DETECTION OF TARGET NUCLEIC ACID SEQUENCE BY PTO CLEAVAGE AND EXTENSION-DEPENDENT NON-HYBRIDIZATION ASSAY

摘要

The present invention relates to the detection of a target nucleic acid sequence by a PCE-NH (PTO Cleavage and Extension-Dependent Non-Hybridization) assay. The present invention adopts the occurrence of the inhibition of the hybridization between the HO with the CTO by the formation of the target-dependent extended duplex. Therefore, the present invention may detect target sequences even when the HO is not cleaved. In this regard, the design of the 5′-tagging portion of PTO, CTO and HO sequences may be readily performed and the conditions for reactions may be also easily established. In addition, the detection of the hybrid between the CTO and the HO may be performed in a different vessel from that for the extension of the CTO.

主权项

1. A method for detecting a target nucleic acid sequence in a nucleic acid sample by a PCE-NH (PTO Cleavage and Extension-Dependent Non-Hybridization) 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; 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 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 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; 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 to produce an extended strand complementary to the templating portion of the CTO and an extended duplex is formed; wherein when the target nucleic acid sequence is not present in the nucleic acid sample, the extended duplex is not formed; (e) performing a melting analysis or a hybridization analysis for the resultant of the step (d) over a range of temperatures with a HO (hybridizing oligonucleotide) comprising a hybridizing nucleotide sequence complementary to the CTO; wherein when the target nucleic acid sequence is not present in the nucleic acid sample, the extended duplex is not formed and the CTO and the HO form a hybrid, thereby providing a signal indicative of the presence of the hybrid between the CTO and the HO; wherein when the target nucleic acid sequence is present in the nucleic acid sample, the extended duplex is formed to prevent the formation of the hybrid between the CTO and the HO, thereby not providing the signal; wherein the signal is provided by (i) a label linked to the HO, (ii) a label linked to the CTO, (iii) a label linked to the HO and a label linked to the CTO, or (iv) an intercalating label; and (f) detecting the signal indicative of the presence of the hybrid between the CTO and the HO; wherein the presence of the hybrid between the CTO and the HO indicates the absence of the target nucleic acid sequence; wherein the absence of the hybrid between the CTO and the HO indicates the presence of the target nucleic acid sequence.