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Reagent for detecting EGFR genetic mutation and application thereof

A technology of reagents and genotypes, applied in the field of reagents for detecting EGFR gene mutations, can solve the problems of undetectable gene mutations, complex operations, and long cycles, and achieve the effects of fast processing, simple sample processing, and low cost

Active Publication Date: 2013-09-04
INST OF CHEM CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These methods have their own characteristics, however, the problems of sensitivity and selectivity, high cost, complicated operation, long period, and special requirements for instruments limit their clinical application.
Especially since clinical cancer samples are mixed with normal non-mutated tissue, gene mutations are often not detected by DNA sequencing methods routinely used in clinical and basic research

Method used

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  • Reagent for detecting EGFR genetic mutation and application thereof
  • Reagent for detecting EGFR genetic mutation and application thereof
  • Reagent for detecting EGFR genetic mutation and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Synthesis of compound shown in embodiment 1, formula (I)

[0037] 9,9-bis-(6'-bromohexyl)-2,7-dibromofluorene: purchased from Synwit Technology Co., Ltd, product number: 570414-33-4.

[0038] 2-Isopropoxy-4,4,5,5-tetramethyl-1,3,2-trioxaborane: purchased from Alfa Aesar Company, the product number is 999770-93-1.

[0039] The synthetic process of compound shown in formula (I) sees Figure 4 .

[0040] 1. Synthesis of 1,2-bis-(4-bromophenyl)-hydrazine

[0041] ① P-bromobenzoic acid (4.02g, 20mmol) is packed into a 25ml one-port bottle, and thionyl chloride (SO 2 Cl 2 , 3.63ml, 50mmol), then a catalytic amount of pyridine was added dropwise, the temperature was raised to 70°C, and stirred for 12h.

[0042] ② Step ① After the reaction is stopped, remove excess thionyl chloride by atmospheric pressure distillation, add dry N-methylpyrrolidone (NMP, 20ml), and add 10ml of dry NMP dissolved in hydrazine hydrate (0.5g, 10mmol) dropwise at room temperature, After the drop...

Embodiment 2

[0083]Embodiment 2, the composition of reagent

[0084] The reagent is composed of the compound represented by formula (I) prepared in Example 1, compound dGTP-F1, primer 1, primer 2, primer 3, primer 4, primer 5 and primer 6, and each component is packaged separately.

[0085] The compound dGTP-F1 is represented by formula (II), purchased from Perkin Elmer, and the product catalog number is: NEL429001EA.

[0086] Formula (II).

[0087] The sequences of the six primers are as follows, all synthesized by Shanghai Biotechnology Co., Ltd.:

[0088] Primer 1: 5'-TCAGCCTGGCAAGTCCAGTAAG-3';

[0089] Primer 2: 5'-AGCTCTGGCTCACACTACCAG-3';

[0090] Primer 3: 5'-CCTCACAGCAGGGTCTTCTC-3';

[0091] Primer 4: 5'-TGCCTCCTTCTGCATGGTA-3';

[0092] Primer 5: 5'-AGATCACAGATTTTGGGCT-3';

[0093] Primer 6: 5'-GATCACAGATTTTGGGCG-3'.

[0094] Primer 1, Primer 2, Primer 3 and Primer 4 were used to amplify the target gene (EFGR gene) by nested PCR, wherein Primer 1 and Primer 2 constituted t...

Embodiment 3

[0095] Embodiment 3, the reagent of application embodiment 2 detects A498 cell line (cancer cell line)

[0096] 1. Cultured cell lines

[0097] The A498 cell line was cultured in DMEM medium containing 10% fetal bovine serum at 37°C, 5% CO 2 ; Cells were collected when the cells covered 70% of the culture plate.

[0098] Two, the reagent of application embodiment 2 detects A498 cell line (cancer cell line)

[0099] 1. Extract the genomic DNA of the cells.

[0100] 2, with the genomic DNA of step 1 as template, carry out PCR amplification with the primer pair that primer 1 and primer 2 form, PCR amplification product carries out agarose gel electrophoresis (see figure 2 ), to obtain a PCR amplification product of 659bp.

[0101] 3, with the PCR amplification product of step 2 as template, carry out PCR amplification with the primer pair that primer 3 and primer 4 are formed, PCR amplification product carries out agarose gel electrophoresis (see figure 2 ), to obtain a PC...

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Abstract

The invention aims to provide a reagent for detecting EGFR genetic mutation and application thereof. The invention provides a compound shown as a formula (I). The reagent provided by the invention comprises the compound shown as the formula (I), a compound dGTP-F1 shown as a formula (II), a primer 1 shown as a sequence 1, a primer 2 shown as a sequence 2, a primer 3 shown as a sequence 3, a primer 4 shown as a sequence 4, a primer 5 shown as a sequence 5 and a primer 6 shown as a sequence 6. The EGFR genetic mutation means that EGFR protein amino acid residue changes from L to R since site No.858 at an N terminal. The method provided by the invention overcomes various insufficiencies in a prior detection technology and has advantages of high sensitivity, rapidness, visibility and simpleness. The invention can be applied to assistance of analysis of sensitivity of a cell line and / or a cancer patient to an anticancer medicament, so as to guide doctors to carry out reasonable anticancer treatment on the patient.

Description

technical field [0001] The invention relates to a reagent for detecting EGFR gene mutation and its application. Background technique [0002] Rapid and accurate detection of cancer-related gene mutations can provide important information for clinical cancer molecular diagnosis and treatment. In non-small cell lung patients, point mutations in exon 21 of the EFGR gene in lung cancer tissues, L858R mutations (ie T / G), account for 44% of all mutations in EFGR. The detection result of the point mutation can guide clinical anticancer treatment. Basic and clinical studies have proved that if lung cancer patients have a point mutation in exon 21 of the EGFR gene, they will have higher sensitivity to anti-lung cancer drugs, gefitinib and erlotinib. Therefore, the point mutation detection result of this gene has become one of the clinically important molecular pathological diagnosis indicators. [0003] Traditional methods for detecting gene mutations mainly include direct DNA seq...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C08G61/12C09K11/06C12Q1/68C12Q1/02G01N21/64
Inventor 王树杨琼刘礼兵吴尉朱春雷冯旭利
Owner INST OF CHEM CHINESE ACAD OF SCI
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