LATE-PCR

• US 20040053254A1
• Filed: 12/17/2002
• Published: 03/18/2004
• Est. Priority Date: 12/19/2001
• Status: Active Grant

• Current Assignee: Brandeis University
• Original Assignee: Brandeis University
• Inventors: Rice, John, Wangh, Lawrence J., Hartshorn, Cristina, Sanchez, J. Aquiles, Pierce, Kenneth
• Granted Patent: US 7,198,897 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6851 Quantitative amplification
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 2527/107 Temperature of melting, i.e. Tm
• CPC Class Codes: C12Q 2531/107 Probe or oligonucleotide ligation
• CPC Class Codes: C12Q 2561/113 Real time assay

Abstract

A non-symmetric polymerase chain reaction (PCR) amplification method employing a limiting primer in low concentration whose concentration-adjusted melting point at least equals, and preferably exceeds, that of the excess primer, the latter in turn not being more than 25° C. below the melting temperature of the amplicon. Assays employing such amplification and labeled hybridization probes, including assays that include a detection step following primer extension or a low-temperature probe, or both. Kits for performing such assays and primer or primer-and-probe sets for performing the foregoing amplifications and assays.

Claims

1. A non-symmetric polymerase chain reaction (PCR) amplification method comprising thermally cycling a PCR reaction mixture containing a deoxyribonucleic acid (DNA) amplification target sequence, a pair of PCR primers, dNTP'"'"'s and a thermostable polymerase repeatedly through PCR steps of strand melting, primer annealing and primer extension, wherein, at the outset of amplification (a) the reaction mixture contains up to 1,000,000 copies of the amplification target sequence, (b) the PCR primer pair comprises a limiting primer and an excess primer, the limiting primer being present at a concentration of up to 200 nM and the excess primer being present at a concentration at least five times higher than the limiting primer, (c) the initial, concentration-adjusted melting temperature of the limiting primer is equal to or greater ii than the initial, concentration-adjusted melting temperature of the excess primer, (d) if the limiting primer is not fully complementary to said target sequences the concentration-adjusted melting temperature of that portion of the limiting primer which hybridizes to said target sequence is not more than 5°

C. below the concentration-adjusted melting temperature of the excess primer, (e) the melting temperature of the amplicon produced by extension of the excess primer exceeds the initial concentration-adjusted melting temperature of the excess primer by not more than 18°

C., and (f) thermal cycling is repeated a number of times sufficient to include multiple cycles of linear amplification using the excess primer following exhaustion of the limiting primer.

2. The method of claim 1 for at least two target sequences wherein the reaction mixture includes a pair of PCR primers for each target, and wherein the initial, concentration-adjusted melting points of all limiting primers are equal to or greater than the initial, concentration-adjusted melting points of all excess primers.

3. The method of claim 1 further comprising reverse transcribing a ribonucleic acid (RNA) molecule to generate said DNA target sequence.

4. The method of claim 1 wherein the reaction mixture contains up to 50,000 copies of the nucleic acid target.

5. The method of claim 1 wherein the initial, concentration-adjusted melting temperature of the limiting primer is at least 3°

C. higher than the initial, concentration-adjusted melting temperature of the excess primer.

6. The method of claim 5 wherein the excess primer is present at a concentration of 500-2000 nM and at least ten times higher than the limiting primer.

7. The method of claim 5 wherein the melting temperature of the amplicon is 7-15°

C. higher than the initial, concentration-adjusted melting temperature of the excess primer.

8. The method of claim 4 wherein the melting temperature of the amplicon is 7-15°

C. higher than the initial, concentration-adjusted melting temperature of the excess primer.

9. The method of claim 4 wherein the initial, concentration-adjusted melting temperature of the limiting primer is at least 3°

