cyanine dyes, and derivatives of any of the foregoing.

90. The composition of claim 89, wherein said cyanine dyes or derivatives comprises Cy3 or Cy5.

91. The composition of claims 86, wherein said solid support comprises beads, tubes, microtiter plates, glass slides, plastic slides, microchip arrays, wells or depressions.

92. A composition of matter that comprises two parts, wherein the first part is a nucleic acid strand comprising two segments, the first segment comprising a nucleic acid primer, wherein said nucleic acid primer comprises one or more fluorescent first energy transfer elements, wherein said first energy transfer element is:

(a) an energy transfer donor and (b) covalently attached directly or via a linker arm to said nucleic acid primer in said first part, and the second segment comprises a primer-extended sequence, and

wherein the second part comprises a nucleic acid binding agent, non-covalently bound to said nucleic acid strand, wherein said nucleic acid binding agent is not a nucleic acid,

wherein said nucleic acid binding agent comprises one or more second energy transfer elements, and wherein said second energy transfer element is an energy transfer acceptor and a protein or a polypeptide.

93. A composition of matter that comprises

(a) two nucleic acid strands hybridized to each other,

wherein at least one of said strands comprises two segments, the first segment comprising a nucleic acid primer, wherein said nucleic acid primer comprises one or more fluorescent first energy transfer elements, wherein said first energy transfer element is:

(i) an energy transfer donor and (ii) covalently attached to said nucleic acid primer, and the second segment comprising a primer-extended sequence, and

(b) a nucleic acid binding agent non-covalently bound to said two nucleic acid strands,

wherein said nucleic acid binding agent is not a nucleic acid, and

wherein said nucleic acid binding agent comprises one or more second energy transfer elements, wherein said second energy transfer element is an energy acceptor.

94. A composition of matter that comprises:

(i) a nucleic acid strand, said strand comprising one or more ribonucleotides, wherein at least one ribonucleotide comprises a first energy transfer element covalently attached directly or via a linker arm to said ribonucleotide, wherein said first energy transfer element is an energy transfer donor; and

(ii) a nucleic acid binding agent non-covalently bound to said nucleic acid strand, said binding agent comprising a T4 gene 32 protein, an SSB protein, histones or a single strand nucleic acid binding dye and one or more second energy transfer elements, wherein said second energy transfer element is an energy transfer acceptor.

95. A composition of matter comprising at least two parts:

a first part comprising at least one first nucleic acid primer that comprises

(i) at least one first energy transfer element, wherein said first energy transfer element is;

(a) an energy acceptor and (h) covalently attached directly or via a linker arm to said first nucleic acid primer; and

(ii) nucleic acid sequences which are complementary to at least a portion of a nucleic acid target sequence;

a second part comprising at least one second nucleic acid primer that comprises;

(i) at least one second energy transfer element, wherein said second energy transfer element is (a) an energy donor and (b) covalently attached directly or via a linker arm to said second nucleic acid primer; and

(ii) nucleic acid sequences which are identical to nucleic acid sequences in at least a portion of said target nucleic acid sequence;

wherein said first nucleic acid primer does not comprise said second energy transfer element and wherein said second nucleic acid primer does not comprise said first energy transfer element;

wherein said first primer hybridizes to said target strand and said second primer hybridizes to the complement of said target strand; and

wherein either said first nucleic acid primer comprises more than one first energy transfer element, said second nucleic acid primer comprises more than one second energy transfer element or said first nucleic acid primer comprises more than one first energy transfer element and said second nucleic acid primer comprises more than one second energy transfer element.

96. The composition of claim 95, wherein said first part comprises more than one nucleic acid primer, or said second part comprises more than one nucleic acid primer, or both said first part and said second part comprise more than one nucleic acid primer.

97. The composition of claim 96, wherein said more than one nucleic acid primer comprise identical or different nucleic acid sequences.

98. The composition of claim 95, wherein said first energy element and said second energy element are independently selected from the group consisting of fluorescein, fluorescein isothiocyanate (FITC), 6-carboxyfluorescein (6-FAM), naphthofluorescein, rhodamine, rhodamine 6G, rhodamine X, rhodal., sulforhodamine 101, tetramethylrhodamine (TAMRA), tetramethylrhodamineisothiocyanate (TRITC), 4,7-dichlororhodamine, eosin, eosinisothiocyanate (EITC), dansyl, hydroxycoumarin, methoxycoumarin or p-(Dimethyl aminophenylazo) benzoic acid (DABCYL), cyanine dyes, and derivatives of any of the foregoing.

