Detection of nucleic acid hybrids

• US 6,391,551 B1
• Filed: 08/25/1999
• Issued: 05/21/2002
• Est. Priority Date: 03/13/1998
• Status: Expired due to Fees

• Current Assignee: Promega Corporation
• Original Assignee: Promega Corporation
• Inventors: Wood, Keith V., Olson, Ryan J., Hartnett, James Robert, Gu, Trent, Mandrekar, Michelle, Rhodes, Richard Byron, Lewis, Martin K., Kephart, Daniel, Leippe, Donna, Shultz, John William, Andrews, Christine Ann, Welch, Roy
• Primary Examiner(s): Fredman, Jeffrey
• Assistant Examiner(s): Chunduru, Suryaprabha
• Application Number:
  US09/383,316
• Time in Patent Office: 1,000 Days
• Field of Search: 435/6, 536/24.31, 536/24.32
• US Class Current: 435/6.11
• CPC Class Codes: C12Q 1/04 Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor C12Q1/6897 takes precedence
• CPC Class Codes: C12Q 1/66 involving luciferase
• CPC Class Codes: C12Q 1/6823 Release of bound markers
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 2521/319 Exonuclease
• CPC Class Codes: C12Q 2535/131 Allele specific probes
• CPC Class Codes: C12Q 2537/101 Homogeneous assay format, e.g. one pot reaction
• CPC Class Codes: C12Q 2565/1015 labels being on the same oligonucleotide
• CPC Class Codes: C12Q 2565/301 Pyrophosphate (PPi)
• CPC Class Codes: C12Q 2565/627 being a mass spectrometer

Abstract

Processes are disclosed using the depolymerization of a nucleic acid hybrid to qualitatively and quantitatively analyze for the presence of a predetermined endogenous nucleic acid. Applications of those processes include the detection of single nucleotide polymorphisms, identification of single base changes, speciation, determination of viral load, genotyping, medical marker diagnostics, and the like.

Claims

1. A method for determining the presence or absence of a predetermined endogenous nucleic acid target sequence in a nucleic acid sample that comprises the steps of:

(A) providing a treated sample that may contain said predetermined nucleic acid target sequence hybridized with a nucleic acid probe that includes an identifier nucleotide in the 3′

-terminal region;

(B) admixing the treated sample with a depolymerizing amount of an enzyme whose activity is to release one or more nucleotides from the 3′

-terminus of a hybridized nucleic acid probe to form a treated reaction mixture;

(C) maintaining the treated reaction mixture for a time period sufficient to permit the enzyme to depolymerize hybridized nucleic acid and release identifier nucleotides therefrom; and

(D) analyzing for the presence of released identifier nucleotides to obtain an analytical output, the analytical output indicating the presence or absence of said endogenous nucleic acid target sequence.

2. The method according to claim 1 wherein said identifier nucleotide is a nucleoside triphosphate.

3. The method according to claim 1 wherein said analytical output is obtained by luminescence spectroscopy.

4. The method according to claim 1 wherein said analytical output is obtained by fluorescence spectroscopy.

5. The method according to claim 4 wherein said released identifier nucleotide includes a fluorescent label.

6. The method according to claim 5 wherein said identifier nucleotide is fluorescently labeled after release from said hybrid.

7. The method according to claim 1 wherein said analytical output is obtained by mass spectrometry.

8. The method according to claim 7 wherein said released identifier nucleotide includes a fluorescent label.

9. The method according to claim 7 wherein said identifier nucleotide is fluorescently labeled after release from said hybrid.

10. The method according to claim 1 wherein said analytical output is obtained by absorbance spectroscopy.

11. The method according to claim 1 including the further steps of forming said treated sample by

(a) admixing a sample to be assayed with one or more nucleic acid probes to form a hybridization composition, wherein the 3′ -terminal region of said nucleic acid probes (i) hybridize with partial or total complementarity to said endogenous nucleic acid target sequence when that sequence is present in the sample and (ii) include an identifier nucleotide;

(b) maintaining said hybridization composition for a time period sufficient to form a treated sample that may contain said one predetermined nucleic acid target sequence hybridized with a nucleic acid probe.