C. higher than the initial, concentration-adjusted melting temperature of the excess primer.

10. The method of claim 1 wherein the melting temperature of the amplicon is 7-15°

C. higher than the initial, concentration-adjusted melting temperature of the excess primer.

11. The method of claim 1 wherein the reaction mixture contains up to 1000 copies of the DNA target.

12. The method of claim 11 wherein the initial, concentration-adjusted melting temperature of the limiting primer is at least 3°

C. higher than the initial, concentration-adjusted melting temperature of the excess primer.

13. The method of claim 11 wherein the melting temperature of the amplicon is 7-15°

C. higher than the initial, concentration-adjusted melting temperature of the excess primer.

14. The method of claim 1 wherein the duration of the primer annealing step is not longer than 30 seconds.

15. The method according to claim 1 further including at least one terminal thermal cycle in which the single-stranded extension product of the excess primer is converted to double-stranded product, wherein the PCR reaction mixture additionally includes a low-temperature primer capable of priming the extension product of the excess primer and having an initial, concentration-adjusted melting temperature at least 5°

C. below the initial, concentration-adjusted melting temperature of the excess primer, and wherein the annealing temperature is maintained above the initial, concentration-adjusted melting temperature of the low-temperature primer except for at least one terminal cycle in which the annealing temperature is lowered to hybridize the low-temperature primer.

16. The method according to claim 15 wherein the initial, concentration-adjusted melting temperature of the low-temperature primer is at least 10°

C. below the initial, concentration-adjusted melting point of the excess primer.

17. The method of claim 1 wherein the reaction mixture additionally contains a complementary oligonucleotide that hybridizes to the 3′

end of the excess primer and has an initial, concentration-adjusted melting temperature at least 3°

C. lower than the initial, concentration-adjusted melting temperature of the excess primer.

18. The method of claim 17 wherein the complementary oligonucleotide is present at the outset in a concentration greater than the concentration of the excess primer.

19. A non-symmetric polymerase chain reaction (PCR) method comprising thermally cycling a PCR reaction mixture containing a deoxyribonucleic acid (DNA) amplification target sequence, a pair of matched limiting PCR primers, an additional excess primer, dNTP'"'"'s and a thermostable polymerase repeatedly through PCR steps of strand melting, primer annealing and primer extension, wherein the matched PCR primers are present in approximately equimolar concentration of up to 200 nM, the excess primer is present at a concentration at least five times higher than the limiting primers, the initial, concentration-adjusted melting temperatures of the excess primer is at least 5°

C. below the initial, concentration-adjusted melting temperature of the limiting primers, and wherein the reaction comprises a first phase wherein the annealing temperature is higher than the initial, concentration-adjusted melting temperature of the excess primer and the matched limiting primers generate a first amplicon, and a second phase wherein the annealing temperature is lowered and the excess primer generates a second amplicon, shorter than the first amplicon, utilizing the first amplicon as a template strand, and wherein the melting temperature of the second amplicon exceeds the initial, concentration-adjusted melting temperature of the excess primer by not more than 25°

C.

20. The method according to claim 19, wherein the initial concentration-adjusted melting temperature of the excess primer is at least 10°

C. below the initial, concentration-adjusted melting temperatures of the limiting primers.

21. The method according to claim 20 wherein the melting temperature of the second amplicon exceeds the initial, concentration-adjusted melting temperature of the excess primer by not more than 18°

C.

22. A non-symmetric polymerase chain reaction (PCR) method with removal of single-stranded amplicon comprising

a) thermally cycling a PCR reaction mixture containing a DNA amplification target sequence, a pair of PCR primers for said amplification target sequence, dNTP'"'"'s and a thermostable DNA polymerase through repeated cycles of strand melting, primer annealing and primer extension, wherein (i) the PCR primer pair comprises a limiting primer and an excess primer, (ii) the limiting primer is present at a concentration of up to 200 nM, and the excess primer is present at a concentration at least five times higher than the limiting primer, (iii) the initial, concentration-adjusted melting temperature of the limiting primer is at least equal to the initial, concentration-adjusted melting temperature of the excess primer, and (iv) thermal cycling is repeated a number of times sufficient to include multiple cycles of linear amplification using the excess primer following exhaustion of the limiting primer;

and

b) during at least some cycles of linear amplification, following the step of primer extension, removing single-stranded extension product of the excess primer from the reaction mixture by hybridizing said product to capture probes.

