# US 11,306,349 B2 · Detection of target nucleic acid sequences by PTO cleavage and extension assay > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 11,306,349 B2 - Document ID: US11306349B2 - Status: Granted - Kind: B2 - Application number: 16/700,229 - Filed: 2019-12-02 - Published: 2020-03-19 - Granted: 2022-04-19 - Earliest claimed: 2011-01-11 - Assignees: Seegene, Inc. - Applicants: Seegene, Inc. - Inventors: Jong Yoon Chun; Young Jo Lee - Web page: https://patentgrep.com/patent/US11306349B2 ## CPC - C12Q 1/6837: using probe arrays or probe chips (C12Q1/6874 takes precedence) - C12Q 1/6818: involving interaction of two or more labels, e.g. resonant energy transfer - C12Q 1/6851: Quantitative amplification - C12Q 1/6853: using modified primers or templates - C12Q 1/6858: Allele-specific amplification - C12Q 1/6869: Methods for sequencing - C12Q 1/683: involving restriction enzymes, e.g. restriction fragment length polymorphism [RFLP] - C12Q 2525/161: incorporating target specific and non-target specific sites - C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES ## Abstract The present invention relates to the detection of a target nucleic acid sequence by a PTOCE (PTO Cleavage and Extension) assay. The present invention detects a target nucleic acid sequence in which the PTO (Probing and Tagging Oligonucleotide) hybridized with the target nucleic acid sequence is cleaved to release a fragment and the fragment is hybridized with the CTO (Capturing and Templating Oligonucleotide) to form an extended duplex, followed by detecting the presence of the extended duplex. The extended duplex provides signals (generation, increase, extinguishment or decrease of signals) from labels indicating the presence of the extended duplex and has adjustable Tm value, which are well adoptable for detection of the presence of the target nucleic acid sequence. ## Claims ### Claim 1 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; 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 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; (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; and wherein the fragment released from the PTO is hybridized with the capturing portion of the CTO; (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 and a extended duplex is formed; 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 CTO or (iii) the sequence and/or length of the fragment and the sequence and/or length of the CTO; and wherein the extended duplex provides a target signal by (i) at least one label linked to the fragment and/or CTO, (ii) a label incorporated into the extended duplex during the extension reaction, (iii) at least one label linked to the fragment and/or CTO and a label incorporated into the extended duplex during the extension reaction or (iv) intercalating label; and (e) detecting the extended duplex by measuring the target signal, whereby the presence of the extended duplex indicates the presence of the target nucleic acid sequence; wherein the method does not use any additional oligonucleotide comprising a nucleotide sequence complementary to either strand of the extended duplex. ### Claim 2 (depends on 1) The method of claim 1, wherein the target signal is provided by at least one label linked to the fragment and/or the CTO. ### Claim 3 (depends on 2) The method of claim 2, wherein the fragment or the CTO has an interactive dual label comprising a reporter molecule and a quencher molecule. ### Claim 4 (depends on 2) The method of claim 2, wherein the fragment has one of an interactive dual label comprising a reporter molecule and a quencher molecule and the CTO has the other of the interactive dual label. ### Claim 5 (depends on 2) The method of claim 2, wherein the fragment or the CTO has a single label. ### Claim 6 (depends on 1) The method of claim 1, wherein the target signal is provided by a single label incorporated into the extended duplex during the extension reaction; and wherein the incorporated single label is linked to a nucleotide incorporated during the extension reaction. ### Claim 7 (depends on 6) The method of claim 6, wherein the nucleotide incorporated during the extension reaction has a first non-natural base and the CTO has a nucleotide having a second non-natural base with a specific binding affinity to the first non-natural base. ### Claim 8 (depends on 1) The method of claim 1, wherein the target signal is provided by a label incorporated into the extended duplex during the extension reaction and a label linked to the fragment and/or the CTO; wherein the label incorporated is linked to a nucleotide incorporated during the extension reaction; wherein the two labels are an interactive dual label of a reporter molecule and a quencher molecule. ### Claim 9 (depends on 8) The method of claim 8, wherein the nucleotide incorporated during the extension reaction has a first non-natural base and the CTO has a nucleotide having a second non-natural base with a specific binding affinity to the first non-natural base. ### Claim 10 (depends on 1) The method of claim 1, wherein the upstream oligonucleotide is an upstream primer or an upstream probe. ### Claim 11 (depends on 10) The method of claim 10, wherein the upstream primer induces through its extended strand the cleavage of the PTO by the enzyme having the 5′ nuclease activity. ### Claim 12 (depends on 1) The method of claim 1, wherein the method further comprises repeating the steps (a)-(b), (a)-(d) or (a)-(e) with denaturation between repeating cycles. ### Claim 13 (depends on 1) The method of claim 1, wherein the steps (a)-(e) are performed in one or more reaction vessels. ### Claim 14 (depends on 1) The method of claim 1, wherein the method is performed to detect at least two types of target nucleic acid sequences; wherein the upstream oligonucleotide comprises at least two types of oligonucleotides, the PTO comprises at least two types of the PTOs, and the CTO comprises at least one type of the CTO. ### Claim 15 (depends on 1) The method of claim 1, wherein the method is performed in the presence of a downstream primer.