Nucleic acid sequence detection employing amplification probes
First Claim
1. A method for detecting a target nucleic acid sequence in a sample, said method employing at least one pair of probes characterized by having sequences homologous to adjacent portions of said target nucleic acid sequence and each probe having at least one side chain which non-covalently binds to a side chain of the other probe of the pair to form a stem upon base pairing of said probes to said target nucleic acid sequence, at least one of said side chains having an activatable group, which upon activation during stem formation forms a covalent cross-link with the other side chain member of said stem, said method comprising:
- a) combining said sample with said at least one pair of said probes under conditions of base pairing between said probes and said target nucleic acid to produce an assay medium, whereby probes binding to said target nucleic acid form said stem;
b) activating said activatable group, whereby a covalent cross-link occurs between said side chain members of said stem; and
c) detecting the presence of cross-linked pairs of probes as indicative of the presence of said target sequence in said sample.
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Accused Products
Abstract
Methods and compositions are provided for detecting nucleic acid sequences. In particular, pairs of probes are employed, where the pair defines a substantially contiguous sequence on a target nucleic acid. Each of the pairs has a side chain which forms a stem of the two side chains which non-covalently binds and is capable of forming a cross-link upon activation, when the probes and sample nucleic acid are base paired. Cross-linking of the stems when unbound to complementary DNA is inhibited. Each of the nucleic acids is initially present as single stranded nucleic acid to allow for base pairing, so that the probes bind to homologous target nucleic acid. The assay mixture is activated to provide cross-linking, the double stranded nucleic acid melted, and the process of base pairing, activation and melting repeated, a sufficient number of cycles, to provide a detectable amount of cross-linked probes. To inhibit background cross-linking, the side chains may provide for duplex formation, where a portion of the side chain binds to a different portion of the side chain or the portion of the probe homologous to the target. Also provided are kits comprising reagents, as well as automatic devices, for carrying out the subject method.
40 Citations
34 Claims
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1. A method for detecting a target nucleic acid sequence in a sample, said method employing at least one pair of probes characterized by having sequences homologous to adjacent portions of said target nucleic acid sequence and each probe having at least one side chain which non-covalently binds to a side chain of the other probe of the pair to form a stem upon base pairing of said probes to said target nucleic acid sequence, at least one of said side chains having an activatable group, which upon activation during stem formation forms a covalent cross-link with the other side chain member of said stem, said method comprising:
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a) combining said sample with said at least one pair of said probes under conditions of base pairing between said probes and said target nucleic acid to produce an assay medium, whereby probes binding to said target nucleic acid form said stem;
b) activating said activatable group, whereby a covalent cross-link occurs between said side chain members of said stem; and
c) detecting the presence of cross-linked pairs of probes as indicative of the presence of said target sequence in said sample. - View Dependent Claims (2, 3)
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4. A method for detecting a target nucleic acid sequence in a sample, said method employing at least one pair of probes characterized by having sequences homologous to adjacent portions of said target nucleic acid sequence and each probe having at least one side chain which non-covalently binds to a side chain of the other probe of the pair to form a stem upon base pairing of said probes to said target nucleic acid sequence, at least one of said side chains having a photoactivatable group, which upon activation during stem formation forms a covalent cross-link with the other side chain member of said stem, each of said side chains having at least two nucleotides capable of base pairing with the other side chain to form said stem, said method comprising:
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a) combining said sample with said at least one pair of said probes under conditions of base pairing between said probes and said target nucleic acid, wherein when said target nucleic acid is single stranded, at least a first pair of probes is added which is homologous to said single stranded nucleic acid, and if said target nucleic acid is double stranded, at least one of first and second pairs of probes are added, which pairs are homologous to one or the other strands of said double stranded target nucleic acid, whereby probes binding to said target nucleic acid form said stem;
b) photoactivating said photoactivatable group, whereby a covalent cross-link is formed between said side chain members of said stem;
c) melting double stranded nucleic acid;
d) repeating the following cycle at least once;
1) incubating for sufficient time for base pairing between homologous sequences to occur, with the proviso that when only said first pair of probes was added, another pair of probes is added having sequences homologous to said first pair of probes;
2) photoactivating said photoactivatable group, whereby a covalent cross-link is formed between said side chain members of said stem; and
3) melting double stranded DNA, which ends a cycle; and
e) detecting the presence of cross-linked pairs of probes as indicative of the presence of said target sequence in said sample. - View Dependent Claims (5, 6, 7, 8, 9, 10, 11, 12)
exciting said first fluorophore; and
reading the fluorescence of said second fluorophore.