23. The method of claim 22 wherein said capture probes are in a thermally isolated product removal zone and said step of removing comprises passing the reaction mixture through said zone.

24. The method according to claim 23 wherein said product removal zone is physically isolatable from said at least one reaction zone, further including periodically isolating said product removal zone and harvesting product hybridized to said capture probes while the reaction mixture is in said at least one reaction zone.

25. The method of claim 22 wherein the initial, concentration-adjusted melting temperature of the limiting primer is at least 3°

C. higher than the initial, concentration-adjusted melting temperature of the excess primer.

26. The method of claim 22 wherein the melting temperature of the amplicon exceeds the initial concentration-adjusted melting temperature of the excess primer by not more than 18°

C.

27. The method of claim 22 wherein the excess primer is present at a concentration of 500-2000 nM and at least ten times higher than the limiting primer.

28. A homogeneous detection assay for at least one DNA amplification target sequence employing non-symmetric polymerase chain reaction (PCR) amplification, comprising (a) thermally cycling through multiple cycles of PCR steps of strand melting, primer annealing and primer extension a PCR reaction mixture containing said at least one amplification target sequence, a thermostable DNA polymerase, dNTP'"'"'s, and for each amplification target sequence a pair of PCR primers for amplifying said amplification target sequence and at least one labeled hybridization probe that hybridizes to the amplicon produced by said primers and (b) detecting signal produced by said at least one probe as an indication of the presence of said at least one amplification target sequence, wherein (i) each PCR primer pair comprises a limiting primer and an excess primer, (ii) the limiting primer is present at a concentration of up to 200 nM, and for each primer pair the excess primer is present at a concentration of at least five times higher than the limiting primer concentration, (iii) the initial, concentration-adjusted melting temperatures of all limiting primers are at least equal to the initial, concentration-adjusted melting temperatures of all excess primers (iv) for each primer pair the melting temperature of the amplicon exceeds the initial, concentration-adjusted melting temperature of the excess primer by not more than 25°

C., (v) thermal cycling is repeated a number of times sufficient to include multiple cycles of linear amplification using the excess primers following exhaustion of the limiting primers, and (vi) said probes are selected from the group consisting of probes that hybridize to the extension product of the limiting primer, and probes that hybridize to the extension product of the excess primer and signal upon hybridization, and (viii) said probes hybridize to said amplicons during the PCR step of primer annealing.

29. The assay according to claim 28 where said step of detecting is an end-point detection.

30. The assay according to claim 28 wherein said step of detection is real-time detection is performed during the annealing step of at least some cycles of linear amplification.

31. The assay according to claim 28 wherein said step of detection is real-time detection performed following the extension step of at least some cycles of linear amplification and prior to strand melting of the following cycles.

32. The assay according to claim 31 wherein the duration of the primer annealing step is not longer than 30 seconds.

33. The assay according to claim 32 wherein, for each primer pair, the melting temperature of the amplicon exceeds the initial, concentration-adjusted melting temperature of the excess primer by not more than 18°

C.

34. The assay according to claim 32 wherein, for each primer pair, the initial, concentration-adjusted melting temperature of the limiting primer is at least 3°

C. higher than the initial, concentration-adjusting melting temperature of the excess primer.

35. The assay according to claim 32 wherein said probes hybridize to the extension products of the excess primers and signal upon hybridization.

36. The assay according to claim 28 wherein said at least one hybridization probe is a dual-labeled fluorescent probe that hybridizes to the extension product of the limiting primer and that is hydrolyzed by the polymerase during extension of the excess primer, thereby generating a detectable signal, and wherein said signal is detected in real time during at least some cycles of said linear amplification.