12. The method according to claim 1 wherein said nucleic acid sample is obtained from a biological sample.

13. A method for determining the presence or absence of at least one predetermined endogenous nucleic acid target sequence in a nucleic acid sample that comprises the steps of:

(A) admixing a sample to be assayed with one or more nucleic acid probes to form a hybridization composition, wherein the 3′

-terminal region of said nucleic acid probes (i) hybridizes with partial or total complementarity to at least one said predetermined endogenous nucleic acid target sequence when that sequence is present in the sample and (ii) includes an identifier nucleotide;

(B) maintaining said hybridization composition for a time period sufficient to form a treated sample that may contain said predetermined endogenous nucleic acid target sequence hybridized with a nucleic acid probe;

(C) admixing the treated sample with a depolymerizing amount of an enzyme whose activity is to release one or more nucleotides from the 3′

-terminus of a hybridized nucleic acid probe to form a treated reaction mixture;

(D) maintaining the treated reaction mixture for a time period sufficient to permit the enzyme to depolymerize hybridized nucleic acid and release identifier nucleotides therefrom; and

(E) analyzing for the presence of released identifier nucleotides to obtain an analytical output, the analytical output indicating the presence or absence of at least one said endogenous nucleic acid target sequence.

14. The method according to claim 13 wherein said identifier nucleotide is a nucleoside triphosphate.

15. The method according to claim 13 wherein said analytical output is obtained by luminescence spectroscopy.

16. The method according to claim 13 wherein said analytical output is obtained by fluorescence spectroscopy.

17. The method according to claim 16 wherein said released identifier nucleotide includes a fluorescent label.

18. The method according to claim 17 wherein said identifier nucleotide is fluorescently labeled after release from said hybrid.

19. The method according to claim 13 wherein said analytical output is obtained by mass spectrometry.

20. The method according to claim 19 wherein said released identifier nucleotide includes a fluorescent label.

21. The method according to claim 20 wherein said identifier nucleotide is fluorescently labeled after release from said hybrid.

22. The method according to claim 13 wherein said analytical output is obtained by absorbance spectroscopy.

23. The method according to claim 13 wherein said sample contains a plurality of predetermined endogenous nucleic acid target sequences and is admixed with a plurality of said nucleic acid probes.

24. The method according to claim 23 wherein the analytical output obtained when one of said nucleic acid probes hybridizes with partial complementarity to one endogenous target nucleic acid sequence is greater than the analytical output when all of the nucleic acid probes hybridize with total complementarity to their respective nucleic acid target sequences.

25. The method according to claim 23 wherein the analytical output obtained when one of said nucleic acid probes hybridizes with partial complementarity to one endogenous target nucleic acid sequence is less than the analytical output when all of the nucleic acid probes hybridize with total complementarity to their respective nucleic acid target sequences.

26. The method according to claim 23 wherein the analytical output obtained when one of said nucleic acid probes hybridizes with, total complementarity to one endogenous nucleic acid target sequence is greater than the analytical output when all of the nucleic acid probes hybridize with partial complementarity to their respective nucleic acid target sequences.

27. The method according to claim 23 wherein the analytical output obtained when one of said nucleic acid probes hybridize with total complementarity to one endogenous target nucleic acid sequence is less than the analytical output when all of the nucleic acid probes hybridize with partial complementarity to their respective nucleic acid target sequences.

28. The method according to claim 13 wherein said enzyme whose activity is to release nucleotides is a template-dependent polymerase that, in the presence of pyrophosphate ions, depolymerizes hybridized nucleic acids whose bases in the 3′

-terminal region are matched with total complementarity.

29. The method according to claim 13 wherein said enzyme whose activity is to release nucleotides exhibits a 3′

→

5′

-exonuclease activity, depolymerizing hybridized nucleic acids having one or more mismatched bases in the 3′

-terminal region of the hybridized probe.

30. A method for determining the presence or absence of a first endogenous nucleic acid target in a nucleic acid sample containing that target or a substantially identical second target that comprises the steps of:

(A) admixing said sample to be assayed with one or more nucleic acid probes to form a hybridization composition, wherein said first and second endogenous nucleic acid targets comprise a region of sequence identity except for at least a single nucleotide at a predetermined position that differs between the endogenous targets, and wherein said nucleic acid probe (i) is substantially complementary to said nucleic acid target region of sequence identity and comprises at least one nucleotide at an interrogation position, said interrogation position of the probe being aligned with said predetermined position of a target when a target and probe are hybridized and (ii) includes an identifier nucleotide in the 3′

-terminal region;

(B) maintaining said hybridization composition for a time period sufficient to form a treated sample wherein the nucleotide at said interrogation position of said probe is aligned with the nucleotide at said predetermined position of said target in said region of identity;

(C) admixing the treated sample with a depolymerizing amount an enzyme whose activity is to release one or more nucleotides from the 3′

-terminus of a hybridized nucleic acid probe to form a treated reaction mixture;

(D) maintaining the treated reaction mixture for a time period sufficient to release identifier nucleotide and depolymerize said hybridized nucleic acid probe; and

(E) analyzing for the presence of released identifier nucleotide to obtain an analytical output, said analytical output indicating the presence or absence of said nucleotide at said predetermined region and thereby the presence or absence of a first or second nucleic acid target.

