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Preparation method of non-small cell lung cancer molecular marker related probes and application thereof

A technology of probes and gene probes, which is used in the preparation and application of non-small cell lung cancer molecular marker-related probes, can solve the problems of strong side effects, poor sensitivity of NSCLC, and inability to perform radical surgery. Achieve the effect of simple operation

Active Publication Date: 2013-08-14
DAAN GENE CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

For NSCLC, if it is found early, radical surgery can be performed, but in fact, 75-85% of them are found to be advanced lung cancer, and radical surgery cannot be performed, so radiotherapy can only be used as the main treatment method, and conventional radiotherapy is 5 years. The survival rate is only 3-10%. Chemotherapy has poor sensitivity to NSCLC and has strong side effects. Molecular targeted therapy uses anti-tumor drugs to specifically act on certain targets of tumor cells to kill tumor cells to achieve therapeutic purposes

Method used

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  • Preparation method of non-small cell lung cancer molecular marker related probes and application thereof
  • Preparation method of non-small cell lung cancer molecular marker related probes and application thereof
  • Preparation method of non-small cell lung cancer molecular marker related probes and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0047] Embodiment 1: Preparation of EGFR gene probe

[0048] (1) Primer design and clone screening: EGFR gene is located in the 7p12 segment of human chromosome. NCBI Mapview database searches all clones containing EGFR gene, and screens out the clone containing this gene, numbered RP11-815K24.

[0049] (2) Cloning culture and identification: purchase the clone RP11-815K24, take 10 μl of the cloning bacteria solution and add it to 5ml TB culture solution (chloramphenicol resistance), and shake the bacteria in a shaker at 37°C for 8 to 12 hours; Add all the solution to 500ml TB culture solution (chloramphenicol resistance), shake the bacteria in a shaker at 37°C for 8-12 hours; use the upstream primer 5'-ACTCCAGACATCTTTCCATCTGC-3' and the downstream primer 5'-AATCATTGCTCTCATGAGTGGTG for the bacterial solution -3' was subjected to PCR amplification, and the amplification conditions were: 95° C. for 5 minutes; (94° C. for 30 seconds, 56° C. for 30 seconds, 72° C. for 45 seconds) ...

Embodiment 2

[0069] Embodiment 2: Preparation of ERCC1 gene probe

[0070] (1) Primer design and clone screening: ERCC1 gene is located in the 19q13 segment of human chromosome. NCBI Mapview database searches all clones containing ERCC1 gene, and screens out the clone containing this gene, numbered RP11-752G9.

[0071] (2) Cloning culture and identification: purchase clone RP11-752G9, take 10 μl of clone bacteria solution and add it to 5ml TB culture solution (chloramphenicol resistance), shake the bacteria in a shaker at 37°C for 8 to 12 hours; Add all the solution to 500ml TB culture solution (chloramphenicol resistance), shake the bacteria in a shaker at 37°C for 8-12 hours; use the upstream primer 5'-GCAATGAGCCGAG for the bacteria solution

[0072] ATAGAA-3' and downstream primer 5'-TGGCTAGCCCATTACTCTA-3' were amplified by PCR, the amplification conditions were: 95°C for 5 minutes; (94°C for 30 seconds, 56°C for 30 seconds, 72°C for 45 seconds) × 40 cycles; 72 °C for 10 minutes. The ...

Embodiment 3

[0092] Example 3: Preparation of fluorescence in situ hybridization detection kit for non-small cell lung cancer

[0093] Take 10 servings / box as an example.

[0094] (1) Preparation of hybridization solution

[0095] Put the labeled probes in order, dissolve the probes first. Use 1 μl of sterilized purified water to dissolve the dry powder of the EGFR gene probe prepared by the method in Example 1, and mix thoroughly. Then another 1 μl of sterilized purified water was dissolved in the dry powder of the ERCC1 gene probe prepared by the method in Example 2, and thoroughly mixed. Prepare the fluorescently labeled probe mixture according to the following scheme:

[0096]

[0097] Prepare the hybridization solution according to the following scheme:

[0098]

[0099] (2) DAPI counterstain preparation

[0100] Anti-fading solution: The whole process must be protected from light. Dissolve 10mg of p-phenylenediamine in 1ml of PBS, adjust the pH to 9.0, add 9ml of glycerin,...

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Abstract

The invention relates to a preparation method of non-small cell lung cancer molecular marker related probes and also relates to a non-small cell lung cancer molecular marker fluorescence in situ hybridization detection kit prepared by the use of the probes. By the utilization of EGFR gene probe and ERCC1 gene probe, which are prepared by the use of the method provided by the invention and which are combined with a hybridization buffer, unlabelled competitive DNA and DAPI counter stain, the non-small cell lung cancer molecular marker fluorescence in situ hybridization detection kit can be prepared. The kit provided by the invention helps to accomplish the combined detection of two indexes of EGFR and ERCC1, is beneficial to the prognosis and effect prediction of the non-small cell lung cancer, and can be used to guide the establishment of an individualized treatment scheme and the selection of an appropriate therapeutic method so as to reduce death rate, decrease recurrence risk and achieve the purpose of optimizing the diagnosis and treatment effect.

Description

technical field [0001] The present invention relates to a method for preparing non-small cell lung cancer molecular marker-related probes, and also relates to the use of these probes to prepare a non-small cell lung cancer molecular marker fluorescence in situ hybridization detection kit. The non-small cell lung cancer molecular marker-related probe of the present invention The probe and the corresponding kit can be used for prognosis judgment and curative effect prediction of non-small cell lung cancer. Background technique [0002] In 2006, the Cancer Prevention and Control Office of the Ministry of Health counted the top ten malignant tumors in China. Lung cancer ranked first in the incidence of male malignant tumors, and the mortality rate of lung cancer ranked first in malignant tumors worldwide. Lung cancer is divided into two types based on its biological characteristics, treatment and prognosis: non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC), of...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68
Inventor 李明何瑰陈华云
Owner DAAN GENE CO LTD
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