Patent ID: 8809239

Claim:
A method for detecting a target nucleic acid sequence from a DNA or a mixture of nucleic acids by a PTOCE (PTO Cleavage and Extension) assay, comprising: (a) hybridizing the target nucleic acid sequence with an upstream oligonucleotide and a PTO (Probing and Tagging Oligonucleotide), wherein the upstream oligonucleotide comprises a hybridizing nucleotide sequence complementary to the target nucleic acid sequence, 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 is hybridized with the target nucleic acid sequence and the 5′-tagging portion is not hybridized with the target nucleic acid sequence, and wherein the upstream oligonucleotide is located upstream of the PTO; (b) contacting the hybridized target nucleic acid sequence of the step (a) with an enzyme having a 5′ nuclease activity under conditions for cleavage of the PTO, wherein the upstream oligonucleotide or its extended strand induces cleavage of the PTO 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 CTO (Capturing and Templating Oligonucleotide), 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; (d) reacting the resultant of the step (c) with a template-dependent nucleic acid polymerase; to extend the fragment hybridized with the capturing portion of the CTO and form an extended duplex, wherein the extended duplex has a T m value adjustable by at least one of (i) a sequence of the fragment, (ii) a sequence of the CTO, (iii) a length of the fragment, and (iv) a length of the CTO; (e) melting the extended duplex over a range of temperatures to give a target signal indicative of the presence of the extended duplex; wherein the melting of the extended duplex generates a target signal comprising at least one of (i) at least one label linked to the fragment and/or the CTO, (ii) a label incorporated into the extended duplex during the extension reaction, (iii) a label incorporated into the extended duplex during the extension reaction and a label linked to the fragment and/or the CTO, and (iv) an intercalating label; and (f) measuring the target signal to detect the presence of the extended duplex, whereby the presence of the extended duplex indicates the presence of the target nucleic acid sequence.