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Kit for detecting BRAF gene V600E trace mutation through pyrosequencing technique and application of kit

A pyrosequencing and technical detection technology, which is applied in the field of pyrosequencing technology to detect BRAF gene V600E trace mutation kits, can solve the problems of low positive detection rate and cumbersome operation, and achieves easy interpretation, simple operation, and reduced sample collection. desired effect

Inactive Publication Date: 2016-03-16
HANGZHOU DIAN BIOTECH CO LTD
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  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the traditional Sanger sequencing method has the advantages of accurate results and intuitive interpretation, it has the problems of cumbersome operation and low positive detection rate; while the ARMS method is simple to operate, but the sensitivity of the detection method is only 1%, and there are still positive detection rates. The problem of low yield

Method used

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  • Kit for detecting BRAF gene V600E trace mutation through pyrosequencing technique and application of kit
  • Kit for detecting BRAF gene V600E trace mutation through pyrosequencing technique and application of kit
  • Kit for detecting BRAF gene V600E trace mutation through pyrosequencing technique and application of kit

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Effect test

Embodiment 1

[0037] Example 1: Reagents.

[0038] (1) DNA extraction reagents:

[0039] Purchased from QIAGEN Company.

[0040] (2) Reaction solution:

[0041] PCRBuffer: purchased from Fermentas, USA;

[0042] Primers SEQIDNO: 1-12, synthesized by Shanghai Yingjun Biotechnology Co., Ltd.;

[0043] MgCl 2 : purchased from the U.S. Fermentas company;

[0044] 0.2mMdNTPs: purchased from Fermentas, USA;

[0045] 2U / μL TaqDNA polymerase: purchased from Fermentas, USA.

[0046] (3) Reagent for single-strand purification:

[0047] 75% (v / v) ethanol solution: purchased from Hangzhou Changzheng Chemical Reagent Co., Ltd.;

[0048] 0.2M NaOH: purchased from Shanghai Shisi Hewei Chemical Co., Ltd.;

[0049] 10mM Tris-Acetate (pH7.6): Tris-base was purchased from Sigma, USA, and anhydrous acetic acid was purchased from Hangzhou Chemical Reagent Co., Ltd.;

[0050] Binding buffer: 10mM Tris-HCl (Tris-base was purchased from Sigma, USA, hydrochloric acid was purchased from Hangzhou Chemical R...

Embodiment 2

[0057] Embodiment 2: detection method.

[0058] Instruments: Bio-RadS1000PCR instrument, BeckmanMicrofuge22R desktop microrefrigerated centrifuge, Beijing Liuyi agarose gel electrophoresis instrument, Shanghai Peiqing gel imaging system, QIAGENPyroMarkQ96ID sequencer.

[0059] (1) Extract DNA from paraffin biopsy tissue samples, the steps are as follows:

[0060] a. Take tissue DNA, add tissue lysate, proteinase K, and incubate at 56°C for 2 hours;

[0061] b. Transfer the lysate to a DNA adsorption column and centrifuge at 12,000 rpm for 1 minute;

[0062] c. Add 500uL rinse solution 1 and centrifuge at 12000 rpm for 30 seconds;

[0063] d. Add 500uL rinse solution 2 and centrifuge at 12000 rpm for 30 seconds;

[0064] e. Transfer the DNA adsorption column to a new 1.5mL centrifuge tube, add 50uL deionized water, and centrifuge at 12000 rpm for 1 minute to obtain genomic DNA.

[0065] (2) using the genomic DNA obtained in step (1) as a template, and using EGFR-specific prim...

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Abstract

The invention mainly relates to the field of molecular diagnosis and provides a kit for accurately detecting BRAF gene V600E trace mutation. The kit mainly comprises two specific amplification primers, one blocking primer, one sequencing primer and affinity magnetic beads marked bystreptomycin. By the application of the kit, trace mutation of the BRAF gene V600E which is lowered to 0.1% in concentration can be accurately detected. A detection method is high in sensitivity, the mutation generated when the concentration is lowered to 0.1% can be detected, the result is visual, interpretation is simpler, more accurate and more rapid, the false negative rate of the detection result is greatly lowered, clinical invalid choice of targeted drugs of a patient is avoided, valuable treatment time is saved for the patient, and the living quality of the patient is improved.

Description

technical field [0001] The invention belongs to the field of molecular pathological diagnosis, and specifically relates to a kit for detecting BRAF gene V600E micro mutation by pyrosequencing technology and its application. Background technique [0002] BRAF gene mutation occurs in nearly 2% of human tumors, mainly in colon cancer, melanoma. According to statistics, about 5% of colorectal cancer patients have somatic BRAF gene mutations, the most common of which is the mutation at the 1799th nucleotide (T mutation to A) that causes the encoded valine to be replaced by glutamic acid. Replace (V600E). [0003] Pyrosequencing (pyrosequencing) technology is a new generation of DNA sequence analysis technology, which is widely used in the field of genotype analysis. This technique does not require electrophoresis, and DNA fragments do not require special fluorescent labels, and the operation is extremely simple. [0004] When using targeted drugs such as Erbitux or panitumumab...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6869C12Q2565/301C12Q2563/107C12Q2537/163
Inventor 任绪义虞闰六张锋
Owner HANGZHOU DIAN BIOTECH CO LTD
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