A method for detecting a target nucleic acid sequence from a DNA or a mixture of nucleic acids by a POCH (PO Cleavage and Hybridization) assay on a solid substrate, comprising: (a) hybridizing the target nucleic acid sequence with an upstream oligonucleotide and a probing oligonucleotide; wherein the upstream oligonucleotide comprises a hybridizing nucleotide sequence complementary to the target nucleic acid sequence; the PO comprises a targeting portion comprising a hybridizing nucleotide sequence complementary to the target nucleic acid sequence; the PO has a single label; 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 target nucleic acid sequence, 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 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 occurrence of the cleavage of the PO by measuring a signal from the single label on the solid substrate; whereby the occurrence of the PO cleavage indicates the presence of the target nucleic acid sequence. (a) hybridizing the target nucleic acid sequence with an upstream oligonucleotide and a probing oligonucleotide; wherein the upstream oligonucleotide comprises a hybridizing nucleotide sequence complementary to the target nucleic acid sequence; the PO comprises a targeting portion comprising a hybridizing nucleotide sequence complementary to the target nucleic acid sequence; the PO has a single label; 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 target nucleic acid sequence, 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 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 occurrence of the cleavage of the PO by measuring a signal from the single label on the solid substrate; whereby the occurrence of the PO cleavage indicates the presence of the target nucleic acid sequence.