99. The composition of claim 98, wherein said cyanine dyes or derivatives comprises Cy3 or Cy5.

100. The composition of claim 95, wherein at least one of said nucleic acid primers is fixed or immobilized to a solid support.

101. The composition of claim 100, wherein said solid support comprises beads, tubes, microtiter plates, glass slides, plastic slides, microchip arrays, wells or depressions.

102. The composition of claim 95, wherein said fixation or immobilization is direct or indirect.

103. A composition of matter comprising two nucleic acid strands, wherein the first of said two strands comprises at least one first energy transfer element, wherein said first energy transfer element is:

(a) an energy acceptor and (b) covalently attached directly or via a linker arm to said first strand, and the second of said two strands comprises at least one second energy transfer element, wherein said second energy transfer element is;

(a) an energy donor and (b) covalently attached directly or via a linker arm to said second strand and wherein said first strand or a portion thereof is hybridized to said second strand or a portion thereof, wherein said first strand lacks said second energy transfer element and said second strand lacks said first energy transfer element; and

wherein either said first nucleic acid strand comprises more than one first energy transfer element, said second nucleic acid strand comprises more than one second energy transfer element or said first nucleic acid strand comprises more than one second energy transfer element and said second nucleic acid strand comprises more than one first energy transfer element.

104. The composition of claim 103, wherein said first energy element and said second energy element are independently selected from the group consisting of fluorescein, fluorescein isothiocyanate (FITC), 6-carboxyfluorescein (6-FAM), naphthofluorescein, rhodamine, rhodamine 6G, rhodamine X, rhodal, sulforhodamine 101, tetramethylrhodamine (TAMRA), tetramethylrhodarnineisothiocyanate (TRITC), 4,7-dichlororhodamine, eosin, eosinisothiocyanate (EITC), dansyl, hydroxycoumarin, methoxycoumarin or p-(Dimethyl aminophenylazo) benzoic acid (DABCYL), cyanine dyes, and derivatives of any of the foregoing.

105. The composition of claim 104, wherein said cyanine dyes or derivatives comprises Cy3 or Cy5.

106. The composition of claim 103, wherein at least one of said strands is fixed or immobilized to a solid support.

107. The composition of claim 106, wherein said solid support comprises beads, tubes, microtiter plates, glass slides, plastic slides, microchip arrays, wells or depressions.

108. The composition of claim 103, wherein said fixation or immobilization is direct or indirect.

109. A composition of matter that comprises a nucleic acid strand, said strand comprising two segments, the first segment comprising a primer;

and said second segment comprising an enzymatically added primer-extended sequence

wherein said primer comprises a first energy transfer element, wherein said first energy transfer element is;

(a) an energy acceptor and (b) covalently attached directly or via a linker arm to a nucleotide in said first segment,

wherein said primer-extended sequence comprises (i) a second energy transfer element, wherein said second energy transfer element is;

(a) an energy donor and (b) covalently attached directly or via a linker arm to a nucleotide in said second segment and (ii) sequences derived from adding more than one nucleotides to said primer using a nucleic acid target sequence as a template.

110. The composition of claim 109, wherein said nucleic acid primer comprises more than one first energy transfer element.

111. The composition of claim 109, wherein said primer-extended sequence comprises more than one second energy transfer element.

112. The composition of claim 109, wherein said first energy element and said second energy element are independently selected from the group consisting of fluorescein, fluorescein isothiocyanate (FITC), 6-carboxyfluorescein (6-FAM), naphthofluorescein, rhodamine, rhodamine 6G, rhodamine X, rhodal, sulforhodamine 101, tetramethylrhodamine (TAMRA), tetramethylrhodamineisothiocyanate (TRITC), 4,7-dichiororhodamine, eosin, eosinisothiocyanate (EITC), dansyl, hydroxycoumarin, methoxycoumarin or p-(Dimethyl aminophenylazo) benzoic acid (DABCYL), cyanine dyes, and derivatives of any of the foregoing.

113. The composition of claim 112, wherein said cyanine dyes or derivatives comprises Cy3 or Cy5.

114. The composition of claim 109, further comprising a second nucleic acid strand hybridized to said nucleic acid strand, said second strand comprising two segments, the first segment comprising a primer;

and said second segment comprising an enzymatically added primer-extended sequence,

wherein said primer comprises a first energy transfer element, wherein said first energy transfer element is;

(a) an energy acceptor and (b) covalently attached directly or via a linker arm to a nucleotide in said first segment, and wherein said primer-extended sequence comprises a second energy transfer element, wherein said second energy transfer element is;

(a) an energy donor and (b) covalently attached directly or via a linker arm to a nucleotide in said second segment.

