Detection of nucleic acid hybrids
First Claim
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1. A method for determining the presence or absence of a predetermined 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 nucleic acid target sequence.
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Abstract
Processes are disclosed using the depolymerization of a nucleic acid hybrid to qualitatively and quantitatively analyze for the presence of a predetermined 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.
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Citations
186 Claims
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1. A method for determining the presence or absence of a predetermined nucleic acid target sequence in a nucleic acid sample that comprises the steps of:
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(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 nucleic acid target sequence. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20)
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21. A method for determining the presence or absence of at least one predetermined nucleic acid target sequence in a nucleic acid sample that comprises the steps of:
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(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 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 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 nucleic acid target sequence. - View Dependent Claims (22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37)
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38. A method for determining the presence or absence of a specific base in a nucleic acid target sequence in a sample to be assayed that comprises the steps of:
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(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 at least one of said nucleic acid probes (i) is substantially complementary to said 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 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. - View Dependent Claims (39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54)
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55. A method for determining the presence or absence of a first nucleic acid target in a nucleic acid sample containing that target or a substantially identical second target that comprises the steps of:
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(A) admixing said sample to be assayed with one or more nucleic acid probes to form a hybridization composition, wherein said first and second nucleic acid targets comprise a region of sequence identity except for at least a single nucleotide at a predetermined position that differs between the 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. - View Dependent Claims (56, 57, 58, 59, 60, 61, 62, 63, 64, 71, 72)
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65. The method according to claim 65 wherein said sample to be assayed comprises a plurality first nucleic acid targets and second substantially identical nucleic acid targets.
- 66. The method according to claim 66 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.
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73. A method for selectively detecting a poly(A)+ RNA that comprises the steps of:
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(A) admixing a sample to be assayed with an oligo(dT) probe to form a hybridization composition, wherein said oligo(dT) probe 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 said poly(A)+ RNA hybridizes to said oligo(dT) probe;
(C) admixing the treated sample with an enzyme whose activity is to release of one or more nucleotides from the 3′
-terminus of a nucleic acid hybrid, including the identifier nucleotide, to form a treated reaction mixture;
(D) maintaining the treated reaction mixture for a time period sufficient to release identifier nucleotides therefrom and depolymerize 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 of said poly(A)+ RNA.
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- 74. The method according to claim 74 wherein said identifier nucleotide is a nucleoside triphosphate.
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83. 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:
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(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.
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- 84. The method according to claim 84 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.
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88. The method according to claim 88 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.
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90. A one-pot method for determining the presence or absence of at least one predetermined nucleic acid target sequence in a nucleic acid sample that comprises the steps of:
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(A) admixing a treated sample that may contain said predetermined 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.
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- 91. The method according to claim 91 wherein said analytical output is obtained by luminescence spectroscopy.
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96. A method for enhancing the discrimination of analytical output in the determination of the presence or absence of a predetermined target nucleic acid sequence in a nucleic acid sample that comprises the steps of:
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(A) providing a plurality of separate treated samples, each sample containing (a) a nucleic acid that may contain said predetermined nucleic acid target sequence, said nucleic acid target sequence being hybridized when present with (b) a nucleic acid probe, a first probe of a first treated sample comprising (i) a 3′
-terminal region sequence that is complementary to said nucleic acid target sequence and includes an identifier nucleotide that is complementary to a first predetermined nucleotide of said nucleic acid target sequence and (ii) a second sequence otherwise complementary to said nucleic acid target sequence except for a second predetermined nucleotide located 2 to about 10 nucleotides upstream from the 3′
-terminus of said probe that is not complementary to a second nucleotide of said nucleic acid target sequence, and a second probe of a second treated sample comprising (i) a 3′
-terminal sequence that is complementary to said nucleic acid target sequence except for an identifier nucleotide that is not complementary to said first-named predetermined nucleotide of said nucleic acid target sequence and (ii) a second sequence otherwise complementary to said nucleic acid target sequence except for said second predetermined nucleotide located 3 to about 10 nucleotides upstream from the 3′
-terminus of said probe that is not complementary to a second predetermined nucleotide of said nucleic acid target sequence;
(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 release an identifier nucleotide; and
(D) analyzing the samples for the presence of released identifier nucleotide to obtain an analytical output, the ratio of the analytical output from the sample containing the first probe relative to that of the second probe being enhanced compared to the ratio of the analytical output from a similar sample containing a third probe of the same length having the same identifier nucleotide and total complementarity to said nucleic acid target sequence relative to that of a fourth probe of the same length whose identifier nucleotide is non-complementary to said first predetermined nucleotide and is otherwise totally complementary to said target nucleic acid sequence.