31. The method according to claim 30 wherein said analytical output is obtained by fluorescence spectroscopy.

32. The method according to claim 31 wherein said identifier nucleotide is fluorescently labeled after release from said hybrid.

33. The method according to claim 30 wherein said analytical output is obtained by mass spectrometry.

34. The method according to claim 30 wherein said released identifier nucleotide includes a fluorescent label.

35. The method according to claim 30 wherein said identifier nucleotide is a nucleoside triphosphate.

36. The method according to claim 35 wherein said analytical output is obtained by luminescence spectroscopy.

37. The method according to claim 36 wherein said analytical output is obtained by absorbance spectroscopy.

38. The method according to claim 30 wherein said nucleic acid target sequence is selected from the group consisting of deoxyribonucleic acid and ribonucleic acid.

39. The method according to claim 30 further comprising a first probe and a second probe.

40. The method according to claim 39 wherein said sample to be assayed comprises a plurality first nucleic acid targets and second substantially identical nucleic acid targets.

41. The method according to claim 40 wherein said first probe comprises a nucleotide at said interrogation position that is complementary to a first target nucleic acid at said predetermined position, and said second probe comprises a nucleotide at the interrogation position that is complementary to a second target nucleic acid at said predetermined position.

42. The method according to claim 40 wherein the analytical output obtained when one of said nucleic acid probes hybridizes with partial complementarity to one target nucleic acid sequence is greater than the analytical output when all of the nucleic acid probes hybridize with total complementarity to their respective nucleic acid target sequences.

43. The method according to claim 40 wherein the analytical output obtained when one of said nucleic acid probes hybridizes with partial complementarity to one target nucleic acid sequence is less than the analytical output when all of the nucleic acid probes hybridize with total complementarity to their respective nucleic acid target sequences.

44. The method according to claim 40 wherein the analytical output obtained when one of said nucleic acid probes hybridizes with total complementarity to one nucleic acid target sequence is greater than the analytical output when all of the nucleic acid probes hybridize with partial complementarity to their respective nucleic acid target sequences.

45. The method according to claim 40 wherein the analytical output obtained when one of said nucleic acid probes hybridizes with total complementarity to one target nucleic acid sequence is less than the analytical output when all of the nucleic acid probes hybridize with partial complementarity to their respective nucleic acid target sequences.

46. The method according to claim 30 wherein said enzyme whose activity is to release nucleotides is a template-dependent polymerase that, in the presence of pyrophosphate ions, depolymerizes hybridized nucleic acids whose bases in the 3′

-terminal region are matched with total complementarity.

47. The method according to claim 30 wherein said enzyme whose activity is to release nucleotides exhibits a 3′

→

5′

-exonuclease activity, depolymerizing hybridized nucleic acids having one or more mismatched bases in the 3′

-terminal region of the hybridized probe.

48. A one-pot method for determining the presence or absence of at least one predetermined endogenous nucleic acid target sequence in a nucleic acid sample that comprises the steps of:

(A) admixing a treated sample that may contain said predetermined endogenous nucleic acid target sequence hybridized to a nucleic acid probe whose 3′

-terminal region is completely complementary to said predetermined nucleic acid target sequence and includes an identifier nucleotide with (i) a depolymerizing amount of an enzyme whose activity in the presence of pyrophosphate is to release identifier nucleotide as a nucleoside triphosphate from the hybridized nucleic acid probe, (ii) adenosine 5′

diphosphate, (iii) pyrophosphate and (iv) NDPK to form a treated reaction mixture;

(b) maintaining the treated reaction mixture at a temperature of about 25 to about 80 degrees C. for a time period sufficient to permit the enzyme to depolymerize hybridized nucleic acid probe, release an identifier nucleotide in the 3′

-terminal region as a nucleoside triphosphate and to convert said nucleoside triphosphate and said adenosine 5′

diphosphate to adenosine 5′

triphosphate; and

(d) analyzing for the presence of adenosine 5′

triphosphate to obtain an analytical output, the analytical output indicating the presence or absence of at least one said nucleic acid target sequence.