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11. A method according to claim 4, wherein each of probes comprises a label, wherein one of said labels is a member of a specific binding pair and the other of said labels provides for a detectable signal, wherein said detecting the presence of cross-linked probes comprises:
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separating said cross-linked probes from said sample on a solid support; and
detecting the presence of said signal on said solid support.
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12. A method according to claim 4, wherein at least three cycles are repeated.
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13. A method for detecting a sequence of target dsDNA in a sample, said method employing first and second pairs of probes characterized by having sequences homologous to adjacent portions of first and second strands of said target dsDNA and each probe having at least one side chain which non-covalently binds to a side chain of the other probe of the pair to form a stem upon base pairing of said probes to said target first and second strands, respectively, at least one of said side chains in each pair having a photoactivatable group, which upon activation during stem formation forms a covalent cross-link with the other side chain member of said stem, each of said side chains having at least two nucleotides capable of base pairing with the other side chain to form said stem, said method comprising:
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a) combining said sample with said first and second pairs of probes under conditions of base pairing between said probes and said target nucleic acid, which first and second pairs of probes are homologous to one or the other strands of said target dsDNA, whereby probes binding to said target nucleic acid form said stem;
b) photoactivating said photoactivatable group, whereby a covalent cross-link isformed between said side chain members of said stem;
c) melting double stranded nucleic acid;
d) repeating the following cycle at least once;
1) incubating for sufficient time for base pairing between homologous sequences to occur;
2) photoactivating said photoactivatable group, whereby a covalent cross-link occurs between said side chain members of said stem; and
3) melting double stranded DNA, which ends a cycle; and
e) detecting the presence of cross-linked pairs of probes as indicative of the presence of said target dsDNA in said sample. - View Dependent Claims (14, 15, 16, 17, 18, 19, 20)
exciting said first fluorophore; and
reading the fluorescence of said second fluorophore.
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20. A method according to claim 13, wherein each of said probes comprises a label, wherein one of said labels is a member of a specific binding pair and the other of said labels provides for a detectable signal, wherein said detecting the presence of cross-linked probes comprises:
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separating said cross-linked probes from said sample on a solid support; and
detecting the presence of said signal on said solid support.
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21. A method for detecting a target nucleic acid sequence in a sample, said method employing at least one pair of probes characterized by having sequences homologous to adjacent portions of said target nucleic acid sequence and each probe having at least one side chain which non-covalently binds to a side chain of the other probe of the pair to form a stem upon base pairing of said probes to said target nucleic acid sequence, at least one of said side chains having an activatable group, which upon activation during stem formation forms a covalent cross-link with the other side chain member of said stem, and at least one of said stems forming a hairpin or stem and loop by one portion of said side chain binding to a different portion of said chain or to the sequence of said probe homologous to said target, said method comprising:
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a) combining said sample with said pair of probes under conditions of melting of said hairpin or stem and loop and of base pairing between said probes and said target nucleic acid to produce an assay medium, whereby probes binding to said target nucleic acid form said stem;
b) activating said activatable group, whereby a covalent cross-link occurs between said side chain members of said stem; and
c) detecting the presence of cross-linked pairs of probes as indicative of the presence of said target sequence in said sample. - View Dependent Claims (22, 23)
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- 24. A kit comprising at least one pair of probes, said probes being characterized by having sequences homologous to adjacent portions of a target nucleic acid sequence and each probe having at least one side chain which non-covalently binds to a side chain of the other probe of the pair to form a stem upon base pairing of said probes to said target nucleic acid sequence, at least one of said side chains having an activatable group, which upon activation during stem formation forms a covalent cross-link with the other side chain member of said stem.
- 28. A nucleic acid compound comprising a nucleic acid sequence of at least 12 nucleotides defining a sequence of interest covalently linked at one end to a side chain characterized by having at least two nucleotides and not more than 8 nucleotides and having a photoactivatable group other than a nucleotide.
- 30. An oligonucleotide comprising from 2 to 60 nucleotides and a deoxyribosyl backbone having intervening in said backbone from 1 to 2 linkers comprising other than nucleosides and pendent from said linkers, a photoactivatable group other than a nucleotide, capable of forming a covalent bond with a nucleotide.
Specification