37. The assay according to claim 36, wherein the reaction mixture contains up to 50,000 of the at least one amplification target sequence.

38. The assay according to claim 37 wherein, for each primer pair, the melting temperature of the amplicon is not more than 18°

C. higher than the initial, concentration-adjusted melting temperature of the excess primer.

39. The assay according to claim 37 wherein, for each primer pair, the initial, concentration-adjusted melting temperature of the limiting primer is at least 3°

C. higher than the initial, concentration-adjusted melting temperature of the excess primer.

40. The assay according to claim 39 wherein the duration of the primer annealing step is not longer than 30 seconds.

41. The assay according to claim 37, wherein said signal from said at least one hybridization probe is detected between the PCR steps of primer extension and strand melting.

42. The assay according to claim 28 wherein said at least one hybridization probe is a dual-labeled fluorescent probe that hybridizes to the extension product of the limiting primer and that is hydrolyzed by the polymerase during extension of the excess primer, thereby generating a detectable signal.

43. The assay according to claim 28 wherein said at least one hybridization probe emits a detectable signal upon hybridization to the extension product of the excess primer.

44. The assay according to claim 43 wherein, for at least one PCR primer pair, the reaction mixture additionally contains a complementary oligonucleotide that hybridizes to the 3′

end of the excess primer and has an initial, concentration-adjusted melting temperature at least 3°

C. lower than the initial, concentration-adjusted melting temperature of the excess primer.

45. The assay according to claim 44 wherein the complementary oligonucleotide is present at the outset in a concentration greater than the concentration of the excess primer.

46. The assay according to claim 28 wherein said at least one hybridization probe comprises a first probe for one allelic variant and a second probe for another allelic variant.

47. The assay according to claim 28 further comprising reverse transcribing of ribonucleic acid (RNA) molecule to generate said DNA target sequence.

48. The assay according to claim 28 wherein the reaction mixture contains up to 50,000 copies of the nucleic acid target.

49. The assay according to claim 28 wherein the initial, concentration-adjusted melting temperature of the limiting primer is at least 3°

C. higher than the in initial, concentration-adjusted melting temperature of the excess primer.

50. The assay according to claim 28 wherein the melting temperature of the amplicon is not more than 18°

C. higher than the initial, concentration-adjusted melting temperature of the excess primer.

51. The assay according to claim 28 wherein said at least one amplification target sequence comprises at least two amplification target sequences.

52. A homogeneous detection assay for at least one DNA amplification target sequence employing non-symmetric polymerase chain reaction (PCR) amplification, comprising (a) thermally cycling through multiple cycles of PCR steps of strand melting, primer annealing and primer extension a PCR reaction mixture containing said at least one amplification target sequence, a thermostable DNA polymerase, dNTP'"'"'s, and for each amplification target sequence a pair of PCR primers for amplifying said amplification target sequence and at least one labeled low-temperature hybridization probe that hybridizes to the amplicon produced by said primers and (b) detecting signal produced by said at least one probe as an indication of the presence of said at least one amplification target sequence, wherein (i) each PCR primer pair comprises a limiting primer and an excess primer, (ii) each limiting primer is present at a concentration of up to 200 nM, and for each primer pair the excess primer is present at a concentration at least five times higher than the limiting primer concentration, (iii) for each amplification target sequence, the low-temperature hybridization probe binds to the extension product of the excess primer and emits a detectable signal upon hybridization, (iv) for each amplification target sequence the initial, concentration-adjusted melting temperature of the low-temperature hybridization probe is at least 5°

C. below the initial, concentration-adjusted melting temperature of the limiting primer, (v) thermal cycling is repeated a number of times sufficient to include multiple cycles of linear amplification using the excess primers following exhaustion of the limiting primers, and (vi) detection is performed at a temperature sufficiently low for said low-temperature probes to hybridize and signal.

