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

摘要

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.

主权项

1. A kit for detecting a target nucleic acid sequence from a DNA or a mixture of nucleic acids, comprising a CTO (Capturing and Templating Oligonucleotide), comprising in a 3′ to 5′ direction:
(a) a capturing portion comprising a nucleotide sequence complementary to a 5′-tagging portion or a part of the 5′-tagging portion of a PTO (Probing and Tagging Oligonucleotide) comprising (i) a 3′-targeting portion comprising a hybridizing nucleotide sequence complementary to a target nucleic acid sequence, and (ii) the 5′-tagging portion comprising a nucleotide sequence non-complementary to the target nucleic acid sequence; and (b) a templating portion comprising a nucleotide sequence non-complementary to the 5′-tagging portion and the 3′-targeting portion of the PTO; wherein the CTO has a single label or an interactive dual label comprising a reporter molecule and a quencher molecule; wherein the 3′-targeting portion of the PTO is hybridized with the target nucleic acid sequence and the 5′-tagging portion is not hybridized with the target nucleic acid sequence; wherein an upstream oligonucleotide or its extended strand induces cleavage of the PTO hybridized with the target nucleic acid sequence by an 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; wherein the fragment released from the PTO is hybridized with the capturing portion of the CTO; wherein the fragment hybridized with the capturing portion of the CTO is extended to form an extended duplex; wherein the extended duplex provides a target signal.