Tumor repair gene mutation fluorescent real time PCR detection method and reagent system

A detection method and detection reagent technology, applied in the field of detection, can solve problems such as unfavorable, increased DNA repair ability, increased drug resistance, etc.

Inactive Publication Date: 2008-09-17
SHANGHAI PULMONARY HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In other words, increased DNA repair capacity increases drug resi

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0105] Example 1: ERCC1, ERCC2, XRCC3 mutation detection of peripheral blood DNA genomic DNA extraction method

[0106] (1) Sample DNA extraction: Collect 2ml of peripheral venous blood, centrifuge at 5000r / min for 3 minutes, and remove the supernatant serum. Remove the lower layer of blood and transfer it to a centrifuge tube, add PBS buffer to dissolve, centrifuge at 12000r / min for 15 minutes, pour off the upper layer, add SDS, proteinase K, shake at 37°C overnight, then extract DNA with phenol-chloroform, precipitate with absolute ethanol, Wash with 75% ethanol, dry naturally, and finally melt into 200 μL TE solution. Store in -20°C refrigerator.

[0107] (2) Taqman-MGB fluorescent real-time quantitative PCR detection of mutations

[0108] The following reaction system was used:

[0109] 5XPCR Buffer 5μl

[0110] dNTP Mixture 0.75μl

[0111] Mg Solution 0.5μl

[0112] XRCC3-241FP 0.5 μl

[0113] XRCC3-241RP 0.5 μl

[0114] XRCC3-241FAM(C) 0.5μl

[0115] XRCC3-241FA...

Embodiment 2

[0128] Example 2: Mutation detection of ERCC1, ERCC2, XRCC3 by genomic DNA extraction method of tumor samples

[0129](1) Sample DNA extraction: phenol / chloroform DNA purification method. Take 2-10 mg of tumor specimen, pour into liquid nitrogen, grind into powder, add 1ml of split buffer (10mmol / L Tris.CL PH7.5, 10mmol / L NaCL, 25mmol / L EDTA); add 0.1ml of 10% SDS, mix Homogenize; add 0.1mg proteinase K, 37 ℃ water bath for 2-4 hours, until the tissue block is completely decomposed. Centrifuge at 2500rpm for 20 seconds, and transfer the supernatant to a new centrifuge tube. DNA was extracted with phenol-chloroform, precipitated with absolute ethanol, washed with 75% ethanol, dried naturally, and finally dissolved in 200 μL TE solution. Store in -20°C refrigerator.

[0130] (2) Taqman-MGB fluorescent real-time quantitative PCR detection of mutations

[0131] The following reaction system was used:

[0132] 5XPCR Buffer 5μl

[0133] dNTP Mixture 0.75μl

[0134] Mg Solutio...

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Abstract

The invention relates to a real-time PCR detecting method of tumor-associated gene mutation and reagent system, belonging to medicine curative effect detecting technical field. The tumour repair genes XRCC3, ERCC1 and ERCC2 associated with the tumour individualized theraphy are used as target genes to detect, and the customed designed special oligonucleotide primer sequence, hydrolysis probe and Taqman-MGB detection agent system are used and using real-time quantitative PCR method, therefore the mutation of special site of the tumor-associated gene is detected wherein the agent system is just the real-time quantitative PCR agent system comprising any oligonucleotide primer sequence and probe. The method can be used for effect detection of the clinical individualized medication scheme.

Description

technical field [0001] The invention belongs to the technical field of detection, and in particular relates to a fluorescent real-time PCR detection method and a reagent system for tumor repair gene mutations. Background technique [0002] Tumors are multifactorial diseases, and many factors can cause DNA damage in cells. Normally, the body has the ability to repair damage by itself. But if this repair ability declines, it will lead to the generation of tumors. With the development of society, the pollution of the environment, and the increase of smoking rate, lung cancer has become the first cause of cancer death in many countries. Current research on lung cancer is mainly focused on gene repair. Many factors can cause gene damage, such as radiation, alkylating agents, etc. The way of gene repair is different for different damages, and at the same time, there are many kinds of repair genes involved in the process of gene repair. DNA repair is the main biological respon...

Claims

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Application Information

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IPC IPC(8): G01N21/00C12Q1/68
Inventor 周彩存张颉张增利
Owner SHANGHAI PULMONARY HOSPITAL
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