49. The method according to claim 48 wherein said analytical output is obtained by luminescence spectroscopy.

50. The method according to claim 48 including the further steps of forming said treated sample by

(a) admixing a sample to be assayed with one or more nucleic acid probes to form a hybridization composition, wherein the 3′ -terminal region of said nucleic acid probe (i) hybridizes with partial or total complementarity to a nucleic acid target sequence when that sequence is present in the sample and (ii) includes an identifier nucleotide;

(b) maintaining said hybridization composition for a time period sufficient to form a treated sample that may contain said one predetermined nucleic acid target sequence hybridized with a nucleic acid probe.

51. The method according to claim 48 wherein said depolymerizing enzyme maintains activity at 60-90°

C.

52. The method according to claim 48 wherein said depolymerizing enzyme is selected from the group consisting of the Tne triple mutant DNA polymerase, Bst DNA polymerase, Ath DNA polymerase, Taq DNA polymerase and Tvu DNA polymerase.

53. The method according to claim 48 wherein said NDPK is that encoded by Pyrococcus furiosis.

54. A method for determining the presence or absence of a specific base in an endogenous nucleic acid target sequence in a sample to be assayed that comprises the steps of:

(A) admixing a sample to be assayed with one or more nucleic acid probes to form an endogenous hybridization composition, wherein the 3′

-terminal region of at least one of said nucleic acid probes (i) is substantially complementary to said endogenous nucleic acid target sequence and comprises at least one predetermined nucleotide at an interrogation position, and (ii) includes an identifier nucleotide, and wherein said nucleic acid target sequence comprises at least one specific base whose presence or absence is to be determined

(B) maintaining said hybridization composition for a time period sufficient to form a treated sample, wherein said interrogation position of the probe is a nucleotide that is aligned with said specific base to be identified in said endogenous target sequence, when present, so that base pairing can occur;

(C) admixing the treated sample with an enzyme whose activity is to release one or more nucleotides from the 3′

-terminus of a hybridized nucleic acid probe to depolymerize the hybrid and form a treated reaction mixture;

(D) maintaining the treated reaction mixture for a time period sufficient to release an identifier nucleotide therefrom; and

(E) analyzing for the presence or absence of released identifier nucleotide to obtain an analytical output that indicates the presence or absence of said specific base to be identified.

55. The method according to claim 54 wherein the identifier nucleotide is at the interrogation position.

56. The method according to claim 54 wherein said analytical output is obtained by fluorescence spectroscopy.

57. The method according to claim 56 wherein said identifier nucleotide is fluorescently labeled after release from said hybrid.

58. The method according to claim 54 wherein said analytical output is obtained by mass spectrometry.

59. The method according to claim 56 wherein said released identifier nucleotide includes a fluorescent label.

60. The method according to claim 58 wherein said released identifier nucleotide includes a fluorescent label.

61. The method according to claim 54, wherein said nucleic acid target sequence is selected from the group consisting of deoxyribonucleic acid and ribonucleic acid.

62. The method according to claim 61, further comprising a first probe, a second probe, a third probe and a fourth probe.

63. The method according to claim 62, wherein said interrogation position of said first probe comprises a nucleic acid residue that is a deoxyadenosine or adenosine residue, said interrogation position of said second probe comprises a nucleic acid residue that is a deoxythymidine or uridine residue, said interrogation position of said third probe comprises a nucleic acid residue that is a deoxyguanosine or guanosine residue, and said fourth nucleic acid probe comprises a nucleic acid residue that is a deoxycytosine or cytosine residue.

64. The method according to claim 54 wherein said sample to be assayed comprises a plurality of endogenous nucleic acid target sequences in which the presence or absence of a plurality of specific bases is interrogated.

65. The method according to claim 64 wherein the analytical output obtained when one of said exogenous nucleic acid probes hybridizes with partial complementarity to one target nucleic acid sequence is greater than the analytical output when all of the nucleic acid probes hybridize with total complementarity to their respective nucleic acid target sequences.

