PCR detecting method of tumour associated gene mutation and reagent system

A technology for tumor-related genes and detection methods, which is applied in the field of PCR detection methods and reagent systems for tumor-related gene mutations, and can solve problems such as low efficiency

Inactive Publication Date: 2005-12-21
SHANGHAI PULMONARY HOSPITAL
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Problems solved by technology

For example, Iressa has an effective rate of nearly 80% for patients with non-small cell lung cancer with EGFR mutations, but for those without EGFR mutations, the effective rate is very low

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0074] Example 1: Genomic EGFR mutation detection and Iressa drug sensitivity prediction of fiberoptic bronchoscopic specimens and surgical specimens

[0075] (1) Sample DNA extraction: phenol / chloroform DNA purification method. Take fiberoptic bronchoscopic tumor specimens or surgical tumor specimens 2-10mg, pour into liquid nitrogen, grind into powder, add 1ml separation buffer (10mmol / L Tris Cl pH7.4, 10mmol / LNaCl, 25mmol / LEDTA); add 0.1ml 10% SDS, mix well, the sample becomes very viscous at this time; add 5ul or 0.1mg proteinase K, keep warm at 37°C for 1-2 hours, until the tissue is completely disintegrated. Add 0.1ml 5mol / L NaCl, mix well, and centrifuge at 5000rpm for a few seconds. Take the supernatant in a new centrifuge tube and extract with an equal volume of phenol:chloroform:isoamyl alcohol (25:24:1). After stratification, centrifuge at 3000rpm for 5 minutes. Take the upper aqueous phase to a clean centrifuge tube, add 2 times the volume of ether for extractio...

Embodiment 2

[0113] Example 2: Detection of EGFR mRNA mutations in bronchofiberoptic specimens and surgical specimens and prediction of Iressa drug sensitivity

[0114] (1) Extraction of total RNA from samples: guanidine isothiocyanate / phenol / chloroform total RNA purification method. Grind the fiberoptic bronchoscopic specimens and surgical specimens into powder in liquid nitrogen, then add 1ml Trizol solution to 2-50mg tissue for grinding, leave the grinding solution at room temperature for 5 minutes, add 0.2ml chloroform, cover the centrifuge tube tightly, and grind by hand. Shake the centrifuge tube vigorously for 15 seconds. Take the upper aqueous phase into a new centrifuge tube, add 0.5ml of isopropanol, let it stand at room temperature for 10 minutes, and centrifuge at 12000g for 10 minutes. Discard the supernatant, add at least 1ml of 75% ethanol, vortex, and centrifuge at 7500g for 5 minutes at 4°C. Carefully discard the supernatant, then dry at room temperature or under vacuum ...

Embodiment 3

[0149] Example 3: EGFR Mutation Detection and Iressa Drug Sensitivity Prediction by Rapid and Simplified Genomic DNA Extraction Method

[0150] (1) Sample DNA extraction: rapid and simplified extraction method. Take fiberoptic bronchoscopic tumor specimens or surgery tumor specimens about 1 mg, add 50 μl TE buffer solution containing 0.38 mg / ml proteinase K, 60 ℃ for 30 minutes, 100 ℃ water bath for 15 minutes, centrifuge at 14000r / min for 2 minutes and take 0.5- 1 μl of the supernatant was used as a template for the PCR reaction.

[0151] (2) Conventional PCR reaction system: the same as in Example 1.

[0152] (3) Detection of mutations by Taqman-MGB real-time fluorescent quantitative PCR: same as in Example 1.

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Abstract

This invention belongs to the medicine curative effect detection technique field; involve a kind of tumor relevant gene mutation PCR detection method and reagent system concretely. Further, involve TaqMan - MGB real-time fluorescence quantitative PCR method of EGFR and ras gene mutation correlated with curative effect of molecule target anticancer drug and reagent system. It mainly includes clicking in the specific sudden change location where the molecule target is correlated with the curative effect of anticancer drug, the peculiar oligonucleotides designed specially primer series and probe series, determine procedure method , reagent system and use. The reagent system mean includes the any peculiar oligonucleotides primer series and real-time fluorescence quantitative PCR reagent systemof the probe.

Description

technical field [0001] The invention belongs to the technical field of drug curative effect detection, and in particular relates to a PCR detection method and a reagent system for tumor-related gene mutations. Furthermore, it relates to a TaqMan-MGB real-time fluorescent quantitative PCR method and reagent system for EGFR and ras gene mutations related to the curative effect of molecularly targeted anticancer drugs. The invention can detect the mutation of a specific site of a tumor-related gene and its mutation type, and is used for predicting the curative effect of molecularly targeted new anti-tumor drugs and guiding clinical individualized drug regimens for tumor patients. Background technique [0002] Modern biomedical research shows that the pathogenesis and therapeutic effect of tumors are closely related to somatic mutations of tumor-related genes, namely oncogenes and tumor suppressor genes. Tumor-related genes include: EGFR (epidermal growth factor receptor); VEGF...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
Inventor 周彩存粟波潘虹
Owner SHANGHAI PULMONARY HOSPITAL
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