Patent NZ-706144-A (NZ)
Title: Detection of target nucleic acid sequences by pto cleavage and extension assay
Inventor: CHUN JONG YOON
IPC: C12Q1/68
Filed: 2012-01-11
Abstract: 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 in a liquid phase or in a solid phase is disclosed. For both phases - the method comprises (a) hybridizing the target nucleic acid sequence with an upstream oligonucleotide and a PTO (Probing and Tagging Oligonucleotide); where the upstream oligonucleotide comprises a hybridizing nucleotide sequence complementary to the target nucleic acid sequence; 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; where 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 upstream 15 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; where 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); where 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; where the fragment released from the PTO is hybridized with the capturing portion of the CTO and (d) performing an extension reaction using the resultant of the step (c) and a template-dependent nucleic acid polymerase; where the fragment hybridized with the capturing portion of the CTO is extended and an extended duplex is formed; where 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; where 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. For the liquid phase the method further comprises (e) detecting the extended duplex by measuring the target signal in a liquid phase at a predetermined temperature that the extended duplex maintains its double-stranded form, whereby the presence of the extended duplex indicates the presence of the target nucleic acid sequence; where the extended duplex is not immobilized on a solid substrate. For the solid phase the method further comprises (e) detecting the extended duplex by measuring the target signal on the solid substrate at a predetermined temperature that the extended duplex maintains its double-stranded form, whereby the presence of the extended duplex indicates the presence of the target nucleic acid sequence; where the extended duplex is immobilized on the solid substrate through the CTO.