Method for detecting target nucleic acid

a nucleic acid and target technology, applied in the field of target nucleic acid detection methods and kits, can solve the problems of poor detection efficiency and difficult design of molecular beacon methods, and achieve the effect of simple manner

Inactive Publication Date: 2007-07-05
INOSE KEN
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

Enables simple and quantitative real-time detection of target nucleic acids, preferentially binding to the target sequence, and providing clear signal differences for quantification, applicable to various reaction systems including PCR amplification.

Problems solved by technology

Although the methods for quantifying PCR amplified products in real time have been known as described above, they have problems; the Taq-Man probe method cannot be applied in the case of amplification methods that use polymerases having no 5′→3′ exonuclease activity, the molecular beacon method is difficult to design a probe and suffers poor detection efficiency due to the intermolecular bond, and the intercalator method has no sequence specificity.

Method used

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  • Method for detecting target nucleic acid
  • Method for detecting target nucleic acid

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example

[0039] Hereinafter, the present invention will be described in more detail by examples.

[0040] Probe 1 (SEQ ID NO: 1), Probe 2 (SEQ ID NO: 2), and a target oligonucleotide (SEQ ID NO: 3) were synthesized. The synthesis of each oligonucleotide was requested to Japan Bio Service Co., Ltd. The nucleotide (T) of the base 21 of Probe 2 was labeled with FITC and the nucleotide (T) of base 52 was labeled with Texas Red. Note that the target oligonucleotide is a partial sequence of human amylin gene.

[0041] The bases 1 to 28 of Probe 1 are complementary to the bases 6 to 33 of the target oligonucleotide. The bases 11 to 3.3 and bases 35 to 55 of Probe 1 are complementary to the bases 52 to 74 and bases 1 to 21 of Probe 2, respectively. Note that the bases 57 to 74 of SEQ ID NO: 2 is homologous to the bases 6 to 23 of SEQ ID NO: 3.

[0042] Each oligonucleotide was dissolved in TE buffer to 5 μM. In a 1.5-ml tube were added 2.2 μl of 10×Ex Taq buffer (Takara Shuzo Co., Ltd., Lot. A6501-1), 19....

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Abstract

A target nucleic acid having a target sequence in a sample is detected according to the steps of: (a) mixing a first probe including a nucleic acid which has a specific region having a sequence complementary to the target sequence and a nonspecific region having a sequence that is not complementary to the target sequence of the target nucleic acid; a second probe including a nucleic acid which has a first region that is complementary to at least a portion of the nonspecific region of the first probe, a loop region that does not have a sequence complementary to the first probe, and a second region that is complementary to at least a portion of the specific region of the first probe, the loop region being capable of forming a loop when it is annealed with the first probe, wherein the nucleic acid is labeled with a labeling material generating a signal by which formation of the aforementioned loop can be detected; and a sample under conditions in which the first probe and the second probe are annealed and the first probe and the target nucleic acid are annealed; and (b) detecting a signal of the labeling material.

Description

TECHNICAL FIELD [0001] The present invention relates to a method and kit for detecting a target nucleic acid having a target sequence in a sample. The method and kit of the present invention permit the target nucleic acid to be detected in real time and are useful in fields of biochemistry and so forth. BACKGROUND ART [0002] A method for detecting a target nucleic acid having a target sequence in a sample, which has been used, includes a hybridization method in which a probe is used, a PCR method in which oligonucleotide primers are used, and other methods. Further, the PCR method is generally used in various fields including the detection and cloning of target nucleic acid, and various improved methods have been developed. [0003] A so-called real time PCR has been known, which is a PCR method that performs amplification of a target sequence and analysis of the amplified product simultaneously. Means for analyzing the amplified products that has been known include, for example, a Ta...

Claims

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Application Information

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Patent Type & AuthorityApplications(United States)
IPC IPC(8): C12Q1/68C07H21/04
CPCC12Q1/6818C12Q2565/101C12Q2525/301C12Q2525/161C12Q2565/1015C12Q2537/1373
InventorINOSE, KEN
OwnerINOSE KEN