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Probe set for identification of nucleotide mutation, and method for identification of nucleotide mutation

A technology of probe group and nucleotide, which is applied in the field of probe group for nucleotide mutation identification and nucleotide mutation identification, which can solve insufficient problems and achieve the effect of simple research

Inactive Publication Date: 2012-05-16
MITSUBISHI TANABE PHARMA CORP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Therefore, in cases where negative results are obtained, the identification of mutations requires additional assays and this method is insufficient

Method used

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  • Probe set for identification of nucleotide mutation, and method for identification of nucleotide mutation
  • Probe set for identification of nucleotide mutation, and method for identification of nucleotide mutation
  • Probe set for identification of nucleotide mutation, and method for identification of nucleotide mutation

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Experimental program
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Embodiment

[0243] Amino acid at position 156 of HCV-1b NS3 protease is Ala in the wild type, and as a result of nucleotide substitution, it is replaced with Val, Phe, Thr or Ser, etc. in the mutant virus. Here, real-time PCR was performed using the following fluorescent probes for the specific detection of mutations leading to amino acid substitutions at position 156 to Val, more specifically, the nucleotide encoding the NS3 protease Mutation of C→T substitution at position 467 of the sequence.

[0244]

[0245] In each of the upstream and downstream sides of the nucleotides encoding the 156-position amino acid, the primers were fixed to the HCVNS3 protease in patients enrolled in clinical trials of Teraprevir and in a public database (The Entrez Nucloetide Database) In the consensus region between the regions (positions 289 to 305 (Fw primer) and 487 to 503 (Re primer) of SEQ ID NO: 1.

[0246] Fw primer 5'-TGCACCTGCGGCAGCTC-3' (SEQ ID NO: 3)

[0247] Tm = 69.2°C (at the salt concen...

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Abstract

The type of a nucleotide located in a mutation site on a target nucleic acid can be identified by carrying out a fluorescence real-time PCR using a probe set comprising a detection probe and a counter probe, wherein the detection probe comprises an oligonucleotide which comprises a nucleotide sequence containing the mutation site on the target nucleic acid and also containing a nucleotide of interest in the mutation site or a nucleotide sequence complementary to the aforementioned nucleotide sequence, has a fluorescent substance added to the 5'-terminal and a quenching substance added to the 3'-terminal, and has, introduced therein, such a modification that the melting temperature of the probe becomes 3 DEG C or more higher than that of the counter probe, and wherein the counter probe comprises an oligonucleotide which comprises a nucleotide sequence containing a mutation site and also containing a nucleotide that is different from the nucleotide of interest in the mutation site or a nucleotide sequence complementary to the aforementioned nucleotide sequence.

Description

technical field [0001] The present invention relates to a probe set of nucleotide type for identifying a nucleotide mutation site, and a method for identifying a nucleotide mutation. The present invention also relates to probe sets for identifying mutations involved in drug resistance of viruses and / or the like, and methods for identifying mutations. Background technique [0002] Nucleotide mutations (including nucleotide polymorphisms) are factors that have a great influence on the phenotype of an organism, and studies of mutation types are often conducted in order to predict the phenotype thereof or predict the effect of drugs. Examples of known methods for identifying the type of mutated nucleotides include direct sequencing, invasion methods, methods using DNA chips on which polymorphism-specific probes are immobilized, and allele-specific PCR methods, but considering the identification These methods are insufficient in terms of labor required and detection sensitivity,...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/09
CPCC12Q1/707C12Q2561/113C12Q1/6825C12Q2600/156C12Q1/6858C12Q2600/136C12Y304/21098C12N9/506A61P31/14A61P43/00C12Q2525/113C12Q2535/131C12Q2537/163C12Q2561/101
Inventor 岸安宏神谷直洋
Owner MITSUBISHI TANABE PHARMA CORP
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