DETECTION OF TARGET NUCLEIC ACID SEQUENCE BY PTO CLEAVAGE AND EXTENSION ASSAY

摘要

PURPOSE: A method for detecting target nucleic acids by PTOCE(PTO cleavage and extension) assay is provided to obtain a target-dependent extension dimer and to completely overcome false positive signal problem. CONSTITUTION: A method for detecting nucleic acids from DNA or a nucleic acid mixture by PTOCE assay comprises: a step 1 of hybridizing a target nucleic acid sequence with an upstream oligonucleotide and PTO(probing and tagging oligonucleotide); a step 2 of contacting the resultant of step 1 to an enzyme with 5' nuclease activity under the condition for cleaving PTO; a step 3 of hybridizing CTO(capturing and templating oligonucleotide) with a fragment released from PTO; a step 4 of performing extension of the resultant of step 3 with template-dependent nucleic acid polymerase; a step 5 of melting the extension monomer at a predetermined temperature and providing a target signal; and a step 6 of measuring the target signal and detecting the extension dimer. [Reference numerals] (A) Hybridization; (AA) Upstream primer; (B) Primer extension and PTO cleaving; (BB) DNA or nucleic acid mixture; (C) Hybridization of PTO fragments and CTO; (CC) Downstream primer; (D) PTO fragment extension; (DD) Fragments emitted from PTO(PTO fragments); (E) Melting analysis; (EE) PTO fragments; (FF) Hybrid of non-cleaved PTO and CTP; (GG) Extension dimer