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Primer used for detecting c-kit gene mutation and application thereof

A genome and primer sequence technology, applied to the primers used to detect c-kit gene mutation and its application field, can solve the problems of slow disease progression and prolongation of average survival period, and achieve the effect of high reference significance

Inactive Publication Date: 2011-05-18
SHANGHAI BIOTECAN PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Some foreign clinical studies have shown that mutations in exon 11 of the kit gene encoding the near-transmembrane domain are the most common, accounting for 50% to 92%, followed by exon 9 of the extracellular domain, accounting for 8 to 13%. Moreover, compared with those without mutations and exon 9 mutations, patients with mutations in exon 11 had a significantly higher partial response rate to imatinib, longer average survival time, and slower disease progression.

Method used

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  • Primer used for detecting c-kit gene mutation and application thereof
  • Primer used for detecting c-kit gene mutation and application thereof
  • Primer used for detecting c-kit gene mutation and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1 A method for identifying mutations in exon 9 of kit

[0031] 1. Extract patient tumor tissue or blood genomic DNA;

[0032] 2. Carry out PCR amplification. The 20 μl PCR reaction system is as follows: 50-100 ng of tumor tissue or blood genomic DNA, 0.125 μl of Taq enzyme, 1 μl of upstream and downstream primers (10 μM), 2 μl of dNTP (2.5 mM), 10×PCR buffer solution (containing Mg2+) 2 μl, and the rest was sterile distilled water; PCR reaction conditions were: denaturation at 95°C for 2 min, followed by denaturation at 95°C for 30 sec, annealing at 55°C for 30 sec, extension at 72°C for 1 min, 45 cycles, and finally extension at 72°C for 7 min. Store at 4°C;

[0033] 3. Gel electrophoresis analysis of PCR amplification products:

[0034] a. Rinse the electrophoresis tank and comb with distilled water. Put it on a level table and set up the comb. 1.5% agarose can be prepared for electrophoresis.

[0035] b. Put 50ml of 1×TAE electrophoresis buffer and 100ml...

Embodiment 2

[0054] Example 2 A method for identifying mutations in exon 11 of kit

[0055] 1. Extract patient tumor tissue or blood genomic DNA;

[0056] 2. Carry out PCR amplification. The 20 μl PCR reaction system is as follows: 50-100 ng of tumor tissue or blood genomic DNA, 0.125 μl of Taq enzyme, 1 μl of upstream and downstream primers (10 μM), 2 μl of dNTP (2.5 mM), 10×PCR buffer solution (containing Mg2+) 2 μl, and the rest was sterile distilled water; PCR reaction conditions were: denaturation at 95°C for 2 min, followed by denaturation at 95°C for 30 sec, annealing at 64°C for 30 sec, extension at 72°C for 1 min, 45 cycles, and finally extension at 72°C for 7 min. Store at 4°C;

[0057] 3. Gel electrophoresis analysis of the PCR amplification product, the specific steps are the same as in Example 1.

[0058] The result is as figure 1 As shown, band 2 represents kit exon 11, and its PCR product is 438bp.

[0059] 4. PCR amplification product sequencing, the specific steps are ...

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Abstract

The invention relates to the technical field of molecule detection for guiding tumor therapy and in particular discloses a primer used for detecting mutation of exons 9 and 11 of a c-kit gene and application thereof to the identification of the sensitivity of a tumor patient to imatinib (gleevec) medicaments. First, a DNA is extracted from a tumor tissue or a blood sample of a patient; secondly, a target fragment is amplified by the designed primer; and finally, the mutation locuses of the kit exons 9 and 11 are detected by sequencing the target segment so as to guide the therapy on the tumor patient.

Description

technical field [0001] The invention relates to the technical field of molecular detection for guiding tumor treatment, in particular to a primer for detecting mutations of exons 9 and 11 of c-kit gene and its application. Background technique [0002] The kit protein encoded by the c-kit gene is a transmembrane receptor protein, which is a member of the receptor tyrosine kinase family. It is expressed in many malignant tumors, especially in 89%-100% of gastrointestinal stromal tumors (GIST). In 1998, it was reported that there was a c-kit gene mutation in GIST, so that the kit protein could still maintain its own tyrosine protein kinase activity in the absence of ligand binding, thereby activating the downstream signal transduction pathway. This gain-of-function mutation is an important mechanism for the occurrence of GIST. [0003] Imatinib (Gleevec) is an orally administered 2-aniline pyrimidine derivative that selectively inhibits tyrosine kinases associated with kit p...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
Inventor 楼敬伟
Owner SHANGHAI BIOTECAN PHARMA
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