Detection of target nucleic acid sequences by PTO cleavage and extension-dependent extension assay

A method for detecting a target nucleic acid sequence from a DNA or a mixture of nucleic acids by a PTOCE-E (PTO Cleavage and Extension-dependent 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 to the target nucleic acid sequence;
• wherein the PTO comprises (i) a 3′-targeting portion comprising a hybridizing nucleotide sequence to the target nucleic acid sequence and (ii) a 5′-tagging portion comprising a non-hybridizing nucleotide sequence 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 of the PTO is not hybridized with the target nucleic acid sequence;
• 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 to release a PTO fragment comprising the 5′-tagging portion or a part of the 5′-tagging portion of the PTO;
• (c) hybridizing the PTO fragment with a first CTO (Capturing and Templating Oligonucleotide);
• wherein the first CTO comprises in a 3′ to 5′ direction (i) a capturing portion comprising a hybridizing nucleotide sequence to the PTO fragment and (ii) a templating portion comprising a non-hybridizing nucleotide sequence to the 5′-tagging portion and the 3′-targeting portion of the PTO;
• wherein the PTO fragment is hybridized with the capturing portion of the first CTO;
• (d) performing an extension reaction using the resultant of the step (c) and a template-dependent nucleic acid polymerase;
• wherein the PTO fragment hybridized with the capturing portion of the first CTO is extended to generate a first extended strand comprising a first extended sequence complementary to the templating portion of the first CTO, thereby forming a first extended duplex between the first extended strand and the first CTO;
• (e) hybridizing the first extended strand with a second CTO;
• wherein the second CTO comprises in a 3′ to 5′ direction (i) a capturing portion comprising a hybridizing nucleotide sequence to the first extended strand and (ii) a templating portion comprising a non-hybridizing nucleotide sequence to the first extended strand;
• wherein the first extended strand is hybridized with the capturing portion of the second CTO;
• (f) performing an extension reaction using the resultant of the step (e) and a template-dependent nucleic acid polymerase;
• wherein the first extended strand hybridized with the capturing portion of the second CTO is further extended to generate a second extended strand comprising a second extended sequence complementary to the templating portion of the second CTO, thereby forming a second extended duplex between the second extended strand and the second CTO;
• and
• (g) detecting the presence of the first extended strand;
• whereby the presence of the first extended strand indicates the presence of the target nucleic acid sequence.

A kit for detecting a target nucleic acid sequence from a DNA or a mixture of nucleic acids by a PTOCE-E (PTO Cleavage and Extension-dependent Extension) assay, comprising:

• (a) an upstream oligonucleotide comprising a hybridizing nucleotide sequence to the target nucleic acid sequence;
• (b) a PTO (Probing and Tagging Oligonucleotide);
• wherein the PTO comprises (i) a 3′-targeting portion comprising a hybridizing nucleotide sequence to the target nucleic acid sequence and (ii) a 5′-tagging portion comprising a non-hybridizing nucleotide sequence 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 of the PTO is not hybridized with the target nucleic acid sequence;
• the upstream oligonucleotide is located upstream of the PTO;
• wherein the upstream oligonucleotide or its extended strand induces cleavage of the PTO by the enzyme having the 5′ nuclease activity to release a PTO fragment comprising the 5′-tagging portion or a part of the 5′-tagging portion of the PTO;
• (c) a first CTO (Capturing and Templating Oligonucleotide);
• wherein the first CTO comprises in a 3′ to 5′ direction (i) a capturing portion comprising a hybridizing nucleotide sequence to the PTO fragment and (ii) a templating portion comprising a non-hybridizing nucleotide sequence to the 5′-tagging portion and the 3′-targeting portion of the PTO;
• wherein the PTO fragment is hybridized with the capturing portion of the first CTO;
• the PTO fragment hybridized with the capturing portion of the first CTO is extended to generate a first extended strand comprising a first extended sequence complementary to the templating portion of the first CTO, thereby forming a first extended duplex between the first extended strand and the first CTO;
• and
• (d) a second CTO;
• wherein the second CTO comprises in a 3′ to 5′ direction (i) a capturing portion comprising a hybridizing nucleotide sequence to the first extended strand and (ii) a templating portion comprising a non-hybridizing nucleotide sequence to the first extended strand;
• wherein the first extended strand hybridized with the capturing portion of the second CTO is further extended to generate a second extended strand comprising a second extended sequence complementary to the templating portion of the second CTO, thereby forming a second extended duplex between the second extended strand and the second CTO.

A kit for detecting a target nucleic acid sequence from a DNA or a mixture of nucleic acids, comprising a second CTO (Capturing and Templating Oligonucleotide), comprising in a 3′ to 5′ direction:

• (i) a capturing portion comprising a hybridizing nucleotide sequence to a first extended strand;
• and
• (ii) a templating portion comprising a non-hybridizing nucleotide sequence to the first extended strand;
• wherein the first extended strand is formed by cleaving a PTO (Probing and Tagging Oligonucleotide) hybridized with the target nucleic acid sequence with the enzyme having the 5′ nuclease activity to release a PTO fragment, hybridizing the PTO fragment with the capturing portion of a first CTO, and extending the PTO fragment by the template-dependent nucleic acid polymerase;
• 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 first CTO (Capturing and Templating Oligonucleotide) comprises in a 3′ to 5′ direction (i) a capturing portion comprising a hybridizing nucleotide sequence to the PTO fragment and (ii) a templating portion comprising a non-hybridizing nucleotide sequence to the 5′-tagging portion and the 3′-targeting portion of the PTO.