Processes for quantitative or qualitative detection of single-stranded or double-stranded nucleic acids

• US 10,144,956 B2
• Filed: 02/26/2016
• Issued: 12/04/2018
• Est. Priority Date: 03/12/2002
• Status: Expired due to Term

• Current Assignee: Enzo Life Sciences Incorporated (Enzo Biochem Incorporated)
• Original Assignee: Enzo Biochem Incorporated
• Inventors: Rabbani, Elazar, Stavrianopoulos, Jannis G., Donegan, James J., Coleman, Jack, Liu, Dakai
• Primary Examiner(s): Chunduru, Suryaprabha
• Application Number:
  US15/054,462
• Publication Number:
  US 20160273028A1
• Time in Patent Office: 1,012 Days
• Field of Search: None
• 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 for 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 process for detecting qualitatively or quantitatively the presence of a single-stranded or double-stranded nucleic acid of interest in a sample, said process comprising the steps of:

(a) providing

(i) a sample suspected of containing said nucleic acid of interest;

(ii) an oligonucleotide primer that comprises

(A) a nucleic acid sequence complementary to at least a portion of said nucleic acid of interest; and

(B) one or more fluorescent first energy transfer elements;

(iii) a nucleic acid binding agent comprising one or more second energy transfer elements, wherein said nucleic acid binding agent is sequence independent;

(iv) reagents for carrying out nucleic acid strand extension, said reagents comprising deoxynucleotides;

(b) forming a reaction mixture comprising (i), (ii), (iii) and (iv) above;

(c) contacting under hybridization conditions said oligonucleotide primer (ii) in said composition with said nucleic acid of interest;

(d) extending said oligonucleotide primer with said deoxynucleotides to form a primer-extended sequence;

(e) binding said nucleic acid binding agent (iii) to said primer-extended sequence or to a complex comprising said nucleic acid of interest and said primer-extended sequence; and

(f) detecting the presence or quantity of said nucleic acid of interest by means of energy transfer between said first energy transfer element of said oligonucleotide primer and said second energy transfer element of said nucleic acid binding agent (a)(iii) bound to said primer-extended sequence or to said complex in step (e).

2. The process of claim 1, wherein in said providing step (a), the nucleic acid binding agent (iii) comprises a protein or a polypeptide.

3. The process of claim 2, wherein said protein or polypeptide comprises a T4 gene 32 protein, an SSB protein, a histone or an antibody.

4. The process of claim 1, wherein in said providing step (a), the nucleic acid binding agent (iii) comprises a single strand nucleic acid binding dye.

5. The process of claim 1, wherein in said providing step (a), the nucleic acid binding agent (iii) comprises an intercalator.

6. The process of claim 5, wherein said intercalator comprises SYBR green I, ethidium bromide, a homodimer or a heterodimer that comprises ethidium bromide or derivatives.

7. The process of claim 1, wherein said nucleic acid of interest (i) has been rendered single stranded before or during said reaction mixture forming step (b).

8. The process of claim 1, wherein said primer-extended sequence formed in said extending step (d) has been rendered single-stranded prior to said binding step (e).

9. The process of claim 1, wherein prior to the detecting step (f), said process further comprises the step of (b′

) separating said extended nucleic acid from the nucleic acid of interest and repeating the contacting step (c), the extending step (d) and the binding step (e).

10. The process of claim 9, wherein said separating step (b′

), said contacting step (c), said extending step (d) and said binding step (e) are repeated one or more times.

11. The process of claim 1, wherein in the providing step (a), said reagents (iv) comprise a thermostable DNA polymerase.

12. The process of claim 8, wherein said rendering or separating step is carried out by thermal denaturation or strand displacement.

13. The process of claim 1, wherein in said providing step (a), either said first energy transfer element is an energy donor and said second energy transfer element is an energy acceptor, or said first energy transfer element is an energy acceptor and said second energy transfer element is an energy donor.

14. The process of claim 1, wherein in said providing step (a), said first energy transfer element and said second energy transfer element independently comprise fluorescein, fluorescein isothiocyanate (FITC), 6-carboxyfluorescein (6-FAM), naphthofluorescein, rhodamine, rhodamine 6 G, rhodamine X, rhodol, sulforhodamine 101, tetramethylrhodamine (TAMRA), tetramethylrhodamineisothiocyanate (TRITC), 4,7-dichlororhodamine, eosin, eosinisothiocyanate (EITC), dansyl, hydroxycoumarin, methoxycoumarin or p-(Dimethyl aminophenylazo) benzoic acid (DABCYL), cyanine dyes, or derivatives of any of the foregoing.