66. The method according to claim 64 wherein the analytical output obtained when one of said nucleic acid probes hybridizes with partial complementarity to one target endogenous nucleic acid sequence is less than the analytical output when all of the nucleic acid probes hybridize with total complementarity to their respective nucleic acid target sequences.

67. The method according to claim 64 wherein the analytical output obtained when one of said nucleic acid probes hybridizes with total complementarity to one endogenous nucleic acid target sequence is greater than the analytical output when all of the nucleic acid probes hybridize with partial complementarity to their respective nucleic acid target sequences.

68. The method according to claim 64 wherein the analytical output obtained when one of said nucleic acid probes hybridize with total complementarity to one target nucleic acid sequence is less than the analytical output when all of the nucleic acid probes hybridize with partial complementarity to their respective nucleic acid target sequences.

69. The method according to claim 54 wherein said enzyme whose activity is to release nucleotides is a template-dependent polymerase that, in the presence of pyrophosphate ions, depolymerizes hybridized nucleic acids whose bases in the 3′

terminal region of the probe are matched with total complementarity.

70. The method according to claim 54 wherein said enzyme whose activity is to release nucleotides exhibits a 3′

→

5′

-exonuclease activity, depolymerizing hybridized nucleic acids having one or more mismatched bases at the 3′

-terminus of the hybridized probe.

71. A method for determining the number of known sequence repeats present in a nucleic acid target sequence in a nucleic acid sample that comprises the steps of:

(A) providing a plurality of separate treated samples, each treated sample containing a nucleic acid target sequence hybridized with a nucleic acid probe wherein

(a) the nucleic acid target sequence contains (i) a plurality of known sequence repeats and (ii) a non-repeated region downstream of the repeats, and

(b) the nucleic acid probe is one of a plurality of different probes wherein said probes differ in the number of complementary sequence repeats contained therein, each nucleic acid probe containing (i) a plurality of sequence repeats complementary to the known sequence repeat of alleles of the target nucleic acid, (ii) an identifier nucleotide in the 3′

-terminal region of the probe and (iii) a 5′

-terminal locker sequence that is complementary to the non-repeated region of the target and comprises 1 to about 20 nucleotides,

(B) admixing each treated sample with a depolymerizing amount of an enzyme whose activity is to release one or more nucleotides from the 3′

-terminus of a hybridized nucleic acid probe to form a treated depolymerization reaction mixture;

(C) maintaining the treated depolymerization reaction mixtures for a time period sufficient to permit the enzyme to depolymerize hybridized nucleic acid probe and release identifier nucleotide therefrom; and

(D) analyzing the samples for the presence of released identifier nucleotide to obtain an analytical output indicative of the number of sequence repeats present in said nucleic acid target sequence.

72. The method according to claim 71 wherein said nucleic acid sample comprises two nucleic acid target sequences representing alleles and is homozygous with respect to the number of sequence repeats in the two alleles.

73. The method according to claim 71 wherein said nucleic acid sample comprises two nucleic acid target sequences representing alleles and is heterozygous with respect to the number of sequence repeats in the two alleles.

74. The method according to claim 71 wherein said identifier nucleotide is a nucleotide that is part of the repeated sequence.

75. The method according to claim 71 wherein said identifier nucleotide of the probe sequence is complementary to a non-repeating sequence located 3′

to the repeated sequences of the target nucleic acid.

76. The method according to claim 75 wherein said identifier nucleotide is present in a sequence containing 1 to about 20 nucleic acids that is complementary to a non-repeating sequence located 3′

to the repeated sequences of the target nucleic acid.

77. The method according to claim 71 wherein a repeated known sequence present in a nucleic acid target sequence has a length of 2 to about 24 bases per repeat.

78. A method for determining whether the nucleic acid target sequence in a nucleic acid sample is an allele from a homozygous or heterozygous locus that comprises the steps of:

(A) providing a plurality of separate treated samples, each sample containing (a) a nucleic acid target sequence hybridized with (b) a nucleic acid probe, said nucleic acid target sequence being that of a first allele, a second allele or a mixture of said first and second alleles from a locus of interest of said nucleic acid target, said alleles differing in sequence at an interrogation position, said nucleic acid probe containing an identifier nucleotide in the 3′

-terminal region that is aligned at an interrogation nucleotide position of the target sequence when said probe and target are hybridized;

(B) admixing each treated sample with a depolymerizing amount of an enzyme whose activity is to release one or more nucleotides from the 3′

-terminus of a hybridized nucleic acid probe to form a treated reaction mixture;

(C) maintaining the treated reaction mixtures for a time period sufficient to permit the enzyme to depolymerize hybridized nucleic acid probe and an release identifier nucleotide; and

(D) analyzing the samples for the presence of released identifier nucleotide to obtain an analytical output, the analytical output indicating whether the nucleic acid target sequence in a nucleic acid sample is an allele from a homozygous or a heterozygous locus.

