Primer, probe, locked nucleic acid probe, kit and detection method for detecting PDGFRA gene hotspot mutation

A technology for locking nucleic acid probes and kits, which is applied in the field of probes, locking nucleotide probes, and primers for detecting PDGFRA gene mutations. It can solve the problems of low sensitivity and achieve high sensitivity, strong affinity, and high thermal stability. Effect

Active Publication Date: 2015-02-04
WUHAN BIOTECH GENE ENG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] In order to solve the problem of low sensitivity of the method for detecting PDGFRA gene mutation in the prior art, the embodimen

Method used

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  • Primer, probe, locked nucleic acid probe, kit and detection method for detecting PDGFRA gene hotspot mutation
  • Primer, probe, locked nucleic acid probe, kit and detection method for detecting PDGFRA gene hotspot mutation
  • Primer, probe, locked nucleic acid probe, kit and detection method for detecting PDGFRA gene hotspot mutation

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Embodiment 1

[0040] The embodiment of the present invention provides a primer and probe for detecting PDGFRA gene mutation, the primer and probe for detecting PDGFRA gene mutation include primers and probe for detecting the No. 12 exon of PDGFRA gene, and using At least one of the primers and probes for detecting the No. 18 exon of the PDGFRA gene, the primers include: forward primers and reverse primers, wherein,

[0041] The forward primer of exon 12 of the PDGFRA gene is shown in SEQ ID NO.1 in the sequence listing;

[0042] The reverse primer of exon 12 of PDGFRA gene is shown as SEQ ID NO.2 in the sequence listing;

[0043] The probe of exon 12 of PDGFRA gene is shown as SEQ ID NO.3 in the sequence listing;

[0044] The forward primer of exon 18 of PDGFRA gene is shown as SEQ ID NO.4 in the sequence listing;

[0045] The reverse primer of exon 18 of PDGFRA gene is shown as SEQ ID NO.5 in the sequence listing;

[0046] The probe of exon 18 of PDGFRA gene is shown as SEQ ID NO.6 in t...

Embodiment 2

[0050] The embodiment of the present invention provides a locked nucleic acid probe for detecting PDGFRA gene mutation, and the modified locked nucleic acid probe includes:

[0051] The locked nucleic acid probe whose mutation type is V561D in exon 12 of PDGFRA gene is shown as SEQ ID NO.7 in the sequence listing;

[0052] The locked nucleic acid probe whose mutation type is D842V in exon 18 of PDGFRA gene is shown as SEQ ID NO.8 in the sequence listing;

[0053] The 3' ends of the locked nucleic acid probes are all connected with PO4 groups.

[0054] Locked Nucleic Acid (LNA) is a kind of artificially synthesized antisense oligonucleotide used for detecting PDGFRA gene mutation lock nucleic acid probe provided by the embodiment of the present invention, in its structure β-D-ribofuranose The 2'-O, 4'-C positions form a ring-shaped oxymethylene bridge, a sulfide methylene bridge or an amine-methylene bridge rigid condensation structure through different shrinkages, and the rin...

Embodiment 3

[0056] The embodiment of the present invention provides a kit for detecting PDGFRA gene mutation, the kit includes: the primers and probes provided in the first embodiment of the present invention, the locked nucleic acid probe provided in the second embodiment of the present invention, PCR reaction solution, Positive quality control, negative quality control, sequencing reaction solution, digestive enzyme system and sequencing PCR product purification reagents.

[0057] Specifically, in the kit, 0.75 μl each of 10 μmol / L forward primers, 0.75 μl each of 10 μmol / L reverse primers, 0.5 μl each of 10 μmol / L probes, and 0.5 μl each of 10 μmol / L locked nucleic acid probes.

[0058] Specifically, the PCR reaction solution includes: 5 μl of 5×PCR buffer containing Mg2+, 1.5 μl of 2.5 mmol / L dNTPs, 0.2 μl of 5U / μl Taq enzyme and 2 μl of 20ng / μl template DNA.

[0059] Specifically, the digestive enzyme system includes alkaline phosphatase and exonuclease I, and the enzyme activity rat...

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Abstract

The invention discloses a primer, a probe, a locked nucleic acid probe, a kit and a detection method for detecting PDGFRA gene hotspot mutation, belonging to the technical field of biology. The primer and probe comprise at least one of the primers and probes for detecting a No.12 exon and a No.18 exon of a PDGFRA gene, the primer comprises a forward primer and a reverse primer, and the kit comprises the primers and probe. The primers and probe disclosed by the invention can be used for highly-sensitively detecting whether the PDGFRA gene has mutation, meanwhile, the sensitivity of detecting PDGFRA gene mutation can be greatly improved by adopting the locked nucleic acid probe, and whether the PDGFRA gene is mutated can be accurately detected by adopting the kit disclosed by the invention.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a primer, a probe, a locked nucleotide probe, a kit and a detection method for detecting PDGFRA gene mutation. Background technique [0002] Platelet-derived growth factor receptor alpha polypeptide (platelet-derived growth factor receptor alpha, PDGFRA) is a single-chain transmembrane protein that belongs to the type III tyrosine protein kinase family. The most common PDGFRA mutation site is the region encoding the activation loop located in the second tyrosine kinase domain (exon18), and a few mutations occur in the proximal membrane region (exon12). Mutations cause changes in the function of the PDGFRA protein, usually resulting in non-ligand β-dependent dimer formation, autophosphorylation of tyrosine residues, and activation of downstream signaling. The end result of this activation is tumorigenesis. [0003] Existing methods for detecting PDGFRA gene mutations include Sanger ...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6869C12Q1/6883C12Q2600/156C12Q2525/113
Inventor 林佳李品霍然唐景峰
Owner WUHAN BIOTECH GENE ENG
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