53. The assay according to claim 52, wherein the initial, concentration-adjusted melting temperature of the low-temperature hybridization probe is at least 10°

C. below the initial, concentration-adjusted melting temperature of the limiting primer.

54. The assay according to claim 52 wherein each probe is present in said reaction mixture at a concentration of at least 1 uM.

55. The assay according to claim 52 wherein the PCR step of primer annealing during at least some cycles of linear amplification is of sufficiently low temperature and of sufficient duration that the low-temperature probe hybridizes during primer annealing, and detection is performed during that step.

56. The assay according to claim 52 wherein the initial, concentration-adjusted melting temperatures of all limiting primers are at least equal to the initial, concentration-adjusted melting temperatures of all excess primers.

57. The assay according to claim 52 wherein the PCR amplification includes an added detection step following primer extension during at least some cycles of linear amplification, said detection step being of sufficiently low temperature and sufficient duration for the low-temperature hybridization probes to hybridize and signal, and wherein the PCR step of primer annealing is not of sufficiently low temperature and sufficient duration for said probes to hybridize and signal.

58. The assay according to claim 57, wherein the initial, concentration-adjusted melting temperature of the low-temperature hybridization probe is at least 10°

C. below the initial, concentration-adjusted melting temperature of the limiting primer

59. The assay according to claim 57, wherein the initial, concentration-adjusted melting temperature of the low-temperature hybridization probe is at least 5°

C. below the mean temperature of the annealing step of the exponential phase of the amplification reaction.

60. The assay according to claim 59, wherein the initial, concentration-adjusted melting temperature of the low-temperature hybridization probe is at least 10°

C. below the mean temperature of the annealing step of the exponential phase of the amplification reaction.

61. The assay according to claim 57, wherein the detection step is performed only beginning a few cycles prior to the threshold cycle of the reaction.

62. The assay according to claim 57, wherein the low-temperature detection step is not more than 30 seconds duration.

63. The assay according to claim 57, wherein detection is end-point detection performed following completion of the amplification reaction.

64. The assay according to claim 52, wherein for each primer pair the melting temperature of the amplicon exceeds the initial, concentration-adjusted melting temperature of the excess primer by not more than 25°

C.

65. A method for amplification of a nucleic acid target sequence present in a sample containing up to about 10,000 copies of said target sequence, the method comprising:

a) contacting the nucleic acid target sequence with a first oligonucleotide primer and a second oligonucleotide primer, wherein the T_m of the first primer is at least 5°

C. greater than the T_m of the second primer and wherein the concentration of the second primer is up to 1000 nM and at least about 10 times greater than the concentration of the first primer; and

b) amplifying the target sequence by a polymerase chain reaction utilizing said first and second oligonucleotide primers, said reaction having an exponential phase of amplicon generation followed by a linear phase of amplicon generation that utilizes only the second primer.

66. The method according to claim 65 wherein said sample contains nucleic acid from a single cell.

67. The method according to claim 65 wherein the T_m of the first primer is 10°

C.-20°

C. greater than the T_m of the second primer.

68. The method according to claim 67 wherein the concentration of the second primer is 20-100 times greater than the concentration of the first primer.

69. The method according to claim 65 wherein the concentration of the second primer is 20-100 times greater than the concentration of the first primer.

70. A method for detecting at least one nucleic acid target sequence in a sample containing up to about 10,000 copies of said at least one target sequence, the method comprising:

a) contacting the at least one nucleic acid target sequence with a first oligonucleotide primer hybridizable thereto and a second oligonucleotide primer hybridizable thereto, wherein the T_m of the first primer is at least 5°

C. greater than the T_m of the second primer and wherein the concentration of the second primer is up to 1000 nM and at least about 10 times greater than the concentration of the second primer;

b) amplifying the at least one target sequence by a polymerase chain reaction utilizing said first and second oligonucleotide primers, said reaction having an exponential phase of amplicon generation followed by a linear phase of amplicon generation that utilizes only the ii second primer; and

c) detecting amplicon generated from said second primer in real time during the polymerase chain reaction by means of a first hybridization probe targeted thereto.

