Detection of target nucleic acid sequence on solid phase by PTO cleavage and extension using HCTO assay

A method for detecting a target nucleic acid sequence from a DNA or a mixture of nucleic acids by a PCE-hCTO (PTO Cleavage and Extension using hCTO) assay on a solid phase, comprising:

• (a) hybridizing the target nucleic acid sequence with an upstream oligonucleotide and a probing and targeting oligonucleotide (PTO);
• said upstream oligonucleotide comprising a hybridizing nucleotide sequence complementary to the target nucleic acid sequence;
• said PTO comprising (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 PTO is 15-80 nucleotides in length, the 3′-targeting portion of the PTO is 10-50 nucleotides in length and the 5′-tagging portion of the PTO is 5-30 nucleotides in length;
• 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;
• and the upstream oligonucleotide is located upstream of the PTO;
• (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 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;
• wherein when the enzyme having a 5′ nuclease activity is a FEN (flap endonuclease) nuclease, the FEN nuclease is a 5′ flap-specific nuclease;
• (c) hybridizing the fragment released from the PTO with a hybridizing-capturing and templating oligonucleotide (hCTO);
• said hCTO comprising (i) a CTO in a 3′ to 5′ direction comprising 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 a templating portion comprising a nucleotide sequence non-complementary to the 5′-tagging portion and the 3′-targeting portion of the PTO, and (ii) at the 3′- or 5′-end of the CTO an JO-hybridizing portion comprising a nucleotide sequence complementary to an immobilized oligonucleotide (IO) immobilized on a solid substrate;
• wherein when the JO-hybridizing portion is located at the 5′-end of the CTO, said hCTO further comprising a blocker portion between the CTO and the JO-hybridizing portion to prevent extension reaction of the fragment such that the fragment is extended before a position opposite of the blocker portion;
• wherein the fragment released from the PTO is hybridized with the capturing portion of the hCTO;
• (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 hCTO is extended to form an extended duplex;
• wherein the extended duplex has a Tm value adjustable by (i) a sequence and/or length of the fragment, (ii) a sequence and/or length of the hCTO or (iii) the sequence and/or length of the fragment and the sequence and/or length of the hCTO;
• wherein the JO-hybridizing portion of the hCTO remains in a single strand state;
• (e) hybridizing the IO with the JO-hybridizing portion of the hCTO in the single strand state;
• said IO immobilized on the solid substrate comprising a nucleotide sequence complementary to the JO-hybridizing portion of the hCTO;
• wherein the IO is hybridized with the JO-hybridizing portion of the hCTO to form an IO duplex;
• wherein the extended duplex provides a target signal by (i) at least one label linked to the fragment and/or the hCTO, (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 hCTO, or (iv) a label linked to the fragment or incorporated into the extended duplex during the extension reaction and a label linked to the IO;
• and
• (f) detecting the extended duplex on the solid phase by measuring the target signal at a pre-determined temperature at which both the extended duplex and the IO duplex are in a double strand state;
• wherein the presence of the extended duplex indicates the presence of the target nucleic acid sequence;
• wherein the hybridization of the step (a) is performed under stringent conditions such that 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 hybridization of the step (c) is performed under stringent conditions such that the fragment released from the PTO is hybridized with the capturing portion of the hCTO to provide a form suitable in extension of the fragment;
• and the hybridization of the step (e) is performed under stringent conditions such that the IO is hybridized with the JO-hybridizing portion of the hCTO in the single strand state to form the IO duplex.