TSG primer target detection

Patent metadata

Field	Value
Publication number	US-9845492-B2
Application number	US-201013515975-A
Country	US
Kind code	B2
Filing date	Mar 26, 2010
Priority date	Dec 21, 2009
Publication date	Dec 19, 2017
Grant date	Dec 19, 2017

Abstract

The present invention relates to the detection of a target nucleic acid sequence in a real-time manner using a target signal generating primer (TSG primer) having dual interactive labels. The present invention allows for both target amplification and signal amplification by introducing dual interactive labels into a primer used in PCR reactions, ensuring real-time target detection by PCR reactions without the use of complicated oligonucleotides. The present invention could be free from the troublesome matters and shortcomings associated with conventional real-time PCR methods. The present invention allows for successful real-time target detection by using only a labeled primer. Also, the present invention can obtain strong signals indicative of the presence of target nucleic acid sequences in both a liquid phase and solid phase.

First claim

What is claimed is:

1. A method for detecting a target nucleic acid sequence from a DNA or a mixture of nucleic acids using a target signal generating primer (TSG primer) in an amplification reaction:
wherein said target signal generating primer (TSG primer) is not immobilized on a solid substrate; and
wherein said amplification reaction is in a liquid phase;
said method comprising the steps:
(a) hybridizing the target nucleic acid sequence with a primer pair composed of two primers as a forward primer and a reverse primer capable of amplifying the target nucleic acid sequence; wherein at least one of the two primers is the TSG primer; wherein the TSG primer comprises (i) a hybridizing nucleotide sequence complementary to the target nucleic acid sequence and (ii) a dual label system consisting of a single reporter molecule and a single non-fluorescent quencher molecule; wherein either or both of the reporter molecule and the quencher molecule is/are located on the hybridizing nucleotide sequence; wherein the reporter molecule and the quencher molecule are positioned at 6-40 nucleotides apart from each other; wherein the TSG primer is a single stranded oligonucleotide and has no substantial self-complementary sequence such that the TSG primer does not comprise hairpin loop structure(s) when it is not hybridized with the target nucleic acid sequence; wherein when the TSG primer is not hybridized with the target nucleic acid sequence, the reporter molecule and the quencher molecule are three-dimensionally adjacent to each other in a twist conformation, without assist by hairpin loop structure(s), to allow the quencher molecule to quench a signal from the reporter molecule, thereby generating no signal indicative of the presence of the target nucleic acid sequence; wherein when the TSG primer is hybridized with the target nucleic acid sequence, the reporter molecule and the quencher molecule are three-dimensionally separated in a stretch conformation to allow the quencher molecule to unquench the signal from the reporter molecule, whereby the signal indicative of the presence of the target nucleic acid sequence is generated and obtained without cleavage of the TSG primer before extension of the primer pair wherein the TSG primer does not comprise a minor groove binder (MGB);
(b) contacting the resultant of step (a) to a template-dependent nucleic acid polymerase under primer extension conditions such that the 3′-extension reaction at the 3′-ends of the two primers is induced;
(c) denaturing the resultant of step (b);
(d) repeating the steps (a)-(c) at least twice to amplify both the target nucleic acid sequence and the signal indicative of the presence of the target nucleic acid sequence; and
(e) detecting the signal indicative of the presence of the target nucleic acid sequence, wherein the detection is performed for each cycle of the repetition of step (d), at the end of the repetition of step (d) or at each of predetermined time intervals during the repetition, such that the signal indicates the presence of the target nucleic acid sequence; wherein at least some of the amplified target nucleic acid products comprises the reporter molecule and the quencher molecule remaining attached to the TSG primer, and the reporter molecule and the quencher molecule on the amplified target nucleic acid products provide the signal indicative of the presence of the target nucleic acid sequence; and wherein the method does not further include any other oligonucleotide comprising a hybridizing nucleotide sequence complementary to the target nucleic acid sequence.

2. The method according to claim 1 , wherein the template-dependent nucleic acid polymerase is a template-dependent nucleic acid polymerase having no 5′ to 3′ nuclease activity.

