Detection of target nucleic acid sequences by PO cleavage and hybridization

A method for detecting at least one target nucleic acid molecule having sequence information from a DNA or a mixture of nucleic acids in a sample by a POCH (PO Cleavage and Hybridization) assay on a solid substrate, comprising:

• (a) hybridizing the sequence of said target nucleic acid molecule with an upstream oligonucleotide and a probing oligonucleotide (PO);
• wherein the upstream oligonucleotide comprises a hybridizing nucleotide sequence complementary to the sequence of said target nucleic acid molecule;
• the PO comprises a targeting portion comprising a hybridizing nucleotide sequence complementary to the sequence of said target nucleic acid molecule;
• the PO has an interactive dual label comprising a donor molecule and an acceptor molecule;
• the upstream oligonucleotide is located upstream of the PO;
• the upstream oligonucleotide or its extended strand induces cleavage of the PO by an enzyme having a 5′ nuclease activity;
• (b) contacting the resultant of the step (a) to the enzyme having the 5′ nuclease activity under conditions for cleavage of the PO;
• wherein when the PO is hybridized with the sequence of said target nucleic acid molecule, the PO is cleaved by the enzyme having the 5′ nuclease activity to separate the interactive dual label, whereby a donor-containing fragment and an acceptor-containing fragment are produced;
• (c) performing a hybridization reaction by contacting the resultant of the step (b) to a capturing oligonucleotide (CO) immobilized onto the solid substrate;
• wherein the CO comprises a nucleotide sequence hybridizable with the PO;
• wherein the hybridization reaction is performed under conditions such that at least one of the donor-containing fragment and the acceptor-containing fragment is not hybridized with the CO and an uncleaved PO is hybridized with the CO to form an uncleaved PO/CO duplex;
• wherein a signal from the uncleaved PO/CO duplex is differentiated from a signal provided at the time that at least one of the donor-containing fragment and the acceptor-containing fragment is not hybridized with the CO;
• and
• (d) detecting a signal from the interactive dual label on the solid substrate;
• wherein (i) when a signal difference is observed by comparing the signal from the interactive dual label on the solid substrate to a signal from a control that does not contain the target nucleic acid sequence, the signal difference indicates the presence of the target nucleic acid sequence, or (ii) when a signal change is observed at cycles with predetermined interval by detecting the signal from the interactive dual label on the solid substrate along with repetition of the steps (a)-(b), (a)-(c) or (a)-(d), the signal change indicates the presence of the target nucleic acid sequence.

A method for detecting at least one target nucleic acid molecule having sequence information from a DNA or a mixture of nucleic acids in a sample by a POCH (PO Cleavage and Hybridization) assay on a solid substrate, comprising:

• (a) hybridizing the sequence of said target nucleic acid molecule with an upstream oligonucleotide and a probing oligonucleotide (PO);
• wherein the upstream oligonucleotide comprises a hybridizing nucleotide sequence complementary to the sequence of said target nucleic acid molecule;
• the PO comprises a targeting portion comprising a hybridizing nucleotide sequence complementary to the sequence of said target nucleic acid molecule;
• the upstream oligonucleotide is located upstream of the PO;
• the upstream oligonucleotide or its extended strand induces cleavage of the PO by an enzyme having a 5′ nuclease activity;
• (b) contacting the resultant of the step (a) to the enzyme having the 5′ nuclease activity under conditions for cleavage of the PO;
• wherein when the PO is hybridized with the sequence of said target nucleic acid molecule, the PO is cleaved by the enzyme having the 5′ nuclease activity to produce a cleaved fragment;
• (c) performing a hybridization reaction in the presence of an intercalating agent by contacting the resultant of the step (b) to a capturing oligonucleotide (CO) immobilized onto the solid substrate;
• wherein the CO comprises a nucleotide sequence hybridizable with the PO;
• wherein the hybridization reaction is performed under conditions such that the cleaved fragment is not hybridized with the CO and an uncleaved PO is hybridized with the CO to form an uncleaved PO/CO duplex;
• and
• (d) detecting a signal from the intercalating agent on the solid substrate;
• wherein (i) when a signal difference is observed by comparing the signal from the intercalating agent on the solid substrate to a signal from a control that does not contain the target nucleic acid sequence, the signal difference indicates the presence of the target nucleic acid sequence, or (ii) when a signal change is observed at cycles with predetermined interval by detecting the signal from the intercalating agent on the solid substrate along with repetition of the steps (a)-(b), (a)-(c) or (a)-(d), the signal change indicates the presence of the target nucleic acid sequence.