71. The method according to claim 70 wherein said at least one nucleic acid sequence comprises a genetic sequence subject to allelic mutation wherein said first hybridization probe is targeted to a first allelic variant.

72. The method according to claim 71 further comprising detecting amplicon generated from said second primer in real time during the polymerase chain reaction by means of a second hybridization probe targeted to a second allelic variant.

73. The method according to claim 71 wherein the T_m of the first primer is 10°

C.-20°

C. greater than the T_m of the second primer.

74. The method according to claim 71 wherein the T_m of the first primer is 10°

C.-20°

C. greater than the T_m of the second primer.

75. The method according to claim 71 wherein the concentration of the second primer is 20-100 times the concentration of the first primer.

76. The method according to claim 71 wherein the concentration of the second primer is 20-100 times the concentration of the first primer.

77. The method according to claim 70 wherein said at least one nucleic acid sequence comprises at least two different nucleic acid sequences, and wherein the method comprises contacting each nucleic acid sequence with a first primer hybridizable thereto and a second primer hybridizable thereto.

78. The method according to claim 70 wherein the concentration of the second primer is 20-100 times the concentration of the first primer.

79. The method according to claim 70 wherein the T_m of the first primer is 10°

C.-20°

C. greater than the T_m of the second primer.

80. The method according to claim 79 wherein the concentration of the second primer is 20-100 times the concentration of the first primer.

81. The method according to claim 70, wherein detection is performed between the PCR steps of primer extension and strand melting.

82. The method according to claim 81, wherein the detection is performed at a temperature below the primer extension temperature.

83. The method according to claim 81 wherein said first hybridization probe is a molecular beacon probe.

84. An oligonucleotide set comprising a pair of primers for amplifying a selected DNA sequence by a non-symmetric polymerase chain reaction (PCR) comprising a first amount of a limiting primer intended to be used at a concentration of not more than 200 nM and a second amount, at least five times greater than said first amount, of an excess primer intended to be used at a concentration at least five times greater than the limiting primer concentration, wherein the intended, concentration-adjusted melting point of the limiting primer is equal to or greater than the intended concentration-adjusted melting point of the excess primer, and wherein the melting point of the amplicon produced by said primer pair does not exceed the intended concentration-adjusted melting point of the excess primer by more than 18°

C.

85. The oligonucleotide set according to claim 84 wherein the intended concentration-adjusted melting point of the limiting primer is at least 3°

C. higher than the intended concentration-adjusted melting point of the excess primer.

86. The oligonucleotide set according to claim 84 wherein said second amount is at least ten times greater than said first amount and wherein said excess primer is intended to be used at a concentration at least ten times greater than the limiting primer concentration.

87. The oligonucleotide set according to claim 86 wherein the melting point of the amplicon exceeds the intended concentration-adjusted melting point of the excess primer by 7-15°

C.

88. The oligonucleotide set according to claim 85 wherein the melting point of the amplicon exceeds the intended concentration-adjusted melting point of the excess primer by 7-15°

C.

89. The oligonucleotide set according to claim 84 wherein the melting point of the amplicon exceeds the intended concentration-adjusted melting point of the excess primer by 7-15°

C.

90. An oligonucleotide set for amplifying a selected DNA sequence by a polymerase chain reaction (PCR) comprising a pair of matched PCR limiting primers for producing a first amplicon and an excess primer for producing a second, single-stranded amplicon utilizing said first amplicon as a template, wherein (i) each of said limiting primers is present in a first amount and is intended to be used at a concentration not exceeding 200 nM, (i) said excess primer is present in a second amount, at least five times greater than said first amount, and is intended to be used at a concentration at least five times higher than the concentration of each limiting primer, and (iii) wherein the intended concentration-adjusted melting temperature of the excess primer is at least 5°

C. lower than the intended concentration-adjusted melting temperature of both limiting primers.

91. The oligonucleotide set according to claim 90 wherein the melting temperature of the second amplicon exceeds the intended concentration-adjusted melting temperature of the excess primer by not more than 18°

C.

