Nucleic acid sequence detection employing amplification probes

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 having nucleic acid side chains which non-covalently bind 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 bonded to a difunctional molecule other than a nucleotide in said nucleic acid side chain which photoactivatable group upon photoactivation during stem formation forms a covalent cross-link with the other side chain member of said stem, said method comprising: combining said sample with said pair of probes under conditions of base pairing between said probes and said target nucleic acid, whereby probes binding to said target nucleic acid form said stem; photoactivating said photoactivatable group, whereby a covalent cross-link occurs between said side chain members of said stem; amplifying the amount of cross-linked probes by performing the following steps 1-3 at least once: (1) melting double stranded nucleic acid; (2) incubating for sufficient time under hybridization conditions for base pairing between homologous sequences to occur; and (3) photoactivating said photoactivatable group, whereby a cross-link occurs between said side chain members of said stem; and detecting the presence of cross-linked pairs of probes as indicative of the presence of said target sequence in said sample.

Claim 2

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 having nucleic acid side chains which non-covalently bind 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 bonded to a difunctional molecule other than a nucleotide in said nucleic acid side chain which photoactivatable group upon photoactivation during stem formation forms a covalent cross-link with the other side chain member of said stem, said method comprising: combining said sample with said pair of probes under conditions of base pairing between said probes and said target nucleic acid, whereby probes binding to said target nucleic acid form said stem; photoactivating said photoactivatable group, whereby a covalent cross-link occurs between said side chain members of said stem; amplifying the amount of cross-linked probes by performing the following steps 1-3 at least once: (1) melting double stranded nucleic acid; (2) incubating for sufficient time under hybridization conditions for base pairing between homologous sequences to occur; and (3) photoactivating said photoactivatable group, whereby a cross-link occurs between said side chain members of said stem; and detecting the presence of cross-linked pairs of probes as indicative of the presence of said target sequence in said sample.

Claim 5

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 having side chains which non-covalently bind 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 bonded to a difunctional molecule other than a nucleotide in said nucleic acid side chain, which photoactivatable group 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: combining said sample with said pair of 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, 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; photoactivating said photoactivatable group, whereby a covalent cross-link is formed between said side chain members of said stem; melting double stranded nucleic acid; repeating the following cycle at least once: 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 a sequence analogous to said target nucleic acid; photoactivating said photoactivatable group, whereby a covalent cross-link is formed between said side chain members of said stem; and melting double stranded DNA, which ends a cycle; and detecting the presence of cross-linked pairs of probes as indicative of the presence of said target sequence in said sample.

Claim 6

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 having side chains which non-covalently bind 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 bonded to a difunctional molecule other than a nucleotide in said nucleic acid side chain, which photoactivatable group 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: combining said sample with said pair of 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, 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; photoactivating said photoactivatable group, whereby a covalent cross-link is formed between said side chain members of said stem; melting double stranded nucleic acid; repeating the following cycle at least once: 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 a sequence analogous to said target nucleic acid; photoactivating said photoactivatable group, whereby a covalent cross-link is formed between said side chain members of said stem; and melting double stranded DNA, which ends a cycle; and detecting the presence of cross-linked pairs of probes as indicative of the presence of said target sequence in said sample.

Claim 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 having side chains which non-covalently bind 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 bonded to a difunctional molecule other than nucleotide in said nucleic acid side chain, which photoactivatable group 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: combining said sample with said first and second pairs of probes under conditions of base pairing between said probes and said target dsDNA, 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 dsDNA nucleic acid form said stem; photoactivating said photoactivatable group, whereby a covalent cross-link is formed between said side chain members of said stem; melting double stranded nucleic acid; repeating the following cycle at least once: 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 a sequence analogous to said target nucleic acid; photoactivating said photoactivatable group, whereby a covalent cross-link is formed between said side chain members of said stem; and melting double stranded DNA, which ends a cycle; and detecting the presence of cross-linked pairs of probes as indicative of the presence of said target dsDNA in said sample.

Claim 14

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 having side chains which non-covalently bind 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 bonded to a difunctional molecule other than nucleotide in said nucleic acid side chain, which photoactivatable group 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: combining said sample with said first and second pairs of probes under conditions of base pairing between said probes and said target dsDNA, 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 dsDNA nucleic acid form said stem; photoactivating said photoactivatable group, whereby a covalent cross-link is formed between said side chain members of said stem; melting double stranded nucleic acid; repeating the following cycle at least once: 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 a sequence analogous to said target nucleic acid; photoactivating said photoactivatable group, whereby a covalent cross-link is formed between said side chain members of said stem; and melting double stranded DNA, which ends a cycle; and detecting the presence of cross-linked pairs of probes as indicative of the presence of said target dsDNA in said sample.

Claim 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 having nucleic acid side chains which form from 3 to 16 base pairs upon non-covalently binding 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 coumarin or furocoumarin as 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: combining said sample with said pair of probes under conditions of base pairing between said probes and said target nucleic acid, whereby probes binding to said target nucleic acid form said stem; activating said activatable group, whereby a covalent cross-link occurs between said side chain members of said stem; melting double stranded nucleic acid: repeating the following cycle at least once; 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 a sequence analogous to said target nucleic acid; photoactivating said photoactivatable group, whereby a covalent cross-link is formed between said side chain members of said stem; and melting double stranded DNA, which ends a cycle: and detecting the presence of cross-linked pairs of probes as indicative of the presence of said target sequence in said sample.

Claim 22

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 having nucleic acid side chains which form from 3 to 16 base pairs upon non-covalently binding 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 coumarin or furocoumarin as 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: combining said sample with said pair of probes under conditions of base pairing between said probes and said target nucleic acid, whereby probes binding to said target nucleic acid form said stem; activating said activatable group, whereby a covalent cross-link occurs between said side chain members of said stem; melting double stranded nucleic acid: repeating the following cycle at least once; 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 a sequence analogous to said target nucleic acid; photoactivating said photoactivatable group, whereby a covalent cross-link is formed between said side chain members of said stem; and melting double stranded DNA, which ends a cycle: and detecting the presence of cross-linked pairs of probes as indicative of the presence of said target sequence in said sample.