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Primer for detecting K-ras genic mutation and application thereof

A technology of primer sequences and codons is applied in the field of primers for detecting K-ras gene codons 12/13 and 61 mutations, which can solve the problems of insufficient development and immature technical conditions, and achieve the effect of high reference significance.

Active Publication Date: 2013-03-20
SHANGHAI BIOTECAN PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] It is understood that although dozens of well-known hospitals in Beijing, Shanghai, Guangzhou and other big cities have introduced this new gene technology, it is not widely used clinically, and its detection is mostly concentrated on K-ras codons 12 and 13. , the detection of codon 61 is rare, and the technical conditions are not mature

Method used

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  • Primer for detecting K-ras genic mutation and application thereof
  • Primer for detecting K-ras genic mutation and application thereof
  • Primer for detecting K-ras genic mutation and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0029] Example 1 A method for identifying K-ras codon 12 / 13 mutations

[0030] 1. Extraction of genomic DNA from tumor patient tissues;

[0031] 2. Perform PCR amplification. The 20 μl PCR reaction system is as follows: 50-100 ng of genomic DNA of the tumor tissue to be tested, 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 ( Contains Mg 2+ ) 2 μl, and the rest was sterile distilled water; the 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, and 45 cycles, and finally extension at 72°C for 7 min, and storage at 4°C;

[0032] 3. Gel electrophoresis analysis of PCR amplification products:

[0033] 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.

[0034] b. Put 50ml of 1×TAE electropho...

Embodiment 2

[0053] Example 2 A method for identifying the mutation of K-ras codon 61

[0054] 1. Extraction of genomic DNA from tumor patient tissues;

[0055] 2. Perform PCR amplification. The 20 μl PCR reaction system is as follows: 50-100 ng of genomic DNA of the tumor tissue to be tested, 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 ( Contains Mg 2+ ) 2 μl, the rest was sterile distilled water; the PCR reaction conditions were: denaturation at 95°C for 2 minutes, followed by denaturation at 95°C for 30 sec, annealing at 50°C for 30 sec, extension at 72°C for 1 min, and 45 cycles, and finally extension at 72°C for 7 min, and storage at 4°C;

[0056] 3. Gel electrophoresis analysis of the PCR amplification product: the specific steps are the same as in Example 1.

[0057] The result is as figure 1 As shown, band 1 represents the PCR product of K-ras codon 61 primer 1, and the product is 426 bp; Band 2 represents the ...

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Abstract

The invention relates to the field of molecular tests guiding oncotherapy and particularly discloses a primer for detecting the mutation condition of K-ras gene codons 12 / 13 and 61 and application of the primer to the identification of cancer patient sensibility to medicines such as geftinat, erlotinib and the like. DNA extraction is performed on tumor tissues of a patient, a target segment is amplified by using the primer designed by the invention, and mutational sites of the K-ras codons 12 / 13 and 61 are detected by sequencing the target segment, so that the treatment on the tumor patients can be guided.

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 the mutation of K-ras gene codon 12 / 13 and 61 and its application. Background technique [0002] At present, targeted therapy has become an important means of clinical tumor treatment. Epidermal growth factor receptor (EGFR) is the main target of targeted therapy. EGFR-targeted drugs include EGFR anti-monoclonal antibody drugs Erbitux and panitumumab, and EGFR tyrosine kinase inhibitors Iressa and Tarceva. These drugs can block the signal transduction of tumor cells by inhibiting the epidermal growth factor receptor tyrosine kinase, which is necessary for the occurrence and development of tumors, so as to inhibit the proliferation, invasion, metastasis, angiogenesis and promote the apoptosis of tumor cells. However, clinical trials have shown that these targeted drugs have a significant effect on only some patients....

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12N15/11
Inventor 楼敬伟张捷卢洁黄慧徐玲丽
Owner SHANGHAI BIOTECAN PHARMA
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