92. The oligonucleotide set according to claim 90 wherein said second amount is at least ten times greater than said first amount and wherein said excess primer is intended to be used at a concentration at least ten times greater than the limiting primer concentration.

93. An oligonucleotide set for a homogenous, non-symmetric polymerase chain reaction (PCR) amplification assay for a selected DNA sequence comprising a first amount of a limiting primer intended to be used at a concentration not exceeding 200 nM, a second amount, at least five times greater than said first amount, of an excess primer intended to be used at a concentration at least five times greater than the limiting primer concentration, and a labeled hybridization probe that emits a signal indicative of the presence of product produced by extension of said excess primer, wherein the intended concentration-adjusted melting temperature of the limiting primer is at least equal to the intended concentration-adjusted melting temperature of the excess primer, and wherein the melting point of the amplicon exceeds the intended concentration-adjusted melting point of the excess primer by not more than 25°

C.

94. The oligonucleotide set according to claim 93 wherein the melting temperature of the amplicon exceeds the intended concentration-adjusted melting temperature of the excess primer by not more than 18°

C.

95. The oligonucleotide set according to claim 93, wherein the intended concentration-adjusted melting temperature of the limiting primer exceeds the intended concentration-adjusted melting temperature of the excess primer by at least 3°

C.

96. The oligonucleotide set according to claim 93, wherein said second amount is at least ten times greater than said first amount and wherein said excess primer is intended to be used at a concentration at least ten times greater than the limiting primer concentration.

97. The oligonucleotide set according to claim 93, wherein said probe hybridizes to the extension product of the excess primer and signals upon hybridization.

98. The oligonucleotide set according to claim 97, wherein said probe comprises a first hybridization probe specific for a first allele and a second hybridization probe specific for a second allele.

99. The oligonucleotide set according to claim 97, wherein said hybridization probe is a molecular beacon probe.

100. The oligonucleotide set according to claim 97, wherein said probe is intended to be used at an intended probe concentration and wherein the intended probe concentration-adjusted melting point of the probe is at least 5°

C. below the intended concentration-adjusted melting point of the limiting primer.

101. The oligonucleotide set according to claim 100, wherein the intended probe concentration-adjusted melting point of the probe is at least 10°

C. below the intended concentration-adjusted melting point of the limiting primer.

102. The oligonucleotide set according to claim 101, wherein said probe is a molecular beacon probe.

103. A kit of reagents for performing a homogeneous polymerase chain reaction assay for at least one pre-selected DNA target sequence, comprising at least one pair of polymerase chain reaction primers including a first primer and a second primer, four deoxyribonucleotide triphosphates, a thermostable DNA polymerase, and a labeled hybridization probe that emits a detectable signal upon hybridization, wherein

a) the T_m of the first primer is at least 5°

C. greater than the T_m of the second primer and the concentration of the second primer is at least ten times greater than the concentration of the first primer, and

b) said labeled hybridization probe binds to the extension product of said second primer.

104. The kit according to claim 103 wherein the T_m of the first primer is 10°

C.-20°

C. greater than the T_m of the second primer.

105. The kit according to claim 104 wherein the concentration of the second primer is 20-100 times greater than the concentration of the first primer.

106. The kit according to claim 103 wherein the concentration of the second primer is 20-100 times greater than the concentration of the first primer.