A method for detecting at least one target nucleic acid molecule having sequence information from a DNA or a mixture of nucleic acids in a sample by a POCH (PO Cleavage and Hybridization) assay on a solid substrate, comprising:

• (a) hybridizing the sequence of said target nucleic acid molecule with a probing oligonucleotide (PO);
• wherein the the PO comprises a targeting portion comprising a hybridizing nucleotide sequence complementary to the sequence of said target nucleic acid molecule;
• the PO has a single label;
• (b) contacting the resultant of the step (a) to the enzyme having the 5′ nuclease activity under conditions for cleavage of the PO;
• wherein when the PO is hybridized with the sequence of said target nucleic acid molecule, the PO is cleaved by the enzyme having the 5′ nuclease activity to produce a single label-containing fragment
• (c) performing a hybridization reaction by contacting the resultant of the step (b) to a capturing oligonucleotide (CO) immobilized onto the solid substrate;
• wherein the CO comprises a nucleotide sequence hybridizable with the PO;
• wherein the hybridization reaction is performed under conditions such that the single label-containing fragment is not hybridized with the CO and an uncleaved PO is hybridized with the CO to form an uncleaved PO/CO duplex;
• and
• (d) detecting a signal from the single label on the solid substrate;
• wherein (i) when a signal difference is observed by comparing the signal from the single label on the solid substrate to a signal from a control that does not contain the target nucleic acid sequence, the signal difference indicates the presence of the target nucleic acid sequence, or (ii) when a signal change is observed at cycles with predetermined interval by detecting the signal from the single label on the solid substrate along with repetition of the steps (a)-(b), (a)-(c) or (a)-(d), the signal change indicates the presence of the target nucleic acid sequence.

A method for detecting at least one target nucleic acid molecule having sequence information from a DNA or a mixture of nucleic acids in a sample by a POCH (PO Cleavage and Hybridization) assay on a solid substrate, comprising:

• (a) hybridizing the sequence of said target nucleic acid molecule with a probing oligonucleotide (PO);
• wherein the PO comprises a targeting portion comprising a hybridizing nucleotide sequence complementary to the sequence of said target nucleic acid molecule;
• the PO has an interactive dual label comprising a donor molecule and an acceptor molecule;
• (b) contacting the resultant of the step (a) to the enzyme having the 5′ nuclease activity under conditions for cleavage of the PO;
• wherein when the PO is hybridized with the sequence of said target nucleic acid molecule, the PO is cleaved by the enzyme having the 5′ nuclease activity to separate the interactive dual label, whereby a donor-containing fragment and an acceptor-containing fragment are produced
• (c) performing a hybridization reaction by contacting the resultant of the step (b) to a capturing oligonucleotide (CO) immobilized onto the solid substrate;
• wherein the CO comprises a nucleotide sequence hybridizable with the PO;
• wherein the hybridization reaction is performed under conditions such that at least one of the donor-containing fragment and the acceptor-containing fragment is not hybridized with the CO and an uncleaved PO is hybridized with the CO to form an uncleaved PO/CO duplex;
• wherein a signal from the uncleaved PO/CO duplex is differentiated from a signal provided at the time that at least one of the donor-containing fragment and the acceptor-containing fragment is not hybridized with the CO;
• and
• (d) detecting a signal from the interactive dual label on the solid substrate;
• wherein (i) when a signal difference is observed by comparing the signal from the interactive dual label on the solid substrate to a signal from a control that does not contain the target nucleic acid sequence, the signal difference indicates the presence of the target nucleic acid sequence, or (ii) when a signal change is observed at cycles with predetermined interval by detecting the signal from the interactive dual label on the solid substrate along with repetition of the steps (a)-(b), (a)-(c) or (a)-(d), the signal change indicates the presence of the target nucleic acid sequence.

A method for detecting at least one target nucleic acid molecule having sequence information from a DNA or a mixture of nucleic acids in a sample by a POCH (PO Cleavage and Hybridization) assay on a solid substrate, comprising:

• (a) hybridizing the sequence of said target nucleic acid molecule with a probing oligonucleotide (PO);
• wherein the PO comprises a targeting portion comprising a hybridizing nucleotide sequence complementary to the sequence of said target nucleic acid molecule;
• (b) contacting the resultant of the step (a) to the enzyme having the 5′ nuclease activity under conditions for cleavage of the PO;
• wherein when the PO is hybridized with the sequence of said target nucleic acid molecule, the PO is cleaved by the enzyme having the 5′ nuclease activity to produce a cleaved fragment;
• (c) performing a hybridization reaction in the presence of an intercalating agent by contacting the resultant of the step (b) to a capturing oligonucleotide (CO) immobilized onto the solid substrate;
• wherein the CO comprises a nucleotide sequence hybridizable with the PO;
• wherein the hybridization reaction is performed under conditions such that the cleaved fragment is not hybridized with the CO and an uncleaved PO is hybridized with the CO to form an uncleaved PO/CO duplex;
• and
• (d) detecting a signal from the intercalating agent on the solid substrate;
• wherein (i) when a signal difference is observed by comparing the signal from the intercalating agent on the solid substrate to a signal from a control that does not contain the target nucleic acid sequence, the signal difference indicates the presence of the target nucleic acid sequence, or (ii) when a signal change is observed at cycles with predetermined interval by detecting the signal from the intercalating agent on the solid substrate along with repetition of the steps (a)-(b), (a)-(c) or (a)-(d), the signal change indicates the presence of the target nucleic acid sequence.