Detection of target nucleic acid sequences by PO cleavage and hybridization

Patent metadata

Field	Value
Publication number	US-9850524-B2
Application number	US-201214114253-A
Country	US
Kind code	B2
Filing date	May 3, 2012
Priority date	May 4, 2011
Publication date	Dec 26, 2017
Grant date	Dec 26, 2017

Abstract

The present invention relates to the detection of a target nucleic acid sequence by a POCH (PO Cleavage and Hybridization) assay on a solid substrate. The present invention detects the target nucleic acid sequence by use of in which the PO (Probing Oligonucleotide) hybridized with the target nucleic acid sequence is cleaved and the cleavage of the PO is detected by hybridization with the CO (Capturing Oligonucleotide). In the present invention, an uncleaved PO is hybridized with the CO immobilized onto the solid substrate. The designs of the PO and the CO are convenient and the optimization of reaction conditions is routinely easy in the present invention. Where the detection of signal on the solid substrate is continuously performed along with repetition of cleavage of the POs in the present invention, the number of the POs cleaved is increased upon the repetition number of the cleavage reaction and the signal is changed in parallel with the number of the POs cleaved. Then, the target nucleic acid sequence can be detected in a real-time manner. In contrast, the change of the signal is not observed in the absence of the target nucleic acid sequence.

First claim

What is claimed is:

1. A method for detecting at least one target nucleic acid molecule having sequence information from a DNA or a mixture of nucleic acids in a sample by a POCH (PO Cleavage and Hybridization) assay on a solid substrate, comprising:
(a) hybridizing the sequence of said target nucleic acid molecule with an upstream oligonucleotide and a probing oligonucleotide (PO); wherein the upstream oligonucleotide comprises a hybridizing nucleotide sequence complementary to the sequence of said target nucleic acid molecule; the PO comprises a targeting portion comprising a hybridizing nucleotide sequence complementary to the sequence of said target nucleic acid molecule; the PO has a single label; the upstream oligonucleotide is located upstream of the PO; the upstream oligonucleotide or its extended strand induces cleavage of the PO by an enzyme having a 5′ nuclease activity;
(b) contacting the resultant of the step (a) to the enzyme having the 5′ nuclease activity under conditions for cleavage of the PO; wherein when the PO is hybridized with the sequence of said target nucleic acid molecule, the PO is cleaved by the enzyme having the 5′ nuclease activity to produce a single label-containing fragment;
(c) performing a hybridization reaction by contacting the resultant of the step (b) to a capturing oligonucleotide (CO) immobilized onto the solid substrate; wherein the CO comprises a nucleotide sequence hybridizable with the PO; wherein the hybridization reaction is performed under conditions such that the single label-containing fragment is not hybridized with the CO and an uncleaved PO is hybridized with the CO to form an uncleaved PO/CO duplex; and
(d) detecting a signal from the single label on the solid substrate; wherein (i) when a signal difference is observed by comparing the signal from the single label on the solid substrate to a signal from a control that does not contain the target nucleic acid sequence, the signal difference indicates the presence of the target nucleic acid sequence, or (ii) when a signal change is observed at cycles with predetermined interval by detecting the signal from the single label on the solid substrate along with repetition of the steps (a)-(b), (a)-(c) or (a)-(d), the signal change indicates the presence of the target nucleic acid sequence.

2. The method according to claim 1 , wherein the nucleotide sequence hybridizable with the PO in the CO comprises a nucleotide sequence hybridizable with the targeting portion of the PO.

3. The method according to claim 1 , wherein the PO is a 3′-tagged PO further comprising in its 3′-portion a tagging portion having a nucleotide sequence non-complementary to the sequence of said target nucleic acid molecule or a 5′-tagged PO further comprising in its 5′-portion a tagging portion having a nucleotide sequence non-complementary to the sequence of said target nucleic acid molecule, and the nucleotide sequence hybridizable with the PO in the CO comprises a nucleotide sequence hybridizable with the tagging portion of the PO.

4. The method according to claim 3 , wherein the single label is positioned such that the single label is not remained on a tagging portion-containing fragment released by cleavage of the PO.