15. The process of claim 14, wherein one or both of said first energy transfer element and said second energy transfer element comprise the cyanine dye Cy3 or Cy5.

16. The process of claim 1, wherein said oligonucleotide primer or said primer-extended sequence is fixed or immobilized to a solid support.

17. The process of claim 16, wherein said solid support comprises beads, tubes, microtiter plates, glass slides, plastic slides, microchip arrays, wells or depressions.

18. The process of claim 16, wherein said fixation or immobilization is direct or indirect.

19. The process of claim 1, wherein any or all of said steps are carried out in a closed container system.

20. The process of claim 19, wherein said closed container system comprises an illumination source and a detection device or unit.

21. A process for detecting qualitatively or quantitatively the presence of a single-stranded or double-stranded nucleic acid of interest in a sample, said process comprising the steps of:

(a) providing

(i) a sample suspected of containing said nucleic acid of interest;

(ii) a first oligonucleotide primer that comprises a nucleic acid sequence complementary to at least a portion of one strand of said nucleic acid of interest;

(iii) a second oligonucleotide primer that comprises a nucleic acid sequence identical to at least a portion of said one strand;

(iv) a nucleic acid binding agent comprising one or more first energy transfer elements, wherein said nucleic acid binding agent is sequence independent; and

(v) reagents for carrying out nucleic acid strand extension, said reagents comprising deoxynucleotides;

wherein (ii), (iii) or both (ii) and (iii) comprise one or more fluorescent second energy transfer elements;

(b) forming a reaction mixture comprising (i), (ii), (iii), (iv) and (v) above;

(c) contacting under hybridization conditions said first oligonucleotide primer in said composition (ii) with one strand of said nucleic acid of interest and contacting under hybridization conditions said second oligonucleotide primer in said composition (iii) with the complementary strand of said nucleic acid of interest if present;

(d) extending said first oligonucleotide primer and said second oligonucleotide primer with said deoxynucleotides to form a first primer-extended nucleic acid sequence and a second primer-extended nucleic acid sequence if the complementary strand is present;

(e) binding said nucleic acid binding agent (iv) to a primer-extended nucleic acid sequence or to a complex comprising said nucleic acid of interest and a primer-extended sequence; and

(f) detecting the presence or quantity of said nucleic acid of interest by means of energy transfer between a second energy transfer element in said first oligonucleotide primer, said second oligonucleotide primer, or both, and a first energy transfer element in said nucleic acid binding agent (a)(iv) bound to said primer-extended sequence or to said complex in step (e).

22. The process of claim 21, wherein in said providing step (a), the nucleic acid binding agent (iv) comprises a protein or a polypeptide.

23. The process of claim 22, wherein said protein or polypeptide comprises a T4 gene 32 protein, an SSB protein, a histone or an antibody.

24. The process of claim 21, wherein in said providing step (a), the nucleic acid binding agent (iv) comprises a single strand nucleic acid binding dye.

25. The process of claim 21, wherein in said providing step (a), the nucleic acid binding agent (iv) comprises an intercalator.

26. The process of claim 25, wherein said intercalator comprises SYBR green I, ethidium bromide, a homodimer or a heterodimer that comprises ethidium bromide or derivatives.

27. The process of claim 21, further comprising the steps of:

(b′

) separating said first primer-extended nucleic acid sequence from said nucleic acid of interest and separating said second primer-extended nucleic acid sequence from said complementary strand of said nucleic acid of interest if present;

(c′

) contacting under hybridization conditions said first oligonucleotide primer in said composition (ii) with said second primer-extended nucleic acid sequence from step (b′

), if said second strand is present, and contacting under hybridization conditions said second oligonucleotide primer in said composition (iii) with said first primer-extended nucleic acid sequence from step (b′

); and

repeating said extending step (d) and said binding step (e) thereafter.

28. The process of claim 27, further comprising the step (b″

) of separating said first primer-extended nucleic acid sequence from said second primer-extended nucleic acid sequence, and the step (c″

) of contacting under hybridization conditions said first oligonucleotide primer in said composition (ii) with said second primer-extended nucleic acid sequence from step (b″

), and contacting under hybridization conditions said second oligonucleotide primer in said composition (iii) with said first primer-extended nucleic acid sequence from step (b″

); and

repeating said extending step (d) and said binding step (e) thereafter.