115. The composition of claim 109 or 114, wherein at least one of said nucleic acid primers is fixed or immobilized to a solid support.

116. The composition of claim 115, wherein said solid support comprises beads, tubes, microtiter plates, glass slides, plastic slides, microchip arrays, wells or depressions.

117. The composition of claim 116, wherein said fixation or immobilization is direct or indirect.

118. A composition of matter comprising a nucleic acid strand, said strand comprising two segments, the first segment comprising a primer;

and the second segment comprising a primer-extended sequence,

wherein said primer-extended sequence comprises (a) sequences derived from enzymatically adding more than one nucleotide to said primer using a nucleic acid target sequence as a template and (b) one or more first energy transfer elements, wherein said first energy element is;

(i) an energy acceptor and (ii) covalently attached directly or via a linker arm to a nucleotide on said first segment and (c) one or more second energy transfer elements, wherein said second energy transfer element is;

(i) an energy donor and (ii) covalently linked directly or via a linker arm to a nucleotide on said second segment.

119. The composition of claim 118, wherein said first energy element and said second energy element are independently selected from the group consisting of fluorescein, fluorescein isothiocyanate (FITC), 6-carboxyfluorescein (6-FAM), naphthofluorescein, rhodamine, rhodamine 6G, rhodamine X, rhodal, sulforhodamine 101, tetramethylrhodamine (TAMRA), tetramethylthodamineisothiocyanate (TRITC), 4,7-dichlororhodamine, eosin, eosinisothiocyanate (EITC), dansyl, hydroxycoumarin, methoxycoumarin or p-(Dimethyl aniinophenylazo) benzoic acid (DABCYL), cyanine dyes, and derivatives of any of the foregoing.

120. The composition of claim 119, wherein said cyanine dyes or derivatives comprises Cy3 or Cy5.

121. The composition of claim 118, further comprising a second nucleic acid strand hybridized to said nucleic acid strand, said second strand comprising two segments, wherein the first segment of said second strand comprises a primer, wherein the second segment of said second strand comprises a primer-extended sequence,

wherein said primer-extended sequence of said second segment comprises one or more first energy transfer elements, wherein said first energy transfer element is:

(a) an energy acceptor and (b) covalently attached directly or via a linker arm to a nucleotide in said primer extended sequence and one or more second energy transfer elements, wherein said second energy transfer element is;

(a) an energy donor and (b) covalently attached directly or via a linker arm to a nucleotide in said primer-extended sequence.

122. The composition of claim 118 or 121, wherein at least one of said nucleic acid primers is fixed or immobilized to a solid support.

123. The composition of claim 122, wherein said solid support comprises beads, tubes, microtiter plates, glass slides, plastic slides, microchip arrays, wells or depressions.

124. The composition of claim 122, wherein said fixation or immobilization is direct or indirect.

125. The composition of claim 118 or 121, wherein at least one of said nucleic acid primers further comprises a quencher.

126. A composition of matter that comprises an RNA transcript, wherein at least one ribonucleotide is covalently attached directly or via a linker arm to a first energy transfer element, wherein said first energy transfer element is an energy acceptor and wherein at least one other ribonucleotide is covalently attached directly or via a linker arm to a second energy transfer element, wherein said second energy transfer element is an energy donor, and wherein said one ribonucleotide and said one other ribonucleotide are different ribonucleotides in the RNA transcript.

127. The composition of claim 126, wherein said first energy element and said second energy element are independently selected from the group consisting of fluorescein, fluorescein isothiocyanate (FITC), 6-carboxyfluorescein (6-FAM), naphthofluorescein, rhodamine, rhodamine 6G, rhodamine X, rhodal, sulforhodarnine 101, tetramethylrhodamine (TAMRA), tetramethylrhodamineisothiocyanate (TRITC), 4,7-dichlororhodamine, eosin, eosinisothiocyanate (EITC), dansyl, hydroxycoumarin, methoxycoumarin or p-(Dimethyl aminophenylazo) benzoic acid (DABCYL), cyanine dyes, and derivatives of any of the foregoing.

128. The composition of claim 127, wherein said cyanine dyes or derivatives comprises Cy3 or Cy5.

129. The composition of claim 127, wherein said nucleic acid strand is fixed or immobilized indirectly to a solid support.

