Detection of target nucleic acid sequence by PTO cleavage and extension-dependent cleavage

Patent metadata

Field	Value
Publication number	US-11078525-B2
Application number	US-201214008096-A
Country	US
Kind code	B2
Filing date	Mar 29, 2012
Priority date	Mar 29, 2011
Publication date	Aug 3, 2021
Grant date	Aug 3, 2021

Abstract

The present invention relates to the detection of a target nucleic acid sequence by a PCEC (PTO Cleavage and Extension-Dependent Cleavage) assay. The present invention is characterized by generating a cleavage site for a nucleolytic enzyme on the extended duplex of which the formation is dependent on the presence of a target nucleic acid sequence. The present invention detects the occurrence of the cleavage of the extended duplex, thereby determining the presence of the target nucleic acid sequence.

First claim

What is claimed is:

1. A method for detecting a target nucleic acid sequence from a DNA sample or a mixture of nucleic acids by a Probing and Tagging Oligonucleotide (PTO) Cleavage and Extension-Dependent Cleavage (PCEC) assay, comprising:
(a) hybridizing the target nucleic acid sequence in a DNA sample or a mixture of nucleic acids with an upstream oligonucleotide and a PTO, thereby forming a target nucleic acid sequence hybridized with the upstream oligonucleotide and the PTO if the target nucleic acid sequence is present in the DNA sample or the mixture of nucleic acids; 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; wherein when the upstream oligonucleotide and the PTO are both hybridized with the target nucleic acid sequence, the upstream oligonucleotide is hybridized upstream of the PTO; wherein the PTO is blocked at its 3′-end to prohibit its extension;
(b) contacting the target nucleic acid sequence hybridized with the upstream oligonucleotide and the PTO of the step (a) to a DNA polymerase having a 5′ nuclease activity under conditions for extension of the upstream oligonucleotide and cleavage of the PTO such that said cleavage of the PTO by the DNA polymerase having the 5′ nuclease activity generates a fragment comprising the 5′-tagging portion or a part of the 5′-tagging portion of the PTO if the target nucleic acid sequence is present in the DNA sample or the mixture of nucleic acids;
(c) hybridizing the fragment comprising the 5′-tagging portion or a part of the 5′-tagging portion of the PTO with a Capturing and Templating Oligonucleotide (CTO) if the target nucleic acid sequence is present in the DNA sample or the mixture of nucleic acids; 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 the part of the 5′-tagging portion of the PTO and non-complementary to the 3′-targeting 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 CTO has no hairpin structure; wherein the CTO has a single fluorescent label or an interactive dual label comprising a fluorescent reporter molecule and a quencher molecule at its templating portion; wherein the fragment comprising the 5′-tagging portion or a part of the 5′-tagging portion of the PTO is hybridized with the capturing portion of the CTO such that a hybridized complex formed by the fragment comprising the 5′-tagging portion or a part of the 5′-tagging portion of the PTO and the CTO is produced; wherein the CTO is blocked at its 3′-end to prohibit its extension;
(d) performing an extension reaction in the presence of the hybridized complex of the step (c) and the DNA polymerase having the 5′ nuclease activity if the target nucleic acid sequence is present in the DNA sample or the mixture of nucleic acids; wherein the hybridized complex of the step (c) is extended such that an extended duplex having the single fluorescent label or the interactive dual label comprising a fluorescent reporter molecule and a quencher molecule is formed; wherein the extended duplex can be cleaved by (i) a restriction enzyme, wherein a cleavage site recognized by the restriction enzyme is present in the templating portion of the CTO; or (ii) a 5′ to 3′ exonuclease capable of cleaving the CTO of the extended duplex but not cleaving the CTO in a single-stranded state;
(e) forming a cleaved fragment by cleaving the extended duplex using the restriction enzyme or the 5′ to 3′ exonuclease if the target nucleic acid sequence is present in the DNA sample or the mixture of nucleic acids;
when the extended duplex has the single fluorescent label, the method is performed on a solid substrate on which the CTO is immobilized, the single fluorescent label is linked to the templating portion of the CTO, the cleaved fragment is with the single fluorescent label, and the cleaved fragment is released from the solid substrate; wherein when the CTO is immobilized through its 5′-end onto the solid substrate, the cleavage site for the restriction enzyme is located between the 5′-end of the CTO and the position of the single fluorescent label of the CTO, and when the CTO is immobilized through its 3′-end onto the solid substrate, the cleavage site for the restriction enzyme is located between the 3′-end of the CTO and the position of the single fluorescent label of the CTO;
when the extended duplex has said interactive dual label comprising a fluorescent reporter molecule and a quencher molecule, the quencher molecule of the CTO quenches a fluorescent signal from the fluorescent reporter molecule of the CTO prior to the formation of the hybridized complex of the step (c) and after formations of the hybridized complex of the step (c) and the extended duplex, the fluorescent reporter molecule and the quencher molecule of the CTO are separated by cleavage at the cleavage site of the CTO by the restriction enzyme or by sequential cleavage from the 5′-end of the CTO by the 5′ to 3′ exonuclease, wherein the cleavage site for the restriction enzyme is located between the fluorescent reporter molecule of the CTO and the quencher molecule of the CTO; and

(f) detecting the target nucleic acid sequence from the DNA sample or the mixture of nucleic acids, wherein detecting a fluorescent signal from the single fluorescent label of the cleaved fragment or the fluorescent reporter molecule separated from the quencher molecule of the extended duplex indicates the presence of the target nucleic acid sequence in the DNA sample or the mixture of nucleic acids.

2. The method according to claim 1 , wherein the DNA polymerase having the 5′ nuclease activity has a 5′ to 3′ exonuclease activity.

3. The method according to claim 1 , wherein the upstream oligonucleotide is an upstream primer or an upstream probe.

4. The method according to claim 1 , wherein the method further comprises repeating the steps (a)-(b), (a)-(d), (a)-(e) or (a) (f) after step (f).

5. The method according to claim 1 , wherein the upstream oligonucleotide is an upstream primer.

6. The method according to any one of claims 1 , 2 , 3 , 4 , and 5 , wherein step (a) is performed in the presence of a downstream primer.

7. A method for detecting a target nucleic acid sequence from a DNA sample or a mixture of nucleic acids by a Probing and Tagging Oligonucleotide (PTO) Cleavage and Extension-Dependent Cleavage (PCEC) assay, comprising:
(a) hybridizing the target nucleic acid sequence in a DNA sample or a mixture of nucleic acids with an upstream primer a downstream primer and a PTO, thereby forming a target nucleic acid sequence hybridized with the upstream primer, the downstream primer and the PTO if the target nucleic acid sequence is present in the DNA sample or the mixture of nucleic acids; wherein each of the upstream primer and the downstream primer 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′-t

Assignees

• Chun Jong Yoon
• Lee Young Jo
• Seegene Inc

Inventors

• Chun Jong Yoon
• Lee Young Jo

Classifications

• C12Q1/6853

  using modified primers or templates · CPC title

• C12Q1/683

  involving restriction enzymes, e.g. restriction fragment length polymorphism [RFLP] · CPC title

• C12Q1/6823 Primary

  Release of bound markers · CPC title

• C12Q1/6834

  Enzymatic or biochemical coupling of nucleic acids to a solid phase · CPC title

• C12Q2521/319

  Exonuclease · CPC title