29. The process of claim 28, wherein said separating step (b″

), said contacting step (c″

), said extending step (d) and said binding step (e) are repeated.

30. The process of claim 21, wherein said complementary strand of the nucleic acid of interest is present.

31. The process of claims 21, wherein said nucleic acid of interest (i) has been rendered single stranded before or during said reaction mixture forming step (b).

32. The process of claim 21, wherein said binding step (e) is carried out after a separation or denaturation step.

33. The process of claim 21, wherein said detecting step (f) is carried out at specific intervals during or after any binding step (e).

34. The process of claim 21, wherein in the providing step (a), said reagents (v) comprise a thermostable DNA polymerase.

35. The process of claim 27, wherein said separating step is carried out by thermal denaturation or strand displacement.

36. The process of claim 21, wherein in said providing step (a), either said first energy transfer element is an energy donor and said second energy transfer element is an energy acceptor, or said first energy transfer element is an energy acceptor and said second energy transfer element is an energy donor.

37. The process of claim 21, wherein in said providing step (a), said first energy transfer element and said second energy transfer element independently comprise fluorescein, fluorescein isothiocyanate (FITC), 6-carboxyfluorescein (6-FAM), naphthofluorescein, rhodamine, rhodamine 6 G, rhodamine X, rhodol, sulforhodamine 101, tetramethylrhodamine (TAMRA), tetramethylrhodamineisothiocyanate (TRITC), 4,7-dichlororhodamine, eosin, eosinisothiocyanate (EITC), dansyl, hydroxycoumarin, methoxycoumarin or p-(Dimethyl aminophenylazo) benzoic acid (DABCYL), cyanine dyes, or derivatives of any of the foregoing.

38. The process of claim 37, wherein one or both of said first energy transfer element and said second energy transfer element comprise the cyanine dye Cy3 or Cy5.

39. The process of claim 21, wherein at least one of said first or second oligonucleotide primers or any primer-extended nucleic acid sequence is fixed or immobilized to a solid support.

40. The process of claim 39, wherein said solid support comprises beads, tubes, microtiter plates, glass slides, plastic slides, microchip arrays, wells or depressions.

41. The process of claim 39, wherein said fixation or immobilization is direct or indirect.

42. The process of claim 21, wherein any or all of said steps are carried out in a closed container system.

43. The process of claim 42, wherein said closed container system comprises an illumination source and a detection device or unit.

44. A process for detecting qualitatively or quantitatively the presence of a single-stranded or double-stranded nucleic acid of interest in a sample, said process comprising the steps of:

(a) providing

(i) a sample suspected of containing said nucleic acid of interest;

(ii) at least one oligonucleotide primer that comprises a nucleic acid sequence complementary to at least a portion of said nucleic acid of interest;

(iii) labeled deoxynucleotide or deoxynucleotides comprising a first energy transfer element;

(iv) a nucleic acid binding agent comprising one or more second energy transfer elements, wherein said nucleic acid binding agent is sequence independent; and

(v) reagents for carrying out nucleic acid strand extension;

(b) forming a reaction mixture comprising (i), (ii), (iii), (iv) and (v) above;

(c) contacting under hybridization conditions said oligonucleotide primer in said composition (ii) with said nucleic acid of interest;

(d) extending said oligonucleotide primer, thereby incorporating said labeled deoxynucleotide or deoxynucleotides;

(e) binding said nucleic acid binding agent to said primer-extended sequence; and

(f) detecting the presence or quantity of said nucleic acid of interest by means of energy transfer between said first energy transfer element and said second energy transfer element.

45. The process of claim 44, wherein in said providing step (a), there is at least one other oligonucleotide primer complementary to a different portion of said nucleic acid of interest.

46. The process of claim 44, wherein in said providing step (a), the nucleic acid binding agent (iv) comprises a protein or a polypeptide.

47. The process of claim 46, wherein said protein or polypeptide comprises a T4 gene 32 protein, an SSB protein, a histone or an antibody.

48. The process of claim 44, wherein in said providing step (a), the nucleic acid binding agent (iv) comprises a single strand nucleic acid binding dye.

49. The process of claim 44, wherein in said providing step (a), the nucleic acid binding agent (iv) comprises an intercalator.

50. The process of claim 49, wherein said intercalator comprises SYBR green I, ethidium bromide, a homodimer or a heterodimer that comprises ethidium a bromide or derivative thereof.

