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Kit for quantificationally detecting BRAF (Block Repeat Active Flag) mutation

A kit and chain reaction technology, applied in the determination/testing of microorganisms, DNA/RNA fragments, recombinant DNA technology, etc., can solve problems such as inability to quantify

Inactive Publication Date: 2011-08-24
BEIJING ACCB BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Other methods, such as allele-specific oligonucleotide probe hybridization, are very sensitive to hybridization conditions and require strict control of experimental conditions; restriction fragment length polymorphism experiments require considerable manpower and cannot be quantified

Method used

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  • Kit for quantificationally detecting BRAF (Block Repeat Active Flag) mutation
  • Kit for quantificationally detecting BRAF (Block Repeat Active Flag) mutation
  • Kit for quantificationally detecting BRAF (Block Repeat Active Flag) mutation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1: Human fresh tumor tissue, paraffin-embedded tissue, peripheral blood, pleural effusion, human Genomic DNA Extraction from Cell Lines

[0028] The human cancer cell lines we tested included non-small cell lung cancer (NSCLC) (A549, H460, H838 and H1703), breast cancer (MCF-7, BT474 and HuL100), malignant multiple mesothelioma cell lines (H513, H2052, H290, MS-1 and H28), thyroid cancer (KAT10), colon cancer cell line (SW480, S1-M1-80), head and neck cancer cell line (U87), cervical cancer (Hela), sarcoma cell line (Mes- SA, Saos-2 and A204).

[0029] Human fresh tumor tissues, peripheral blood, and paraffin-embedded tissues that we tested included NSCLC, mesothelioma, colon cancer, malignant melanoma, renal cancer, esophageal cancer, thyroid cancer, malignant tumor, and ovarian cancer.

[0030] Specimen DNA Extraction

[0031] Can use the DNA extraction kit of Qiagen Company, Promega Company, Roche Company to extract sample genomic DNA, use Gene Company ...

Embodiment 2

[0086] Example 2: Preparation of plasmid standards containing mutant and wild-type detection sequences

[0087] 1. Wild-type plasmid construction ( figure 1 ， figure 2 )

[0088] 1.1 Preparation of the carrier

[0089] The TA cloning vector pMD18-T was purchased from TAKARA Company.

[0090] 1.2 Preparation of insert fragments

[0091] Use the PCR method to prepare the insert fragment. The template for PCR is the sample genomic DNA extracted in step 1. The reaction system and amplification conditions are as follows (Table 1, Table 2, Table 3):

[0092] Table 1: PCR reaction system (50μl)

[0093]

[0094] Table 2: PCR Primers

[0095]

[0096] Table 3: PCR Amplification Conditions

[0097]

[0098] 1.3 After using the QIAgen Gel Recovery Kit to recover the target fragment, it was ligated into the pMD18-T vector (purchased from TAKARA Company) by TA cloning.

[0099] 1.4 The newly constructed plasmid was massively amplified in Escherichia coli DH5α strain, an...

Embodiment 3

[0111] Example 3: Taking thyroid cancer and colorectal cancer samples as examples: human cell lines, human fresh BRAF mutation detection in tumor tissue, peripheral blood, and paraffin-embedded tissue genomic DNA

[0112] 1. The fluorescent quantitative PCR reaction template is the genomic DNA of thyroid cancer and colorectal cancer samples extracted in Example 1 and the standard prepared in Example 2, and double distilled water is used as a negative control. In order to make a standard curve, standard dilutions were 1ng / μl, 0.5ng / μl, 0.25ng / μl, 0.125ng / μl, 0.0625ng / μl, 0.03125ng / μl.

[0113] 2. Reaction system and reaction conditions (table 2, table 5, table 6, table 7), wherein labeling probe fluorescent emission group is selected from: FAM, TET, HEX, ROX; Fluorescence quencher group is selected from: BHQ, TAMARA.

[0114] Table 5: Real-time quantitative PCR reaction system (20μl / tube)

[0115]

[0116] To detect the 600 codon mutation of the BRAF gene, two systems ...

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Abstract

The invention relates to a method and kit for detecting BRAF (Block Repeat Active Flag) mutation relevant to the curative effect of a molecular-targeting anti-cancer medicament, in particular relating to a fluorescence quantificational PCR (Polymerase Chain Reaction) detecting method and kit for detecting mutation in BRAF gene mutation hot spot regions and application thereof. According to the invention, mutation at special positions of BRAF genes is detected, the curative effect of the molecular-targeting anti-cancer medicament such as an EGFR (Epidermal Growth Factor Receptor) tyrosine kinase inhibitor, and the like can be forecasted, and furthermore, the individual medicament use schemes of patients with tumors are directed.

Description

Background of the invention [0001] The BRAF gene belongs to the RAF gene family, is an oncogene encoding serine / threonine kinase, and is also one of the important members of the RAS-RAF-MEK-ERK signal transduction pathway, which plays an important role in the regulation of cell proliferation, differentiation and apoptosis Effect (Ikenoue T, Cancer Res, 2003, 63(23): 8132-37). Therefore, it plays an important role in the occurrence and development of tumors, and is a potential diagnostic marker and therapeutic target. [0002] The BRAF gene is located at 7q34 and encodes a protein of 783 amino acids. Studies have shown that there are mutations in different proportions in various human malignant tumors, such as malignant melanoma, colorectal cancer, lung cancer, thyroid cancer, liver cancer and pancreatic cancer (Davies H , et al, Nature, 2002, 417(6892): 949-54). Among them, about 90% of BRAF mutations are located at the 1799th nucleotide, and T is mutated into A, causing the...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C12N15/63C12Q1/68G01N21/64
CPCC12Q2600/156C12Q1/6886
Inventor 许军普陈钊李隽
Owner BEIJING ACCB BIOTECH
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