130. The composition of claim 129, wherein said solid support comprises beads, tubes, microtiter plates, glass slides, plastic slides, microchip arrays, wells or depressions.

131. A composition of matter that comprises

(a) a nucleic acid strand comprising two segments, said first segment comprising a primer;

and said second segment comprising a primer-extended sequence, wherein said primer-extended sequence comprises one or more first energy transfer elements, wherein said first energy transfer element is;

(i) an energy transfer acceptor and (ii) covalently attached directly or via a linker arm to a nucleotide in said second segment; and

(b) a nucleic acid binding agent non-covalently bound to said nucleic acid strand,

wherein said nucleic acid binding agent is not a nucleic acid, and wherein said nucleic acid binding agent comprises one or more second energy transfer elements, wherein said second energy transfer element is an energy transfer donor.

132. The composition of claim 131, wherein said first energy element and said second energy element are independently selected from the group consisting of fluorescein, fluorescein isothiocyanate (FITC), 6-carboxyfluorescein (6-FAM), naphthofluorescein, rhodamine, rhodamine 6G, rhodamine X, rhodal, sulforhodamine 101, tetramethylrhodamine (TAMRA), tetramethylrhodamineisothiocyanate (TRITC), 4,7-dichlororhodamine, eosin, eosinisothiocyanate (EITC), dansyl, hydroxycoumarin, methoxycoumarin or p-(Dimethyl aniinophenylazo) benzoic acid (DABCYL), cyanine dyes, and derivatives of any of the foregoing.

133. The composition of claim 132, wherein said cyanine dyes or derivatives comprises Cy3 or Cy5.

134. The composition of claim 131, wherein said nucleic acid binding agent (b) comprises a protein or a polypeptide.

135. The composition of claim 134, wherein said protein or polypeptide comprises T4 gene 32 protein, SSB protein or antibodies.

136. The composition of claim 131, wherein said nucleic acid binding agent (b) comprises a single strand nucleic acid binding dye.

137. The composition of claim 131, wherein said nucleic acid strand (a) is fixed or immobilized to a solid support.

138. The composition of claim 137, wherein said solid support comprises beads, tubes, microtiter plates, glass slides, plastic slides, microchip arrays, wells or depressions.

139. The composition of claim 138, wherein said fixation or immobilization is direct or indirect.

140. A composition of matter that comprises

(a) two nucleic acid strands hybridized to each other,

wherein at least one of said strands comprises two segments, the first segment comprising a nucleic acid primer, and the second segment comprising a primer-extended sequence, wherein said primer-extended sequence comprises one or more first energy transfer elements, wherein said first energy transfer element is:

(i) an energy transfer acceptor and (ii) covalently attached directly or via a linker arm to a nucleotide in said second segment, and

(b) a nucleic acid binding agent non-covalently bound to said two nucleic acid strands, wherein said nucleic acid binding agent is not a nucleic acid, and wherein said nucleic acid binding agent comprises one or more second energy transfer elements, wherein said second energy transfer element is an energy transfer donor.

141. The composition of claim 140, wherein said first energy element and said second energy element are independently selected from the group consisting of fluorescein, fluorescein isothiocyanate (FITC), 6-carboxyfluorescein (6-FAM), naphthofluorescein, rhodamine, rhodamine 6G, rhodamine X, rhodal, sulforhodamine 101, tetramethylrhodamine (TAMRA), tetramethylrhodamineisothiocyanate (TRITC), 4,7-dichlororhodamine, eosin, eosinisothiocyanate (EITC), dansyl, hydroxycoumarin, methoxycoumarin or p-(Dimethyl aminophenylazo) benzoic acid (DABCYL), cyanine dyes, and derivatives of any of the foregoing.

142. The composition of claim 141, wherein said cyanine dyes or derivatives comprises Cy3 or Cy5.

143. The composition of claim 140, wherein said nucleic acid binding agent (b) comprises a protein or a polypeptide.

144. The composition of claim 143, wherein said protein or polypeptide comprises histones or antibodies.

145. The composition of claim 140, wherein said nucleic acid binding agent (b) comprises an intercalator.

146. The composition of claim 145, wherein said intercalator comprises SYBR green I, ethidium bromide, a homodimer or a heterodimer that comprises ethidium bromide or derivatives.

147. The composition of claim 140, wherein both nucleic acid strands (a) comprise two segments, the first segment comprising a nucleic acid primer, and the second segment comprising a primer-extended sequence.

