Pyrophosphorolysis and incorporation of nucleotide method for nucleic acid detection
First Claim
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1. A method for determining the presence or absence of a nucleic acid hybrid in a sample comprising the following steps:
- (a) providing a reaction mixture comprising (i) a sample that may contain a nucleic acid hybrid that comprises a 3′
-terminus, (ii) pyrophosphate, (iii) an enzyme that catalyzes the release of a nucleotide from a nucleic acid hybrid, by pyrophosphorolysis of the 3′
-terminus of a strand of the nucleic acid hybrid in the presence of pyrophosphate, and (iv) a suitable nucleotide that can be incorporated in the place of said released nucleotide;
(b) maintaining said reaction mixture for a time period and under conditions that permit (i) pyrophosphorolysis of the 3′
-terminus of a strand of a nucleic acid hybrid to produce a released nucleotide and a modified 3′
-terminus as well as (ii) the incorporation of said suitable nucleotide into the modified 3′
-terminus of the nucleic acid hybrid to produce an incorporated modified 3′
-terminus, thereby forming a treated sample; and
(c) assaying the treated sample to determine whether incorporation of said suitable nucleotide into the hybrid occurred and thereby determining the presence or absence of a nucleic acid hybrid in said sample.
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Abstract
Processes are disclosed using the depolymerization of a nucleic acid hybrid and incorporation of a suitable nucleotide to qualitatively and quantitatively analyze for the presence of predetermined nucleic acid target sequences. Applications of those processes include the detection of single nucleotide polymorphisms, identification of single base changes, genotyping, medical marker diagnostics, mirosequencing, and.
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Citations
28 Claims
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1. A method for determining the presence or absence of a nucleic acid hybrid in a sample comprising the following steps:
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(a) providing a reaction mixture comprising (i) a sample that may contain a nucleic acid hybrid that comprises a 3′
-terminus, (ii) pyrophosphate, (iii) an enzyme that catalyzes the release of a nucleotide from a nucleic acid hybrid, by pyrophosphorolysis of the 3′
-terminus of a strand of the nucleic acid hybrid in the presence of pyrophosphate, and (iv) a suitable nucleotide that can be incorporated in the place of said released nucleotide;
(b) maintaining said reaction mixture for a time period and under conditions that permit (i) pyrophosphorolysis of the 3′
-terminus of a strand of a nucleic acid hybrid to produce a released nucleotide and a modified 3′
-terminus as well as (ii) the incorporation of said suitable nucleotide into the modified 3′
-terminus of the nucleic acid hybrid to produce an incorporated modified 3′
-terminus, thereby forming a treated sample; and
(c) assaying the treated sample to determine whether incorporation of said suitable nucleotide into the hybrid occurred and thereby determining the presence or absence of a nucleic acid hybrid in said sample. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 24, 25, 26)
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12. A method for determining the presence or absence of a nucleic acid target in a sample comprising the following steps:
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(a) providing a reaction mixture comprising (i) a sample that may contain a nucleic acid target, (ii) a nucleic acid probe corresponding to said nucleic acid target, (iii) pyrophosphate, (iv) an enzyme that catalyzes the release of a nucleotide from a nucleic acid hybrid, which comprises a 3′
-terminus, by pyrophosphorolysis of the 3′
-terminus of a strand of the nucleic acid hybrid in the presence of pyrophosphate, and (v) a suitable nucleotide that can be incorporated in the place of said released nucleotide;
(b) maintaining said reaction mixture for a time period and under conditions that permit (i) hybridization of the nucleic acid target with the nucleic acid probe to form a nucleic acid hybrid that comprises a 3′
-terminus, (ii) pyrophosphorolysis of the 3′
-terminus at a strand of a nucleic acid hybrid to produce a released nucleotide and a modified 3′
-terminus as well as (iii) the incorporation of said suitable nucleotide into the modified 3′
-terminus of the nucleic acid hybrid to produce an incorporated modified 3′
-terminus, thereby forming a treated sample; and
(c) assaying the treated sample to determine whether incorporation of said suitable nucleotide occurred and thereby determining the presence or absence of the nucleic acid target in said sample. - View Dependent Claims (13, 14, 15, 16, 17, 18, 19)
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20. A method for determining the presence or absence of a specific nucleic acid base at an interrogation position of a nucleic acid target in a sample comprising the following steps:
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(a) providing a reaction mixture comprising (i) a sample that may contain a nucleic acid target having a nucleic acid residue at an interrogation position, (ii) a nucleic acid probe comprising a nucleic acid residue in its 3′
-terminus that base pairs with the interrogation position of the nucleic acid target when the nucleic acid target and the nucleic acid probe are hybridized to form a nucleic acid hybrid, (ii) pyrophosphate, (iii) an enzyme that catalyzes the release of a nucleotide from a nucleic acid hybrid, which comprises a 3′
-terminus, by pyrophosphorolysis of the 3′
-terminus of a strand of the nucleic acid hybrid in the presence of pyrophosphate, and (iv) a suitable nucleotide that can be incorporated in the place of said released nucleotide;
(b) maintaining said reaction mixture for a time period and under conditions that permit (i) formation of a nucleic acid hybrid between the nucleic acid probe and the nucleic acid target, (ii) pyrophosphorolysis of the 3′
-terminus of a strand of a nucleic acid hybrid to produce a released nucleotide and a modified 3′
-terminus and (iii) the incorporation of said suitable nucleotide into the modified 3′
-terminus of the nucleic acid hybrid to produce an incorporated modified 3′
-terminus, thereby forming a treated sample; and
(c) assaying the treated sample to determine whether incorporation of said suitable nucleotide occurred and thereby determining the presence or absence of a specific nucleic acid base at an interrogation position of a nucleic acid target in said sample. - View Dependent Claims (21, 22)
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23. A reaction mixture comprising (i) a sample that may contain a nucleic acid hybrid that comprises a 3′
- -terminus, (ii) pyrophoaphate, (iii) an enzyme that catalyzes the release of a nucleotide from a nucleic acid hybrid, which comprises a 3′
-terminus, by pyrophosphorolysis of the 3′
-terminus of a strand of the nucleic acid hybrid in the presence of pyrophosphate, and (iv) a suitable nucleotide that can be incorporated in the place of said released nucleotide.
- -terminus, (ii) pyrophoaphate, (iii) an enzyme that catalyzes the release of a nucleotide from a nucleic acid hybrid, which comprises a 3′
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27. A method of determining a nucleotide sequence of a nucleic acid hybrid that comprises the following steps:
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(a) providing a reaction mixture comprising (i) a sample contains a nucleic acid hybrid that comprises a 3′
-terminus, (ii) pyrophosphate, (iii) an enzyme that catalyzes the release of a nucleotide from a nucleic acid hybrid, by pyrophosphorolysis of the 3′
-terminus of a strand of the nucleic acid hybrid in the presence of pyrophosphate, and (iv) a suitable nucleotide that can be incorporated in the place of said released nucleotide;
(b) maintaining said reaction mixture for a time period and under conditions that permit (i) pyrophosphorolysis of the 3′
-terminus of a strand of a nucleic acid hybrid to produce a released nucleotide and a modified 3′
-terminus as well as (ii) the incorporation of said suitable nucleotide into the modified 3′
-terminus of the nucleic acid hybrid to produce an incorporated modified 3′
-terminus, thereby forming a treated sample; and
(c) assaying the treated sample to determine where incorporation of said suitable nucleotide into the hybrid occurred and thereby determining nucleotide sequence of the nucleic acid hybrid. - View Dependent Claims (28)
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Specification