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97. 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:
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(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.
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- 98. The method of claim 98 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.
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102. 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:
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(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.
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103. A method for determining the presence or absence of a RNA target sequence in a nucleic acid sample that comprises the steps of:
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(A) providing a treated sample that may contain said predetermined RNA 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 probe and release identifier nucleotide therefrom;
(D) forming a reaction mixture by admixture of a second nucleic acid probe that hybridizes with said first-named probe, said second probe containing a 3′
-terminal region identifier nucleotide that can be released by the enzyme of step (B) but is not released unless one or more nucleotides have been released from the 3′
-terminus of said first-named probe;
(E) maintaining the reaction mixture for a time period sufficient to permit the enzyme to depolymerize hybridized nucleic acid from said second nucleic acid probe and release identifier nucleotide; and
(F) analyzing for the presence of released identifier nucleotide to obtain an analytical output, the analytical output indicating the presence or absence of said RNA target sequence.
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104. The method according to claim 104 wherein said second nucleic acid probe is admixed with said treated sample and steps (D) and (E) are carried out together with steps (A) and (B).
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105. A method for determining the presence or absence of a restriction endonuclease recognition sequence in a nucleic acid sample that comprises the steps of:
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(A) providing a treated sample that may contain a hybridized nucleic acid target that is a cleaved restriction endonuclease recognition sequence that includes an identifier nucleotide in the restriction endonuclease recognition sequence;
(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 restriction endonuclease recognition sequence 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 nucleotide therefrom; and
(D) analyzing for the presence of released identifier nucleotide to obtain an analytical output, the analytical output indicating the presence or absence of said restriction endonuclease recognition sequence.
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106. The process of claim 106 including the further steps of forming a treated sample by:
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(A) providing an endonuclease cleavage reaction solution comprising a nucleic acid sample and a restriction endonuclease enzyme specific for the restriction endonuclease recognition sequence; and
(B) maintaining the endonuclease cleavage reaction solution for a time period sufficient for the restriction endonuclease enzyme to cleave the restriction endonuclease recognition sequence to form a treated sample.
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107. The process of claim 107 including the further step of amplifying a nucleic acid target sequence in a nucleic acid sample prior to providing said restriction endonuclease recognition sequence.
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108. The process of claim 108 wherein said nucleic acid target sequence is amplified by the further steps of:
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(A) admixing a crude nucleic acid sample with PCR amplification primers that are complementary to regions upstream and downstream of the nucleic acid target sequence and a template-dependent polymerase to form an amplification sample mixture wherein either the nucleic acid target sequence or the PCR amplification primers includes a restriction endonuclease recognition sequence;
(B) maintaining the amplification sample mixture for a time period sufficient to denature the nucleic acid target sequence to form a denatured amplification reaction mixture;
(C) annealing the denatured amplification reaction mixture for a time period sufficient for PCR amplification primers to anneal to the nucleic acid target sequence to form an amplification reaction mixture; and
(D) maintaining the amplification reaction mixture for a time period sufficient to permit the template-dependent polymerase to extend the nucleic acid from the PCR primers to form an amplified nucleic acid sample.
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109. A method for determining the loss of heterozygosity of a locus of an allele that comprises the steps of:
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(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. - View Dependent Claims (110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 140, 141, 143, 144, 146, 147, 149, 150, 152, 154, 155, 157, 158, 159, 160, 161, 162, 163, 166, 167, 168, 169, 170, 171, 172, 173, 175)
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123. A method for determining the presence of trisomy of an allele that comprises the steps of:
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(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.
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- 139. An isolated and purified nucleotide diphosphate kinase (NDPK) enzyme that exhibits higher NDPK activity at a temperature of about 50 to about 90 degrees C. relative to the NDPK activity at 37 degrees C.