79. The method of claim 78 wherein said analyzing comprises analyzing the samples for the quantity of released identifier nucleotide to obtain an analytical output, the analytical output indicating whether the nucleic acid target sequence in the nucleic acid sample is homozygous or heterozygous when compared to the analytical output of an appropriate control.

80. The method according to claim 78 wherein said analytical output indicates which allele is present when the nucleic acid target sequence in a nucleic acid sample is homozygous at the locus of interest.

81. The method according to claim 78 wherein said enzyme whose activity is to release nucleotides is a template-dependent polymerase that, in the presence of pyrophosphate ions, depolymerizes hybridized nucleic acids whose bases in the 3′

-terminal region are completely complementary to bases of said nucleic acid target.

82. The method according to claim 79 wherein said analytical output is obtained by luminescence spectroscopy.

83. A method for determining the loss of heterozygosity of a locus of an allele that comprises the steps of:

(A) providing a plurality of separate treated samples, each sample containing (a) a nucleic acid target sequence hybridized with (b) a nucleic acid probe, said nucleic acid target sequence being that of a first allele or a mixture of said first allele and a second allele of said nucleic acid target, said alleles differing in sequence at an interrogation position, said nucleic acid probe containing a 3′

-terminal region that hybridizes to a region of said nucleic acid target sequence containing said interrogation nucleotide position when said probe and target are hybridized and an identifier nucleotide;

(B) admixing each treated sample with a depolymerizing amount of an enzyme whose activity is to release one or more nucleotides from the 3′

-terminus of a hybridized nucleic acid probe to form a treated reaction mixture;

(C) maintaining the treated reaction mixtures for a time period sufficient to depolymerize hybridized nucleic acid probe and an release identifier nucleotide; and

(D) analyzing the samples for the quantity of released identifier nucleotide to obtain an analytical output, the analytical output indicating whether the nucleic acid target sequence in a nucleic acid sample has lost heterozygosity at the locus of the allele.

84. The method of claim 83 wherein the quantity of said released identifier nucleotide for said first allele is substantially less that the quantity of said released identifier nucleotide for said first allele of a known heterozygous control, and the quantity of said released identifier nucleotide for said second allele is substantially similar to the quantity of said released identifier nucleotide for said second allele of a known heterozygous control, indicating a loss of heterozygosity at the locus of said first allele.

85. The method of claim 83 wherein the quantity of said released identifier nucleotide for said second allele is substantially less that the quantity of said released identifier nucleotide for said second allele of a known heterozygous control, and the quantity of said released identifier nucleotide for said first allele is substantially similar to the quantity of said released identifier nucleotide for said first allele of a known heterozygous control, indicating a loss of heterozygosity at the locus of said second allele.

86. The method according to claim 83 wherein said analytical output is obtained by luminescence spectroscopy.

87. The method according to claim 83 wherein said analytical output is obtained by absorbance spectrometry.

88. The method according to claim 83 wherein said analytical output is obtained by fluorescence spectroscopy.

89. The method according to claim 88 wherein said released identifier nucleotide includes a fluorescent label.

90. The method according to claim 89 wherein said identifier nucleotide is fluorescently labeled after release from said hybrid.

91. The method according to claim 83 wherein said analytical output is obtained by mass spectrometry.

92. The method according to claim 91 wherein said released identifier nucleotide includes a fluorescent label.

93. The method according to claim 92 wherein said identifier nucleotide is fluorescently labeled after release from said hybrid.

94. The method according to claim 83 wherein said enzyme whose activity is to release nucleotides is a template-dependent polymerase that, in the presence of pyrophosphate ions, depolymerizes hybridized nucleic acids whose bases in the 3′

-terminal region are completely complementary to bases of said nucleic acid target.

95. The method according to claim 94 wherein the quantity of said released identifier nucleotide for said first allele is substantially less than the quantity of said released identifier nucleotide for said first and second alleles, indicating a loss of heterozygosity at the locus of said first allele.