3. The method according to claim 1 , wherein the reporter molecule or the quencher molecule on the TSG primer is located at its 5′-end or at 1-5 nucleotides apart from its 5′-end.

4. The method according to claim 1 , wherein the template-dependent nucleic acid polymerase is a template-dependent nucleic acid polymerase having a 3′ to 5′ exonuclease activity.

5. The method according to claim 4 , wherein the TSG primer has at least one mismatch nucleotide having a backbone resistant to the 3′ to 5′ nuclease activity of template-dependent nucleic acid polymerases at its 3′-end portion.

6. A method for detecting at least two types of target nucleic acid sequences from a DNA or a mixture of nucleic acids using a target signal generating primer (TSG primer) not immobilized on a solid substrate in an amplification reaction in a liquid phase, comprising the steps of:
(a) hybridizing the at least two types of target nucleic acid sequences with at least two primer pairs, each primer pair being composed of two primers as a forward primer and a reverse primer capable of amplifying each of the target nucleic acid sequences; wherein at least one of the two primers is the TSG primer; wherein the TSG primer comprises (i) a hybridizing nucleotide sequence complementary to the target nucleic acid sequence and (ii) a dual label system consisting of a single reporter molecule and a single non-fluorescent quencher molecule; wherein either or both of the reporter molecule and the quencher molecule is located on the hybridizing nucleotide sequence; wherein the reporter molecule and the quencher molecule are positioned at 6-40 nucleotides apart from each other; wherein the TSG primer is a single-stranded oligonucleotide, and has no substantial self-complementary sequence such that the TSG primer does not comprise hairpin loop structure(s) when it is not hybridized with the target nucleic acid sequence; wherein when the TSG primer is not hybridized with the target nucleic acid sequence, the reporter molecule and the quencher molecule are three-dimensionally adjacent to each other in a twist conformation with no help of hairpin loop structures to allow the quencher molecule to quench a signal from the reporter molecule, thereby generating no signal indicative of the presence of the target nucleic acid sequence; wherein when the TSG primer is hybridized with the target nucleic acid sequence, the reporter molecule and the quencher molecule are three-dimensionally separated in a stretch conformation to allow the quencher molecule to unquench the signal from the reporter molecule, whereby the signal indicative of the presence of the target nucleic acid sequence is generated and obtained without cleavage of the TSG primer before extension of the primer pair; wherein the TSG primer does not comprise a minor groove binder (MGB);
(b) contacting the resultant of step (a) to a template-dependent nucleic acid polymerase under primer extension conditions such that the 3′-extension reaction at the 3′-ends of the two primers is induced;
(c) denaturing the resultant of step (b);
(d) repeating the steps (a)-(c) at least twice to amplify both the target nucleic acid sequence and the signal indicative of the presence of the target nucleic acid sequence; and
(e) detecting the signal indicative of the presence of the target nucleic acid sequence, wherein the detection is performed for each cycle of the repetition of step (d), at the end of the repetition of step (d) or at each of predetermined time intervals during the repetition, such that the signal indicates the presence of the target nucleic acid sequence; wherein at least some of the amplified target nucleic acid products comprises the reporter molecule and the quencher molecule remaining attached to the TSG primer, and the reporter molecule and the quencher molecule on the amplified target nucleic acid products provide the signal indicative of the presence of the target nucleic acid sequence; wherein the method does not further include any other oligonucleotide comprising a hybridi

Assignees

• Chun Jong Yoon
• Hwang In Taek
• Seegene Inc

Inventors

• Chun Jong Yoon
• Hwang In Taek

Classifications

• C12Q2565/1015

  labels being on the same oligonucleotide · CPC title

• C12Q1/6816 Primary

  characterised by the detection means (C12Q1/6804 takes precedence) · CPC title

• C12Q2565/137

  Chromatographic separation · CPC title

• C12Q2565/107

  Alteration in the property of hybridised versus free label oligonucleotides · CPC title

• C12Q1/34

  involving hydrolase · CPC title