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142. 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:
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(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 to the extended first probe 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. - View Dependent Claims (148)
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151. A process to determine the presence or absence of a predetermined single-stranded nucleic acid target sequence comprising the steps of:
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(A) providing a depolymerization reaction mixture comprising (i) a pair of first and second complementary nucleic acid probes that form 3′
-overhangs on both ends of the duplex formed when each of said pair of complementary nucleic acid probes is hybridized with the other, the first of said probes being complementary to the nucleic acid target sequence, (ii) a hybrid between a third probe and the nucleic acid target sequence when the nucleic acid target sequence is present in the nucleic acid sample, and (iii) a depolymerizing amount of an enzyme whose activity is to release nucleotides from the 3′
-terminus of a hybridized nucleic acid, wherein each of said first and third probes has an identifier nucleotide in its 3′
-terminal region;
(B) maintaining the depolymerization reaction mixture for a time period sufficient to permit the enzyme to depolymerize the 3′
-terminal region of said hybridized third probe to release identifier nucleotide and form a first treated reaction mixture;
(C) denaturing the products of the first treated reaction mixture to form a denatured treated reaction mixture;
(D) maintaining the denatured treated reaction mixture for a time period sufficient to form a second depolymerization reaction mixture that comprises (i) a hybrid formed between said first probe and said nucleic acid target sequence when the nucleic acid target sequence is present in the nucleic acid sample and (ii) a hybrid formed between the 3′
-terminal-depolymerized third probe and said second nucleic acid probe, one end of said hybrid having a blunt end or a 5′
-overhang as well as an identifier nucleotide in the 3′
-terminal region;
(E) depolymerizing hybrids (i) and (ii) of step(D) to release identifier nucleotide from the 3′
-terminal regions of said hybrids to form a second treated reaction mixture; and
(F) analyzing said second treated reaction mixture 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.
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153. A process to determine the presence or absence of a predetermined double-stranded nucleic acid target sequence comprising the following steps:
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(A) providing a first reaction mixture comprising (i) first and second complementary nucleic acid probes that form 3′
-overhangs on both ends of the duplex formed when each of said complementary nucleic acid probes is hybridized with the other, wherein said each of probes is complementary to one or the other strand of the nucleic acid target sequence and has an identifier nucleotide in its 3′
-terminal region, (ii) hybrids between a third and fourth probe and each of the two strands of the nucleic acid target sequence when the nucleic acid target sequence is present in the nucleic acid sample, said third and fourth probes each having an identifier nucleotide in its 3′
-terminal region and (iii) a depolymerizing amount of an enzyme whose activity is to release nucleotides from the 3′
-terminus of a hybridized nucleic acid;
(B) maintaining the first reaction mixture for a time period sufficient to permit the enzyme to depolymerize hybridized nucleic acid to release identifier nucleotide from the 3′
-terminal region of said hybridized third and fourth probes and form a treated first reaction mixture;
(C) denaturing the products of the treated first reaction mixture to form a denatured treated reaction mixture;
(D) maintaining the denatured treated reaction mixture for a time period sufficient to form a second reaction mixture that comprises a (i) hybrids that lack a 3′
-overhang between each of the strands of the target nucleic acid and each of the first and second probes when the nucleic acid target sequence is present in the nucleic acid sample, and (ii) hybrids between each of the first and seconds probes and 3′
-terminal region-depolymerized third and fourth probes, wherein each of said hybrids comprises one end that is blunt or has a 5′
-overhang as well as an identifier nucleotide in the 3′
-terminal region; and
(E) depolymerizing the hybrids (i) and (ii) of step (D) to release identifier nucleotide from the 3′
-terminus of said hybridized probes to form a second treated reaction mixture; and
(F) analyzing said second treated reaction mixture 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. - View Dependent Claims (164)
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156. An amplification and interrogation process to determine the presence or absence of a predetermined nucleic acid target sequence comprising the steps of:
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(A) providing a ligation reaction solution comprising (i) ligating amount of a ligase, (ii) a nucleic acid sample that may contain a predetermined nucleic acid target sequence wherein the nucleic acid target sequence has a 3′
-portion and a 5′
-portion, (iii) an open circle probe comprising three regions;
an open circle probe 3′
-terminal region, a linker region, and an open circle probe 5′