96. The method according to claim 94 wherein the quantity of said released identifier nucleotide for said second allele is substantially less than the quantity of said released identifier nucleotide for said first and second alleles, indicating a loss of heterozygosity at the locus of said second allele.

97. A method for determining the presence of trisomy of an allele that comprises the steps of:

(A) providing a plurality of separate treated samples, each sample containing (a) a nucleic acid target sequence hybridized with (b) a nucleic acid probe, said nucleic acid target sequence being that of a first allele, a second allele or a mixture of said first and second alleles of said nucleic acid target, said alleles differing in sequence at an interrogation position, said nucleic acid probe containing a 3′

-terminal region that hybridizes to a region of said nucleic acid target sequence containing said interrogation nucleotide position when said probe and target are hybridized and an identifier nucleotide;

(B) admixing each treated sample with a depolymerizing amount of an enzyme whose activity is to release one or more nucleotides from the 3′

-terminus of a hybridized nucleic acid probe to form a treated reaction mixture;

(C) maintaining the treated reaction mixtures for a time period sufficient to depolymerize hybridized nucleic acid probe and an release identifier nucleotide; and

(D) analyzing the samples for released identifier nucleotide to obtain an analytical output, the quantity of said analytical output relative to an analytical output of a control sample indicating whether a trisomy is present in the nucleic acid target sequence.

98. The method of claim 97 wherein the ratio of the quantity of said released identifier nucleotide for said first and second allele is about 3 to zero, compared to the ratio of the quantity of said released identifier nucleotide for said first and second allele of a known heterozygous control of about 1 to 1, indicating a trisomy at the locus of said first allele.

99. The method of claim 97 wherein the ratio of the quantity of said released identifier nucleotide for said first and second allele is about zero to 3, compared to the ratio of the quantity of said released identifier nucleotide for said first and second allele of a known heterozygous control of about 1 to 1, indicating a trisomy at the locus of said second allele.

100. The method of claim 97 wherein the ratio of the quantity of said released identifier nucleotide for said first and second allele is about 2 to 1, compared to the ratio of the quantity of said released identifier nucleotide for said first and second allele of a known heterozygous control of about 1 to 1, indicating a trisomy having two copies of the locus of said first allele and one copy of the locus of said second allele.

101. The method of claim 97 wherein the ratio of the quantity of said released identifier nucleotide for said first and second allele is about 1 to 2, compared to the ratio of the quantity of said released identifier nucleotide for said first and second allele of a known heterozygous control of about 1 to 1, indicating a trisomy having one copy of the locus of said first allele and two copies of the locus of said second allele.

102. The method according to claim 97 wherein said analytical output is obtained by luminescence spectroscopy.

103. The method according to claim 97 wherein said analytical output is obtained by absorbance spectrometry.

104. The method according to claim 97 wherein said analytical output is obtained by fluorescence spectroscopy.

105. The method according to claim 104 wherein said released identifier nucleotide includes a fluorescent label.

106. The method according to claim 105 wherein said identifier nucleotide is fluorescently labeled after release from said hybrid.

107. The method according to claim 97 wherein said analytical output is obtained by mass spectrometry.

108. The method according to claim 107 wherein said released identifier nucleotide includes a fluorescent label.

109. The method according to claim 108 wherein said identifier nucleotide is fluorescently labeled after release from said hybrid.

110. The method according to claim 97 wherein said enzyme whose activity is to release nucleotides is a template-dependent polymerase that, in the presence of pyrophosphate ions, depolymerizes hybridized nucleic acids whose bases in the 3′

-terminal region are completely complementary to bases of said nucleic acid target.

111. The method according to claim 97 wherein the quantity of said released identifier nucleotide for said first allele is substantially greater than the quantity of said released identifier nucleotide of a homozygous control, indicating that said nucleic acid target sequence has a trisomy.

112. The method according to claim 97 wherein the quantity of said released identifier nucleotide for said second allele is substantially greater than the quantity of said released identifier nucleotide of a homozygous control, indicating that said nucleic acid target sequence has a trisomy.