148. The composition of claim 147, wherein said primer-extended sequences comprise first energy transfer elements.

149. The composition of claim 140, wherein at least one of said nucleic acid strands (a) is fixed or immobilized to a solid support.

150. The composition of claim 149, wherein said solid support comprises beads, tubes, microtiter plates, glass slides, plastic slides, microchip arrays, wells or depressions.

151. The composition of claim 149, wherein said fixation or immobilization is direct or indirect.

152. The composition of claim 140, wherein said nucleic acid primer further comprises a quencher.

153. A composition of matter that comprises:

(i) a nucleic acid strand, said strand comprising one or more ribonucleotides, wherein at least one ribonucleotide comprises a first energy transfer element covalently attached directly or via a linker arm to said ribonucleotide, wherein said first energy transfer element is an energy transfer acceptor; and

(ii) a nucleic acid binding agent non-covalently bound to said nucleic acid strand, said binding agent comprising a T4 gene 32 protein, an SSB protein, histones or a single strand nucleic acid binding dye and one or more second energy transfer elements, wherein said second energy transfer element is an energy transfer donor.

154. A composition of matter that comprises a hybridized first and second nucleic acids,

said first strand comprising two segments, the first segment comprising a primer;

and the second segment comprising an enzymatically added primer-extended sequence, wherein said primer-extended sequence comprises one or more first energy transfer elements wherein said first energy transfer element is;

(a) an energy transfer acceptor and (b) covalently attached directly or via a linker arm to a nucleotide in said primer extended sequence,

said second strand comprising a nucleic acid probe hybridized to said primer-extended sequence or a portion thereof, wherein said nucleic acid probe comprises one or more second energy transfer elements, wherein said second energy transfer element is;

(a) an energy transfer donor and (b) covalently attached directly or via a linker arm to a nucleotide in said nucleic acid probe,

wherein at least one of said nucleic acid strands is fixed or immobilized to a solid support.

155. The composition of claim 154, wherein said first energy element and said second energy element are independently selected from the group consisting of fluorescein, fluorescein isothiocyanate (FITC), 6-carboxyfluorescein (6-FAM), naphthofluorescein, rhodamine, rhodamine 6G, rhodamine X, rhodal, sulforhodamine 101, tetramethylrhodamine (TAMRA), tetramethylrhodamineisothiocyanate (TRITC), 4,7-dichlororhodamine, eosin, eosinisothiocyanate (EITC), dansyl, hydroxycoumarin, methoxycoumarin or p-(Dimethyl aminophenylazo) benzoic acid (DABCYL), cyanine dyes, and derivatives of any of the foregoing.

156. The composition of claim 155, wherein said cyanine dyes or derivatives comprises Cy3 or Cy5.

157. A composition of matter that comprises a hybridized first and second nucleic acids, said first strand comprising an RNA transcript, wherein at least one ribonucleotide comprises a first energy transfer element, wherein said first energy transfer element is:

(a) an energy transfer acceptor and (b) covalently attached directly or via a linker arm to said ribonucleotide;

said second strand comprising;

a nucleic acid probe hybridized to said first strand or a portion thereof, wherein said nucleic acid probe comprises one or more second energy transfer elements, wherein said energy transfer element is;

(a) an energy transfer donor and (b) covalently attached directly or via a linker arm to said nucleic acid probe,

wherein said first strand or said nucleic acid probe is fixed or immobilized indirectly to a solid support.

158. The composition of claim 157, wherein said nucleic acid probe comprises modified nucleotides or nucleotide analogs.

159. The composition of claim 157, wherein said nucleic acid probe comprises ribonucleotides or deoxyribonucleotides, or both.

160. The composition of claim 157 wherein said first energy element and said second energy element are independently selected from the group consisting of fluorescein, fluorescein isothiocyanate (FITC), 6-carboxyfluorescein (6-FAM), naphthofluorescein, rhodamine, rhodamine 6G, rhodamine X, rhodal, sulforhodamine 101, tetramethylrhodamine (TAMRA), tetramethylrhodamineisothiocyanate (TRITC), 4,7-dichlororhodamine, eosin, eosinisothiocyanate (EITC), dansyl, hydroxycoumarin, methoxycoumarin or p-(dimethyl aminophenylazo) benzoic acid (DABCYL), cyanine dyes, and derivatives of any of the foregoing.

161. The composition of claim 160, wherein said cyanine dyes or derivatives comprises Cy3 or Cy5.