107. A kit of reagents for performing a homogenous polymerase chain reaction (PCR) assay for at least one pre-selected DNA target sequence comprising a thermostable DNA polymerase, dNTP'"'"'s and, for each target sequence, a first amount of a limiting primer to be used at a concentration of up to 200 nM, a second amount, at least five times greater than said first amount, of an excess primer to be used at a concentration at least give times greater than the limiting primer concentration, and a labeled hybridization probe that emits a signal indicative of the presence of product produced by extension of said excess primer, wherein the initial, concentration-adjusted melting temperature of the limiting primer is at least equal to the initial concentration-adjusted melting temperature of the excess primer, and wherein the melting temperature of the amplicon exceeds the initial concentration-adjusted melting temperature of the excess primer by not more than 25°

C.

108. The kit according to claim 107 for performing a multiplex assay for at least two target sequences, wherein the initial, concentration-adjusted melting temperatures of all limiting primers are at least equal to the initial concentration-adjusted melting temperatures of all excess primers.

109. The kit according to claim 107, wherein the initial concentration-adjusted melting temperature of the limiting primer is at least 3°

C. higher than the initial, concentration-adjusted melting temperature of the excess primer.

110. The kit according to claim 107, wherein said probe hybridizes to the extension product of the excess primer and signals upon hybridization.

111. The kit according to claim 110, wherein said probe for said at least one target comprises a first probe for one allelic variant and a second probe for a second allelic variant.

112. The kit according to claim 110 for performing a multiplex assay for at least two target sequences, wherein the initial, concentration-adjusted melting temperatures of all limiting primers are at least equal to the initial concentration-adjusted melting temperatures of all excess primers.

113. The kit according of claim 110, wherein the initial, concentration-adjusted melting temperature of said limiting primer is at least 3°

C. higher than the initial, concentration-adjusted melting temperature of said excess primer.

114. The kit according to claim 110 wherein the initial, concentration-adjusted melting temperature of said probe is at least 5°

C. lower than the initial, concentration-adjusted melting temperature of said limiting primer.

115. The kit according to claim 114 wherein the initial, concentration-adjusted melting temperature of said probe is at least 10°

C. lower than the initial, concentration-adjusted melting temperature of said limiting primer.

116. The kit according to claim 110 further comprising directions specifying a detection step following primer annealing for at least some PCR cycles and specifying an annealing temperature or temperatures to be used for annealing the limiting primer, wherein the initial, concentration-adjusted melting temperature of said probe is at least 5°

C. below the mean of said annealing temperature or temperatures.

117. The kit according to claim 116, wherein the initial, concentration-adjusted melting temperature of said probe is at least 10°

C. below the mean of said annealing temperature or temperatures.

118. The kit according to claim 116, wherein the melting temperature of said amplicon exceeds the initial, concentration-adjusted melting temperature of the excess primer by not more than 18°

C.

119. The kit according to claim 118, wherein the concentration-adjusted melting temperature of said amplicon exceeds the initial, concentration-adjusted melting temperature of the excess primer by 7-15°

C.

120. A kit of reagents for performing a homogenous polymerase chain reaction (PCR) assay for at least one pre-selected DNA target sequence comprising a thermostable DNA polymerase, dNTP'"'"'s and, for each target sequence, a pair of matched PCR limiting primers for producing a first amplicon, an excess primer for producing a second, single-stranded amplicon utilizing said first amplicon as a template, and a labeled hybridization probe that emits a signal indicative of the presence of said second amplicon, wherein said limiting primers are to be used in a first, equimolar, amount at a concentration of up to 200 nM, said excess primer is to be used in a second amount, at least five times greater than said first amount, and at a concentration at least five times greater than the concentration of the limiting primers, and wherein the initial, concentration-adjusted melting temperature of the excess primer is at least 5°

C. lower than the ii initial, concentration-adjusted melting temperatures of the limiting primers.

121. The kit according to claim 120 wherein the melting temperature of the second amplicon exceeds the initial, concentration-adjusted melting temperature of the excess primer by not more than 25°

C.

122. The kit according to claim 120 wherein said second amount is at least ten times greater than said first amount and wherein said excess primer is to be used at a concentration at least ten times greater than the limiting primers concentration.