5. The method according to claim 1 , wherein the PO is a 3′-tagged PO further comprising in its 3′-portion a tagging portion having a nucleotide sequence non-complementary to the sequence of said target nucleic acid molecule or a 5′-tagged PO further comprising in its 5′-portion a tagging portion having a nucleotide sequence non-complementary to the sequence of said target nucleic acid molecule, and the nucleotide sequence hybridizable with the PO in the CO comprises a nucleotide sequence hybridizable with a part of the tagging portion and a part of the targeting portion of the PO.

6. The method according to claim 1 , wherein the PO and/or CO is blocked at its 3′-end to prohibit its extension.

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

8. The method according to claim 1 , wherein the upstream oligonucleotide has a partial-overlapped sequence with the targeting portion of the PO.

9. The method according to claim 1 , wherein the enzyme having the 5′ nuclease activity is a thermostable DNA polymerase having a 5′ nuclease activity or FEN nuclease.

10. The method according to claim 1 , wherein the upstream oligonucleotide is a upstream primer and the enzyme having the 5′ nuclease activity is a template-dependent nucleic acid polymerase having a 5′ nuclease activity.

11. The method according to claim 1 , wherein the method further comprises repeating the steps (a)-(b), (a)-(c) or (a)-(d) with denaturation between repeating cycles.

12. The method according to claim 1 , wherein the steps (a)-(d) are performed in a reaction vessel or in separate reaction vessels.

13. The method according to claim 1 , wherein the sequence of said target nucleic acid molecule comprises at least two types of target nucleic acid molecule.

14. The method according to claim 1 , wherein the sequence of said target nucleic acid molecule comprises a nucleotide variation.

15. The method according to claim 1 , wherein the method is performed in the presence of a downstream primer.

16. A method for detecting at least one target nucleic acid molecule having sequence information from a DNA or a mixture of nucleic acids in a sample by a POCH (PO Cleavage and Hybridization) assay on a solid substrate, comprising:
(a) hybridizing the sequence of said target nucleic acid molecule with an upstream oligonucleotide and a probing oligonucleotide (PO); wherein the upstream oligonucleotide comprises a hybridizing nucleotide sequence complementary to the sequence of said target nucleic acid molecule; the PO comprises a targeting portion comprising a hybridizing nucleotide sequence complementary to the sequence of said target nucleic acid molecule; the PO has an interactive dual label comprising a donor molecule and an acceptor molecule; the upstream oligonucleotide is located upstream of the PO; the upstream oligonucleotide or its extended strand induces cleavage of the PO by an enzyme having a 5′ nuclease activity;
(b) contacting the resultant of the step (a) to the enzyme having the 5′ nuclease activity under conditions for cleavage of the PO; wherein when the PO is hybridized with the sequence of said target nucleic acid molecule, the PO is cleaved by the enzyme having the 5′ nuclease activity to separate the interactive dual label, whereby a donor-containing fragment and an acceptor-containing fragment are produced;
(c) performing a hybridization reaction by contacting the resultant of the step (b) to a capturing oligonucleotide (CO) immobilized onto the solid substrate; wherein the CO comprises a nucleotide sequence hybridizable with the PO; wherein the hybridization reaction is performed under conditions such that at least one of the donor-containing fragment and the acceptor-containing fragment is not hybridized with the CO and an uncleaved PO is hybridized with the CO to form an uncleaved PO/CO duplex; wherein a signal from the uncleaved PO/CO duplex is differentiated from a signal provided at the time that at least one of the donor-containing fragment and the acceptor-containing fragment is not hybridized with the CO; and
(d) detecting a signal from the interactive dual label on the solid substrate; wherein (i) when a signal difference is observed by comparing the signal from the interactive dual label on the solid substrate to a signal from a control that does not contain the target nucleic

Assignees

• Chun Jong Yoon
• Lee Young Jo
• Seegene Inc

Inventors

• Chun Jong Yoon
• Lee Young Jo

Classifications

• C12Q1/6823 Primary

  Release of bound markers · CPC title

• C12Q2565/519

  characterised by the capture moiety being a single stranded oligonucleotide · CPC title

• C12Q2521/319

  Exonuclease · CPC title