| 摘要 |
Disclosed is 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) hybridising the target nucleic acid sequence with an upstream oligonucleotide and a PTO (Probing and Tagging Oligonucleotide); wherein the upstream oligonucleotide comprises a hybridising nucleotide sequence complementary to the target nucleic acid sequence; the PTO comprises (i) a 3’-targeting portion comprising a hybridising 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 hybridised with the target nucleic acid sequence and the 5’-tagging portion is not hybridised 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 such that the cleavage releases a fragment comprising the 5’-tagging portion or a part of the 5’-tagging portion of the PTO; (c) hybridising 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 hybridised 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 hybridised with the capturing portion of the CTO is extended and an 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; (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 target signal is provided by (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, or (iv) an intercalating label; and (f) 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 |
