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Quantitative standard substance for BRAF and EGFR gene mutation detection, preparation method and valuing method thereof

A quantitative standard product and genome technology, applied in the direction of biochemical equipment and methods, microbial determination/inspection, etc., to achieve the effect of improving efficiency and reducing the cost of testing quality control

Inactive Publication Date: 2017-11-21
NAT INST OF METROLOGY CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, there are no quantitative standards for human BRAF and EGFR gene detection in China

Method used

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  • Quantitative standard substance for BRAF and EGFR gene mutation detection, preparation method and valuing method thereof
  • Quantitative standard substance for BRAF and EGFR gene mutation detection, preparation method and valuing method thereof
  • Quantitative standard substance for BRAF and EGFR gene mutation detection, preparation method and valuing method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0084] Preparation and determination of quantitative standards for BRAF gene V600E mutation

[0085] 1. Materials and methods:

[0086] 1. Cell Lines and Media

[0087] The HT29 cell line was purchased from ATCC, and the required medium and culture conditions were carried out according to the instructions of ATCC. BRAF wild-type cell line PG-LCL8 was provided by Fudan University, the medium was 1640+10% FBS, 5% CO 2 , cultivated at 37 degrees.

[0088] 2. DNA extraction and PCR amplification

[0089] The genomic DNA of the cell line was extracted using the Genome Purification Kit of Kangwei Century Company, and the genomic DNA of the cell line containing the target mutation was used as a template for PCR amplification to obtain the PCR product containing the V600E target mutation. 20 μL of PCR amplification system, including 2 μL of upstream and downstream primers with a concentration of 5 uM, 2 μL of DNA template, 10 μL of 2× PCR master mix, and 6 μL of sterile water. Am...

Embodiment 2

[0122] Preparation and determination of quantitative standards for EGFR gene L858R mutation

[0123] 1. Materials and methods:

[0124] 1. Cell Lines and Media

[0125] The cell line containing the L858R mutation was purchased from ATCC, and the required medium and culture conditions were carried out according to the instructions of ATCC. The wild-type cell line PG-LCL8 was provided by Fudan University, the medium was 1640+10% FBS, 5% CO 2 , cultivated at 37 degrees.

[0126] 2. DNA extraction and PCR amplification

[0127] The genomic DNA of the cell line was extracted using the Genome Purification Kit of Kangwei Century Company, and the genomic DNA of the cell line containing the target mutation was used as a template for PCR amplification to obtain the PCR product containing the L858R target mutation. 20 μL of PCR amplification system, including 2 μL of upstream and downstream primers with a concentration of 5 uM, 2 μL of DNA template, 10 μL of 2× PCR master mix, and 6 ...

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Abstract

The invention discloses a quantitative standard substance for BRAF and EGFR gene mutation detection, a preparation method and a valuing method thereof. The method comprises the following steps: respectively establishing plasmids containing BRAF gene V600E, EGFR gene T790M, L858R and deletion mutant E746_A750del mutation site, taking the plasmids as a template for performing PCR amplification, acquiring a PCR product and performing Sanger sequencing verification, and then using a scale for weighting ultrasonic fragmented wild type human genomic DNA and mixing at a certain ratio, thereby acquiring the quantitative standard substance with BRAF gene V600E mutant, EGFR gene T790M, L858R and deletion mutant E746_A750del mutant. The invention provides the preparation method for the quantitative standard substance. The fragmented wild type human genomic DNA is used as background DNA, so that the background for practically detecting a free DNA sample can be simulated. The valuing method for the quantitative standard substance is high in precision and accuracy and is independent from DNA standard substance and the measuring result can be traced to international basic unit (weight), so that the reliability and the traceability of the measuring result can be guaranteed.

Description

technical field [0001] The invention relates to the field of gene detection, in particular to a quantitative standard for BRAF and EGFR gene mutations. Background technique [0002] BRAF is one of the most important proto-oncogenes in humans, and about 8% of human tumors have BRAF mutations. The vast majority of BRAF mutations are BRAF V600E mutations, which mainly occur in melanoma, colon cancer and thyroid cancer. This mutation leads to continuous activation of the downstream MEK-ERK signaling pathway, which is critical to tumor growth, proliferation, invasion and metastasis, and is one of the effective targets for anti-melanoma and other V600E mutant tumors. In 2011, Vemurafenib, the first BRAFV600E-targeted inhibitor, was approved by the FDA for the treatment of advanced melanoma patients with BRAFV600E mutation, effectively prolonging the progression-free survival and overall survival of patients, and achieved a breakthrough therapeutic effect , is also a typical targ...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
CPCC12Q1/6851C12Q2545/113C12Q2531/113
Inventor 董莲华王晶王尚君傅博强唐治玉
Owner NAT INST OF METROLOGY CHINA
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