51. The process of claim 44, wherein said nucleic acid of interest (i) has been rendered single stranded before or during said reaction mixture forming step (b).

52. The process of claim 44, wherein prior to the detecting step (f), said process further comprises the step of (b′

) separating said extended nucleic acid from the nucleic acid of interest and repeating the contacting step (c), the extending step (d) and the binding step (e).

53. The process of claim 52, wherein said separating step (b′

), said contacting step (c), said extending step (d) and said binding step (e) are repeated one or more times.

54. The process of claim 44, wherein said primer-extended sequence formed in said extending step (d) has been rendered single-stranded prior to said binding step (e).

55. The process of claim 44, wherein in the providing step (a), said reagents (v) comprises a thermostable DNA polymerase.

56. The process of claim 52, wherein said separating step is carried out by thermal denaturation or strand displacement.

57. The process of claim 44, wherein in said providing step (a), either said first energy transfer element is an energy donor and said second energy transfer element is an energy acceptor, or said first energy transfer element is an energy acceptor and said second energy transfer element is an energy donor.

58. The process of claim 44, wherein in said providing step (a), said first energy transfer element and said second energy transfer element independently comprise fluorescein, fluorescein isothiocyanate (FITC), 6-carboxyfluorescein (6-FAM), naphthofluorescein, rhodamine, rhodamine 6 G, rhodamine X, rhodol, sulforhodamine 101, tetramethylrhodamine (TAMRA), tetramethylrhodamineisothiocyanate (TRITC), 4,7-dichlororhodamine, eosin, eosinisothiocyanate (EITC), dansyl, hydroxycoumarin, methoxycoumarin or p-(Dimethyl aminophenylazo) benzoic acid (DABCYL), cyanine dyes, or a derivatives of any of the foregoing.

59. The process of claim 58, wherein one or both of said first energy transfer element and said second energy transfer element comprise the cyanine dye Cy3 or Cy5.

60. The process of claim 44, wherein said oligonucleotide primer, or said primer-extended sequence is fixed or immobilized to a solid support.

61. The process of claim 60, wherein said solid support comprises beads, tubes, microtiter plates, glass slides, plastic slides, microchip arrays, wells or depressions.

62. The process of claim 60, wherein said fixation or immobilization is direct or indirect.

63. The process of claim 44, wherein any or all of said steps are carried out in a closed container system.

64. The process of claim 63, wherein said closed container system comprises an illumination source and a detection device or unit.

65. A process for detecting qualitatively or quantitatively the presence of a single-stranded or double-stranded nucleic acid of interest in a sample, said process comprising the steps of:

(a) providing;

(i) a sample suspected of containing said nucleic acid of interest;

(ii) a first oligonucleotide primer that comprises a nucleic acid sequence complementary to at least a portion of one strand of said nucleic acid of interest;

(iii) a second oligonucleotide primer that comprises a nucleic acid sequence identical to at least a portion of said one strand;

(iv) one or more labeled deoxynucleotides comprising a first energy transfer element;

(v) a nucleic acid binding agent comprising one or more energy transfer elements, wherein said nucleic acid binding agent is sequence independent; and

(vi) reagents for carrying out nucleic acid strand extension;

(b) forming a reaction mixture comprising (i), (ii), (iii), (iv), (v) and (vi) above;

(c) contacting under hybridization conditions said first oligonucleotide with one strand of said nucleic acid of interest and contacting under hybridization conditions said second oligonucleotide primer with the complementary strand of said nucleic acid of interest if present;

(d) extending said first oligonucleotide primer and said second oligonucleotide primer to form a first primer-extended nucleic acid sequence and a second primer-extended nucleic acid sequence if the complementary strand is present, thereby incorporating said labeled deoxynucleotide or deoxynucleotides;

(e) binding said nucleic acid binding agent (v) to said first primer-extended nucleic acid sequence, and to said second primer-extended nucleic acid sequence if the complementary strand is present; and

(f) detecting the presence or quantity of said nucleic acid of interest by means of energy transfer between said first energy transfer element and said second energy transfer element.

66. The process of claim 65, wherein in said providing step (a), the nucleic acid binding agent (v) comprises a protein or a polypeptide.

67. The process of claim 66, wherein said protein or polypeptide comprises a T4 gene 32 protein, an SSB protein, a histone or an antibody.

68. The process of claim 65, wherein said providing step (a), the nucleic acid binding agent (v) comprises a single strand nucleic acid binding dye.

