Nucleotide sequence for detecting BRAF gene V600E mutation and application thereof

A nucleic acid sequence and nucleotide sequence technology, which is applied in the nucleotide sequence field of BRAFV600E gene mutation detection, can solve the problems of small difference in Tm value and non-specific amplification, and achieves good effect, high degree of automation and suitable for clinical practice. The effect of application and promotion

Pending Publication Date: 2019-04-05
HANGZHOU D A GENETIC ENG
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, when detecting rare mutations, the biggest difficulty lies in the very high requirements for the design of TaqMan probes, mainly because there are often only 1-2 base differences between the mutant type and the wild type, and the difference in Tm value is small, which is prone to non-specific mutations. heterosexual expansion

Method used

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  • Nucleotide sequence for detecting BRAF gene V600E mutation and application thereof
  • Nucleotide sequence for detecting BRAF gene V600E mutation and application thereof
  • Nucleotide sequence for detecting BRAF gene V600E mutation and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1: The primers and probes for detecting BRAF were synthesized by Yingwei Jieji (Shanghai) Trading Co., Ltd., and the sequence is as follows:

[0038] Primers for amplifying BRAF:

[0039] F1 (SEQ ID NO: 1): 5'-catgaagacctcacagtaaaa-3';

[0040] R1 (SEQ ID NO: 2): 5'-cagacaactgttcaaactga-3';

[0041] Probes to detect BRAF wild-type:

[0042] P BRAFV600-WT (SEQ ID NO: 3): 5'-cta+ca+g+t+ga+aat+ct-3';

[0043] Among them, the 5' end of the BRAF wild-type probe P BRAFV600-WT (SEQ ID NO: 3) sequence is labeled with a FAM fluorescent group, and the 3' end is labeled with a BHQ1 quencher group. In addition, the 4th, 6th, and The 7th, 8th, 10th, and 13th nucleotides are modified by LNA (indicated by + sign).

[0044] Probes for detecting BRAF mutant genes:

[0045] P V600E-Mut (SEQ ID NO: 4): 5'-cta+ca+g+a+ga+aat+ct-3';

[0046] Among them, the 5' end of the BRAF mutant gene probe P V600E-Mut (SEQ ID NO: 6) is marked with a VIC fluorescent group, and the 3' end is ...

Embodiment 2

[0047] Example 2: Preparation method of BRAF V600E gene mutation detection kit.

[0048] (1) PCR reaction solution: 2*supermix (purchased from Bio-rad Company in the United States, item number 186-3026), which is a 2*PCR reaction premix, which contains DNA polymerase, Mg 2+ , PCR reaction buffer, dATP, dCTP, dTTP and dGTP and other components, stored at -20°C;

[0049] (2) Primer-probe premix solution: Dissolve the primers and probes shown in SEQ ID NO: 1 to 4 in double distilled water, the concentration of each primer is 100 μM, and the concentration of each probe is 10 μM; SEQ ID NO: The mother solutions of ID NO: 1 to 3 nucleic acid sequences were mixed at a volume ratio of 4.5:4.5:25; the mother solutions of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 4 were mixed at a volume ratio of 4.5:4.5: The volume ratio ratio of 25 was mixed; the final primer / probe concentration in the reaction system was 900 / 250nM. Store at -20°C;

[0050] (3) Positive control: a solution contain...

Embodiment 3

[0052] Embodiment 3: detection method.

[0053] Instruments: Eppendorf Mastercycler pro S qualitative PCR instrument, Bio-rad QX200 droplet digital PCR system (including droplet generator, sealing device, droplet reader), BECKMAN 22R desktop micro-refrigerated centrifuge, WH-866 vortex oscillator (Taicang Hualida), low-speed plate centrifuge (Anhui Zhongjia).

[0054] 1. Preparation of BRAF V600E sample, positive control, and negative control DNA template: use QIAamp Circulating Nucleic Acid Kit from QIAGEN (Cat. No. 55114), and operate according to the kit instructions.

[0055] 2. Use 1 pair of specific primers and 2 specific probes to detect the BRAF mutation gene, specifically including the following steps;

[0056] (1) Preparation of PCR reaction solution: Take out the components of the kit from the -20°C refrigerator, thaw at room temperature, and put them on an ice box for later use. Within 10 minutes before adding the sample, configure the PCR reaction solution X ul...

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Abstract

The invention discloses a nucleotide sequence for detecting BRAF gene V600E mutation and application thereof and belongs to the technical field of molecular diagnosis. A primer and a probe which are used for detecting BRAF can be specifically amplified and are used for detecting the BRAF V600E gene mutation. The invention further discloses a detection kit for detecting the BRAF V600E gene mutationbased on a micro-droplet type digital PCR (Polymerase Chain Reaction) system and the mutation of a BRAF gene can be rapidly, sensitively and accurately detected. The nucleotide sequence has the advantages of convenient operation, strong specificity, high sensitivity, low cost, high flux and the like, can be used for rapidly detecting the clinical BRAF V600E gene mutation and provides reference for diagnosis and treatment of the BRAF V600E mutation.

Description

technical field [0001] The invention belongs to the technical field of molecular diagnosis, and in particular relates to a nucleotide sequence for BRAF V600E gene mutation detection and application thereof. Background technique [0002] BRAF gene is one of the most important proto-oncogenes in humans, about 8% of human tumors have BRAF mutations. The vast majority of BRAF mutations are BRAFV600E 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 for tumor growth, proliferation, invasion and metastasis, and is one of the effective targets for V600E mutant tumors such as melanoma [0003] BRAF is a gene that affects the effect of tumor drug use in Jiaxue Gene’s tumor drug guidance. A class of BRAF V600E inhibitors has been found, which has a high inhibitory activity on BRAF, especially BRAF V600E, represented by vemurafenib (vemurafenib, PLX403...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6886C12Q1/6851C12N15/11
CPCC12Q1/6851C12Q1/6886C12Q2600/156C12Q2531/113C12Q2563/159C12Q2561/101
Inventor 任绪义虞闰六宣文静
Owner HANGZHOU D A GENETIC ENG
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