Detection of target nucleic acid sequence by PTO cleavage and extension-dependent signaling oligonucleotide hybridization assay

A method for detecting a target nucleic acid sequence from a DNA or a mixture of nucleic acids by a PCE-SH (PTO Cleavage and Extension-Dependent Signaling Oligonucleotide Hybridization) assay in a liquid phase, comprising:

• (a) hybridizing the target nucleic acid sequence with a primer pair comprising an upstream primer and a downstream primer and a PTO (Probing and Tagging Oligonucleotide);
• wherein each of the upstream primer and the downstream primer comprise a hybridizing nucleotide sequence complementary to the target nucleic acid sequences;
• the PTO comprises (i) a 3′-targeting portion comprising a hybridizing nucleotide sequence complementary to the target nucleic acid sequence and (ii) a 5′-tagging portion comprising a nucleotide sequence non-complementary to the target nucleic acid sequence;
• wherein the 3′-targeting portion is hybridized with the target nucleic acid sequence and the 5′-tagging portion is not hybridized with the target nucleic acid sequence;
• the PTO is located between the upstream primer and the downstream primer;
• wherein the PTO is blocked at its 3′-end to prohibit its extension;
• (b) contacting the resultant of the step (a) to a template-dependent nucleic acid polymerase having a 5′ nuclease activity under conditions for extension of the primers and for cleavage of the PTO;
• wherein when the PTO is hybridized with the target nucleic acid sequence, the upstream primer is extended and the extended strand induces cleavage of the PTO by the template-dependent nucleic acid polymerase having the 5′ nuclease activity such that the cleavage releases a fragment comprising the 5′-tagging portion or a part of the 5′-tagging portion of the PTO;
• (c) hybridizing the fragment released from the PTO with a capturing and templating oligonucleotide (CTO);
• wherein the CTO comprises in a 3′ to 5′ direction (i) a capturing portion comprising a nucleotide sequence complementary to the 5′-tagging portion or a part of the 5′-tagging portion of the PTO and (ii) a templating portion comprising a nucleotide sequence non-complementary to the 5′-tagging portion and the 3′-targeting portion of the PTO;
• wherein the fragment released from the PTO is hybridized with the capturing portion of the CTO;
• wherein the CTO is not labeled;
• (d) performing an extension reaction using the resultant of the step (c) and a template-dependent nucleic acid polymerase;
• wherein the fragment hybridized with the capturing portion of the CTO is extended to form an extended strand comprising an extended sequence complementary to the templating portion of the CTO, thereby forming an extended duplex;
• (e) hybridizing the extended strand with a signaling oligonucleotide (SO);
• wherein the SO comprises a complementary sequence to the extended strand and at least one label;
• wherein the at least one label is located on the SO to provide a detectable signal directly by hybridization with the extended strand;
• and
• (f) detecting the signal in said liquid phase;
• wherein the CTO and SO are not immobilized on a solid substrate;
• whereby the detection of the signal indicates the presence of the extended strand and the presence of the target nucleic acid sequence;
• wherein the detectable signal is provided by (i) the label linked to the SO, (ii) a combination of the label linked to the SO and a label linked to the fragment from the PTO, (iii) a combination of the label linked to the SO and a label to be incorporated into the extended strand during the extension reaction of the step (d), or (iv) a combination of the label linked to the SO and an intercalating dye.

A method for detecting a target nucleic acid sequence from a DNA or a mixture of nucleic acids by a PCE-SH (PTO Cleavage and Extension-Dependent Signaling Oligonucleotide Hybridization) assay in a liquid phase, comprising:

• (a) hybridizing the target nucleic acid sequence with a probing and targeting oligonucleotide (PTO);
• wherein the PTO comprises (i) a 3′-targeting portion comprising a hybridizing nucleotide sequence complementary to the target nucleic acid sequence and (ii) a 5′-tagging portion comprising a nucleotide sequence non-complementary to the target nucleic acid sequence;
• wherein the 3′-targeting portion of the PTO is hybridized with the target nucleic acid sequence and the 5′-tagging portion is not hybridized with the target nucleic acid sequence;
• (b) contacting the resultant of the step (a) to an enzyme having a 5′ nuclease activity under conditions for cleavage of the PTO;
• wherein the PTO is cleaved by the enzyme having the 5′ nuclease activity such that the cleavage releases a fragment comprising the 5′-tagging portion or a part of the 5′-tagging portion of the PTO;
• (c) hybridizing the fragment released from the PTO with a capturing and templating oligonucleotide (CTO);
• wherein the CTO comprises in a 3′ to 5′ direction (i) a capturing portion comprising a nucleotide sequence complementary to the 5′-tagging portion or a part of the 5′-tagging portion of the PTO and (ii) a templating portion comprising a nucleotide sequence non-complementary to the 5′-tagging portion and the 3′-targeting portion of the PTO;
• wherein the fragment released from the PTO is hybridized with the capturing portion of the CTO;
• wherein the CTO is not labeled;
• (d) performing an extension reaction using the resultant of the step (c) and a template-dependent nucleic acid polymerase;
• wherein the fragment hybridized with the capturing portion of the CTO is extended to form an extended strand comprising an extended sequence complementary to the templating portion of the CTO, thereby forming an extended duplex;
• (e) hybridizing the extended strand with a signaling oligonucleotide (SO);
• wherein the SO comprises a complementary sequence to the extended strand and at least one label;
• wherein the at least one label is located on the SO to provide a detectable signal directly by hybridization with the extended strand;
• and
• (f) detecting the signal in a liquid phase;
• wherein the CTO and SO are not immobilized on a solid substrate;
• whereby the detection of the signal indicates the presence of the extended strand and the presence of the target nucleic acid sequence;
• wherein the detectable signal is provided by (i) the label linked to the SO, (ii) a combination of the label linked to the SO and a label linked to the fragment from the PTO, (iii) a combination of the label linked to the SO and a label to be incorporated into the extended strand during the extension reaction of the step (d), or (iv) a combination of the label linked to the SO and an intercalating dye.