69. The process of claim 65, wherein said providing step (a), the nucleic acid binding agent (v) comprises an intercalator.

70. The composition of claim 69, wherein said intercalator comprises SYBR green I, ethidium bromide, a homodimer or a heterodimer that comprises ethidium bromide or derivatives.

71. The process of claim 65, further comprising the steps of:

(b′

) separating said first primer-extended nucleic acid sequence from said nucleic acid of interest and separating said second primer-extended nucleic acid sequence from said complementary strand of said nucleic acid of interest if present;

(c′

) contacting under hybridization conditions said first oligonucleotide primer in said composition (ii) with said second primer-extended nucleic acid sequence from step (b′

), if said second strand is present, and contacting under hybridization conditions said second oligonucleotide primer or nucleic acid construct (iii) with said first primer-extended nucleic acid sequence from step (b′

); and

repeating said extending step (d) and said binding step (e) thereafter.

72. The process of claim 71, further comprising the step (b″

) of separating said first primer-extended nucleic acid sequence from said second primer-extended nucleic acid sequence, and the step (c″

) of contacting under hybridization conditions said first oligonucleotide primer or nucleic acid construct (ii) with said second primer-extended nucleic acid sequence from step (b″

), and contacting under hybridization conditions said second oligonucleotide primer or nucleic acid construct (iii) with said first primer-extended nucleic acid sequence from step (b″

); and

repeating said extending step (d) and said binding step (e) thereafter.

73. The process of claim 72, wherein said separating step (b″

), said contacting step (c″

), said extending step (d) and said binding step (e) are repeated.

74. The process of claim 65, wherein said complementary strand of the nucleic acid of interest is present.

75. The process of claims 65, wherein said nucleic acid of interest (i) has been rendered single stranded before or during said reaction mixture forming step (b).

76. The process of claim 65, wherein said detecting step (f) is carried out at specific intervals during or after any binding step (e).

77. The process of claim 65, wherein in the providing step (a), said reagents (vi) comprise a thermostable DNA polymerase.

78. The process of claims 71, wherein said separating step is carried out by thermal denaturation or strand displacement.

79. The process of claim 65, wherein in said providing step (a), either said first energy transfer element is an energy donor and said second energy transfer element is an energy acceptor, or said first energy transfer element is an energy acceptor and said second energy transfer element is an energy donor.

80. The process of claim 65, wherein in said providing step (a), said first energy transfer element and said second energy transfer element independently comprise fluorescein, fluorescein isothiocyanate (FITC), 6-carboxyfluorescein (6-FAM), naphthofluorescein, rhodamine, rhodamine 6 G, rhodamine X, rhodol, sulforhodamine 101, tetramethylrhodamine (TAMRA), tetramethylrhodamineisothiocyanate (TRITC), 4,7-dichlororhodamine, eosin, eosinisothiocyanate (EITC), dansyl, hydroxycoumarin, methoxycoumarin or p-(Dimethyl aminophenylazo) benzoic acid (DABCYL), cyanine dyes, or a derivatives of any of the foregoing.

81. The process of claim 80, wherein one or both of said first energy transfer element and said second energy transfer element comprise the cyanine dye Cy3 or Cy5.

82. The process of claim 65, wherein at least one of said first oligonucleotide primer, second oligonucleotide primer and any primer-extended nucleic acid sequence is fixed or immobilized to a solid support.

83. The process of claim 82, wherein said solid support comprises beads, tubes, microtiter plates, glass slides, plastic slides, microchip arrays, wells or depressions.

84. The process of claim 82, wherein said fixation or immobilization is direct or indirect.

85. The process of claim 65, wherein any or all of said steps are carried out in a closed container system.

86. The process of claim 85, wherein said closed container system comprises an illumination source and a detection device or unit.

87. A process for detecting qualitatively or quantitatively the presence of a single-stranded or double-stranded nucleic acid of interest in a sample, said process comprising the steps of:

(a) providing

(i) a sample suspected of containing said nucleic acid of interest;

(ii) either (a) a first oligonucleotide primer wherein said first oligonucleotide primer comprises a sequence for a phage RNA promoter or (b) a first oligonucleotide primer and a second oligonucleotide primer, wherein either the first oligonucleotide primer or the second oligonucleotide primer comprises a sequence for a phage RNA promoter;

(iii) one or more labeled ribonucleotides comprising a first energy transfer element;

(iv) a nucleic acid binding agent comprising a second energy transfer element, wherein said nucleic acid binding agent is sequence independent;