-terminal region, said open circle probe further including a detection primer target and an amplification primer target, the amplification primer target being downstream of the detection primer target,wherein upon hybridization between the open circle probe and the nucleic acid target sequence, the open circle probe 3′
-terminal region is complementary to a sequence of the 3′
-portion of said predetermined nucleic acid target sequence, and the open circle probe 5′
-terminal region is complementary to a sequence of the 5′
-portion of said predetermined nucleic acid target sequence,and (iv) optionally further comprising a polymerizing amount of a DNA polymerase and deoxynucleoside triphosphates when the hybridized open circle probe 3′
-terminus is not adjacent and ligatable to the hybridized open circle probe 5′
-terminus and a gap is present between those termini;
(B) maintaining the ligation reaction solution for a time period sufficient to permit filling-in of said gap, when present, and ligation of the termini of the open circle probe to form a closed circular probe and a treated ligation reaction solution;
(C) admixing said closed circular probe with an amplification primer that hybridizes with said amplification primer target, nucleoside triphosphates, and a polymerizing amount of a DNA polymerase to form a replication reaction mixture;
(D) maintaining the replication reaction mixture for a time period sufficient to permit extension of a nucleic acid strand from the amplification primer, wherein the extension product nucleic acid strand comprises a interrogation target to form a treated replication mixture;
(E) admixing an interrogation probe with the treated replication mixture, wherein the interrogation probe is complementary to said interrogation target and comprises an identifier nucleotide in the 3′
-terminal region;
(F) denaturing the treated replication mixture to form a denatured mixture;
(G) annealing the denatured mixture to form hybrid between the interrogation probe and the interrogation target when present to form an interrogation solution;
(H) admixing 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 with the interrogation solution to form a depolymerization reaction mixture;
(I) maintaining the depolymerization reaction mixture for a time period sufficient to permit the enzyme to depolymerize hybridized nucleic acid and release identifier nucleotide therefrom; 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.
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165. An amplification and interrogation process to determine the presence or absence of a predetermined nucleic acid target sequence having a 3′
- -portion and a 5′
-portion comprising the steps of;
(A) providing a ligation reaction solution comprising (i) ligating amount of a ligase, (ii) a nucleic acid sample that may contain a predetermined nucleic acid target sequence wherein the nucleic acid target sequence has a 3′
-portion and a 5′
-portion, (iii) a pair of ligation probes, said ligation probe further including a detection primer target and an amplification primer target, the amplification primer target being downstream of the detection primer target,wherein upon hybridization between the open circle probe and the nucleic acid target sequence, the open circle probe 3′
-terminal region is complementary to a sequence of the 3′
-portion of said predetermined nucleic acid target sequence, and the open circle probe 5′
-terminal region is complementary to a sequence of the 5′
-portion of said predetermined nucleic acid target sequence,and (iv) optionally further comprising a polymerizing amount of a DNA polymerase and deoxynucleoside triphosphates when the hybridized open circle probe 3′
-terminus is not adjacent and ligatable to the hybridized open circle probe 5′
-terminus and a gap is present between those termini;
(B) maintaining the ligation reaction solution for a time period sufficient to permit filling-in of said gap, when present, and ligation of the termini of the open circle probe to form a closed circular probe and a treated ligation reaction solution;
(C) admixing said closed circular probe with an amplification primer that hybridizes with said amplification primer target, nucleoside triphosphates, and a polymerizing amount of a DNA polymerase to form a replication reaction mixture;
(D) maintaining the replication reaction mixture for a time period sufficient to permit extension of a nucleic acid strand from the amplification primer, wherein the extension product nucleic acid strand comprises a interrogation target to form a treated replication mixture;
(E) admixing an interrogation probe with the treated replication mixture, wherein the interrogation probe is complementary to said interrogation target and comprises an identifier nucleotide in the 3′
-terminal region;
(F) denaturing the treated replication mixture to form a denatured mixture;
(G) annealing the denatured mixture to form hybrid between the interrogation probe and the interrogation target when present to form an interrogation solution;
(H) admixing 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 with the interrogation solution to form a depolymerization reaction mixture;
(I) maintaining the depolymerization reaction mixture for a time period sufficient to permit the enzyme to depolymerize hybridized nucleic acid and release identifier nucleotide therefrom; 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.
- -portion and a 5′
-
174. A kit containing a pyrophosphorylation enzyme for use in DNA detection for luminescence spectroscopic analysis comprising
(A) a vessel containing an enzyme capable of catalyzing pyrophosphorolysis; -
(B) a vessel containing NDPK; and
(C) a vessel containing ADP.