113. A method for determining the presence or absence of a nucleic acid target sequence containing an interrogation position in a nucleic acid sample that comprises the steps of:

(A) providing a treated sample that contains a nucleic acid sample that may include said nucleic acid target sequence hybridized with a nucleic acid probe that is comprised of three sections, (i) a first section that contains the probe 3′

-terminal about 10 to about 30 nucleotides that are complementary to the nucleic acid target sequence at positions beginning about 1 to about 30 nucleic acids downstream of said interrogation position of the target sequence, (ii) a 5′

-terminal region of about 10 to about 200 nucleic acids in length and having the identical sequence of said nucleic acid target sequence, and (iii) an optional third section that contains zero to about 50 nucleic acids that are not complementary to said nucleic acid sample, and;

(B) extending said nucleic acid probe in a 3′

direction to form a second probe hybridized to the nucleic acid sample as a second hybrid;

(D) denaturing said second hybrid to separate said second probe from said nucleic acid target sequence;

(E) renaturing said aqueous composition to form hairpin structures from said second probe;

(F) admixing the hairpin structure-containing composition with a depolymerizing amount of an enzyme whose activity is to release one or more nucleotides from the 3′

-terminus of a nucleic acid hybrid to form a treated reaction mixture;

(G) maintaining the treated reaction mixture for a time period sufficient to permit the enzyme to depolymerize hybridized nucleic acid and release one or more nucleotides from the 3′

-terminus therefrom; and

(H) analyzing for the presence of released identifier nucleotide to obtain an analytical output, the analytical output indicating the presence or absence of said nucleic acid target sequence.

114. A method for determining the presence or absence of a nucleic acid target sequence, or a specific base within the target sequence, in a nucleic acid sample, that comprises the steps of:

(A) providing a treated sample that contains a nucleic acid sample that may include a nucleic acid target sequence hybridized with a first nucleic acid probe as a first hybrid, said first probe being comprised of at least two sections, a first section containing the probe 3′

-terminal about 10 to about 30 nucleotides that are complementary to the target nucleic acid sequence at a position beginning about 5 to about 30 nucleotides downstream of the target interrogation position, a second section of the first probe containing about 5 to about 30 nucleotides that are a repeat of the target sequence from the interrogation position to about 10 to about 30 nucleotides downstream of the interrogation position that does not hybridize to said first section of the probe, and an optional third section of the probe located between the first and second sections of the probe that is zero to about 50 nucleotides in length and comprises a sequence that does not hybridize to either the first or second section of the probe;

(B) extending the first hybrid in the treated sample at the 3′

-end of the first probe, thereby extending the first probe past the interrogation position and forming an extended first hybrid that includes an interrogation position;

(C) denaturing an aqueous composition of the extended first hybrid to separate the two nucleic acid strands and form an aqueous composition containing a separated target nucleic acid and a separated extended first probe;

(D) annealing the extended first probe to a second probe that is about 10 to about 30 nucleotides in length and is complementary to the extended first probe at a position beginning about 5 to about 2000 nucleotides downstream of the interrogation position in the extended first probe, thereby forming a second hybrid;

(E) extending the second hybrid at the 3′

-end of the second probe until that extension reaches the 5′

-end of the extended first probe, thereby forming a second extended hybrid containing a second extended probe whose 3′

-region includes an identifier nucleotide;

(F) denaturing an aqueous composition of the extended second hybrid to separate the two nucleic acid strands and form an aqueous composition containing separated extended first and second probes;

(G) cooling the aqueous composition to form a hairpin structure from the separated extended second probe to form a hairpin structure-containing composition;

(H) admixing the hairpin structure-containing composition with a depolymerizing amount of an enzyme whose activity is to release one or more nucleotides from the 3′

-terminus of a nucleic acid hybrid to form a treated reaction mixture;

(I) maintaining the reaction mixture for a time period sufficient to release 3′

-terminal region identifier nucleotides; and

(J) analyzing for the presence of released identifier nucleotide to obtain an analytical output, the analytical output indicating the presence or absence of said predetermined nucleic acid target sequence or a specific base within the target sequence.

115. The method according to claim 114 wherein said analytical output is obtained by luminescence spectroscopy.

116. The method according to claim 114 wherein said analytical output is obtained by fluorescence spectroscopy.

117. The method according to claim 116 wherein said released identifier nucleotide includes a fluorescent label.

118. The method according to claim 117 wherein said identifier nucleotide is fluorescently labeled after release from said hybrid.

119. The method according to claim 114 wherein said analytical output is obtained by mass spectrometry.

120. The method according to claim 119 wherein said released identifier nucleotide includes a fluorescent label.

121. The method according to claim 119 wherein said identifier nucleotide is fluorescently labeled after release from said hybrid.

122. The method according to claim 114 wherein said analytical output is obtained by absorbance spectroscopy.