(v) reagents for carrying out nucleic acid strand extension of an oigonucleotide primer with deoxynucleotides and reagents for carrying out RNA transcription from a phage RNA promoter with ribonucleotides;

(b) forming a reaction mixture comprising (i), (ii), (iii), (iv) and (v) above;

(c) contacting under hybridization conditions said first oligonucleotide primer with one strand of said nucleic acid of interest;

(d) extending said first oligonucleotide primer to form a primer-extended nucleic acid sequence;

(e) synthesizing a second nucleic acid strand complementary to said primer-extended nucleic acid sequence or a portion thereof, thereby forming a double-stranded nucleic acid that comprises said phage RNA promoter;

(f) transcribing said double-stranded nucleic acid formed in step (e) with a cognate phage RNA polymerase to incorporate said labeled ribonucleotides (iii) into labeled transcripts;

(g) binding said nucleic acid binding agent (iv) to said labeled transcripts;

(h) detecting the presence or quantity of said nucleic acid of interest by means of energy transfer between said first energy transfer element and said second energy transfer element;

wherein said transcribing step (f) is carried out by an RNA promoter in either the first primer-extended nucleic acid sequence or the second nucleic acid strand synthesized in step (e).

88. The process of claim 87, wherein said first oligonucleotide primer (a)(ii)(a) is provided, and wherein in said synthesizing step (e), the second nucleic acid strand is synthesized by primer extension of RNA fragments produced by the action of RNase H.

89. The process of claim 87, wherein said first oligonucleotide primer and said second oligonucleotide primer (a)(ii)(b) are provided, and wherein in said synthesizing step (e), the second nucleic acid strand is synthesized by primer extension of said optional second oligonucleotide primer.

90. The process of claim 87, wherein said first oligonucleotide primer and said second oligonucleotide primer (a)(ii)(b) are provided, and wherein in said synthesizing step (e), the second nucleic acid strand is synthesized by terminal addition or terminal ligation to said first oligonucleotide primer-extended nucleic acid sequence, which terminal addition or ligation is followed by contacting said terminally added or ligated sequences with one or more second oligonucleotide primers complementary to said terminally added or ligated sequences, followed by extension of said second oligonucleotide primers.

91. The process of claim 87, wherein said RNA promoter comprises a T3 promoter, a T7 promoter or an SP6 promoter.

92. The process of claims 87, wherein said nucleic acid of interest (i) has been rendered single stranded before or during said reaction mixture forming step (b).

93. The process of claim 87, wherein said detecting step (h) is carried out one or more times or at specific intervals.

94. The process of claim 87, wherein in said providing step (a), either said first energy transfer element is an energy donor and said second energy transfer element is an energy acceptor, or said first energy transfer element is an energy acceptor and said second energy transfer element is an energy donor.

95. The process of claim 87, wherein in said providing step (a), said first energy transfer element and said second energy transfer element independently comprise fluorescein, fluorescein isothiocyanate (FITC), 6-carboxyfluorescein (6-FAM), naphthofluorescein, rhodamine, rhodamine 6 G, rhodamine X, rhodol, sulforhodamine 101, tetramethylrhodamine (TAMRA), tetramethylrhodamineisothiocyanate (TRITC), 4,7-dichlororhodamine, eosin, eosinisothiocyanate (EITC), dansyl, hydroxycoumarin, methoxycoumarin or p-(Dimethyl aminophenylazo) benzoic acid (DABCYL), cyanine dyes, or derivatives of any of the foregoing.

96. The process of claim 95, wherein one or both of said first energy transfer element and said second energy transfer element comprise the cyanine dye Cy3 or Cy5.

97. The process of claim 87, wherein in said providing step (a), the nucleic acid binding agent (iv) comprises a protein or a polypeptide.

98. The process of claim 97, wherein said protein or polypeptide comprises a T4 gene 32 protein, an SSB protein, a histone or an antibody.

99. The process of claim 87, wherein said providing step (a), the nucleic acid binding agent (iv) comprises a single strand nucleic acid binding dye.

100. The process of claim 87, wherein said transcripts obtained in step (f) are fixed or immobilized to a solid support.

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

102. The process of claim 100, wherein said fixation or immobilization is direct or indirect.

103. The process of claims 87, wherein any or all of said steps are carried out in a closed container system.

104. The process of claim 103, wherein said closed container system comprises an illumination source and a detection device or unit.