-
-
176. A kit for determining the presence or absence of a predetermined nucleic acid target sequence in a nucleic acid sample comprising:
-
(A) an enzyme whose activity is to release one or more nucleotides from the 3′
terminus of a hybridized nucleic acid probe; and
(B) at least one nucleic acid probe, said nucleic acid probe being complementary to nucleic acid target sequence.
-
-
177. A kit for determining the presence or absence of at least one predetermined nucleic acid target sequence in a nucleic acid sample comprising:
-
(A) an enzyme whose activity in the presence of pyrophosphate is to release identifier nucleotide as a nucleoside triphosphate from hybridized nucleic acid probe;
(B) adenosine 5′
diphosphate;
(C) pyrophosphate;
(D) a nucleoside diphosphate kinase; and
(E) at least one nucleic acid probe, said nucleic acid probe being complementary to said predetermined nucleic acid target sequence.
-
-
178. A kit for determining the presence or absence of a predetermined nucleic acid target sequence in a nucleic acid sample comprising:
-
(A) an enzyme whose activity is to release one or more nucleotides from the 3′
terminus of a hybridized nucleic acid probe; and
(B) instructions for use.
-
-
179. A composition for use in DNA detection for luminescence spectroscopic analysis comprising an aqueous solution that contains:
-
(A) a purified and isolated enzyme that catalyzes pyrophosphorolysis;
(B) a purified and isolated NDPK; and
(C) ADP.
-
-
180. The composition according according to claim 192 further comprising (D) a purified and isolated PRPP synthetase.
-
181. A composition for determining the presence or absence of a predetermined nucleic acid target sequence in a nucleic acid sample comprising an aqueous solution that contains:
-
(A) a purified and isolated enzyme whose activity is to release one or more nucleotides from the 3′
terminus of a hybridized nucleic acid probe; and
(B) at least one nucleic acid probe, said nucleic acid probe being complementary to said predetermined nucleic acid target sequence.
-
-
182. The composition according to claim 194 further comprising a purified and isolated nucleoside diphosphate kinase.
-
183. The composition according to 195, wherein said nucleoside diphosphate kinase is that encoded by Pyrococcus furiosis.
-
184. A composition of matter for determining the presence or absence of at least one predetermined nucleic acid target sequence in a nucleic acid sample comprising an aqueous solution that contains:
-
(A) a purified and isolated enzyme whose activity in the presence of pyrophosphate is to release identifier nucleotide as a nucleoside triphosphate from hybridized nucleic acid probe;
(B) adenosine 5′
diphosphate;
(C) pyrophosphate;
(D) a purified and isolated nucleoside diphosphate kinase; and
(E) at least one nucleic acid probe, said nucleic acid probe being complementary to said predetermined nucleic acid target sequence.
-
-
185. The composition of matter according to claim 197, wherein said purified and isolated enzyme whose activity in the presence of pyrophosphate is to release identifier nucleotides is selected from the group consisting of the Tne triple mutant DNA polymerase, Bst DNA polymerase, Ath DNA polymerase, Tag DNA polymerase and Tvu DNA polymerase.
-
186. The composition of matter according to claim 197, wherein said purified and isolated nucleoside diphosphate kinase is that encoded by Pyrococcus furiosis.
Specification
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Current AssigneePromega Corporation
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Original AssigneePromega Corporation
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InventorsRhodes, Richard Byron, Lewis, Martin K., Kephart, Daniel, Leippe, Donna, Shultz, John William, Andrews, Christine Ann, Welch, Roy, Wood, Keith V., Olson, Ryan J., Hartnett, James Robert, Gu, Trent, Mandrekar, Michelle
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Application NumberUS09/790,457Publication NumberTime in Patent OfficeDaysField of SearchUS Class Current435/6CPC Class CodesC12Q 1/04 Determining presence or kin...C12Q 1/66 involving luciferaseC12Q 1/68 involving nucleic acidsC12Q 1/6823 Release of bound markersC12Q 1/6827 for detection of mutation o...C12Q 2521/319 ExonucleaseC12Q 2535/107 Maxam and Gilbert method, i...C12Q 2535/131 Allele specific probesC12Q 2537/101 Homogeneous assay format, e...C12Q 2565/1015 labels being on the same ol...C12Q 2565/301 Pyrophosphate (PPi)C12Q 2565/627 being a mass spectrometerY10T 436/24 Nuclear magnetic resonance,...
