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A primer, probe and kit for detecting braf gene k601e mutation

A technology for detecting primers and kits, which is used in DNA/RNA fragments, recombinant DNA technology, and microbial determination/inspection. problem, to avoid false positives, short detection time, and strong specificity

Active Publication Date: 2020-04-14
上海润安医学科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At the same time, because there are many other types of mutations near the K601E site of the BRAF gene, such as V600E, V600K, V600R, etc., the mutations at these adjacent sites also cause great interference to the detection of the K601E mutation of the BRAF gene
Due to the limitation of the design conditions of the above primers and probes, and the results of multiple rounds of primers and probes designed for this region in the experiment showed that the combination of primers and probes designed according to conventional principles has poor specificity, obvious non-specific amplification, and cannot effectively distinguish BRAF. Gene wild type and K601E mutant

Method used

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  • A primer, probe and kit for detecting braf gene k601e mutation
  • A primer, probe and kit for detecting braf gene k601e mutation
  • A primer, probe and kit for detecting braf gene k601e mutation

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Experimental program
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Effect test

Embodiment 1

[0045] The present invention relates to a primer and probe for detecting BRAF gene K601E mutation, and its design process is as follows:

[0046] 1) According to the NCBI database BRAF gene K601E mutation sequence information, it is found that the 25bp sequence before and after the BRAF gene K601E mutation site contains 5 consecutive identical base arrangements, namely AAAAA, TTT, AAA, GGG, CCC, at the mutation site The sequence is AAAT. If we follow the design of conventional primers and probes, it is difficult to obtain an effective combination of primers and probes to distinguish between the mutant and wild-type BRAF gene K601E. Therefore, it is considered to introduce 2 consecutive mismatch bases into the 3'end of the forward primer at the mutation site, and after multiple rounds of testing verification and optimization, we finally find a primer and probe combination that can effectively distinguish the BRAF gene K601E mutant and wild-type , The nucleotide sequences are show...

Embodiment 2

[0051] The present invention relates to a kit for detecting BRAF gene K601E mutation, which mainly includes:

[0052] 1) PCR reaction solution and 50×ROX: purchased from Nanjing Novozan Biotechnology Co., Ltd., item number Q113-02. 2×AceQ ® U + Probe Master Mix is ​​2×PCR reaction solution, including DNA polymerase and Mg 2+ , PCR reaction buffer, dATP, dTTP, dCTP, dGTP and other components, stored at -20℃, 50×ROX Reference Dye is 50×ROX, containing ROX fluorescent reference dye, stored at -20℃ away from light;

[0053] 2) Mutation detection primer probe master mix: a mixture of primers and probes with nucleotide sequences as shown in SEQ ID NO.: 1, 2, 3, where the concentration of each primer is 10 μM, and the concentration of the probe It is 10 μM. The two mother liquids of mutation detection primers and probes are mixed in a volume ratio of 2:2:1, and stored at -20°C in the dark;

[0054] 3) Quality control detection primer probe master mix: The nucleotide sequence of the probe...

Embodiment 3

[0058] A detection method of a kit for detecting BRAF gene K601E mutation, including the following steps:

[0059] 1) Sample DNA extraction:

[0060] According to the nature of the sample, use the corresponding DNA extraction kit to extract the sample DNA. Use nuclease-free water to dilute the extracted DNA to 10ng / μL for use. The extracted DNA should be used immediately or placed at 4°C. Store at -20°C.

[0061] 2) Prepare mutation detection PCR reaction system:

[0062] Remove the components of the kit from the refrigerator at -20°C, melt at room temperature, and put on the ice box for later use. Prepare PCR reaction solution according to the number of test samples (another +1 negative control+1 positive control+1 blank control). One of the PCR reaction systems includes:

[0063] Reagent volume PCR reaction solution 10μL 50×ROX Dye 0.4μL Mutation detection primer probe master mix 1μL gDNA (10ng / μL) 2μL Nuclease-free water 6.6μL

[0064] 3) Preparation of PCR reaction system...

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Abstract

The invention relates to the technical field of molecular organisms, and discloses a primer, probe and reagent kit for detecting K601E mutation of a BRAF gene. The primer, probe and reagent kit for detecting K601E mutation of a BRAF gene are characterized in that 2 mispairing alkali bases are continuously designed at a 3' end of a K601E mutation detecting primer of the BRAF gene, so that the detecting specificity for a mutation site is greatly improved, the detection interference for surrounding adjacent mutation sites is reduced, and good detection effects are obtained. The invention furtherdiscloses the reagent kit for detecting the K601E mutation of the BRAF gene by using the primer and the probe, so that the K601E mutation of the BRAF gene can be rapidly, acutely and accurately detected. The primer, probe and reagent kit for detecting K601E mutation of a BRAF gene have the advantages of being good in specificity, high in sensitivity, simple to operate, low in cost, accurate in results, convenient for interpretation and the like, and have favorable clinical application prospects.

Description

Technical field [0001] The present invention relates to the field of molecular biology technology, in particular to a primer, probe and kit for detecting the BRAF gene K601E mutation. Background technique [0002] BRAF gene mutations are common in some malignant tumors, such as melanoma, non-small cell lung cancer, non-Hodgkin’s lymphoma and colorectal cancer. In thyroid cancer, the frequency of BRAF gene mutation is also very high, especially BRAF gene V600E mutation frequency is the highest, because this mutation is often associated with poor clinical prognosis, so BRAF gene V600E mutation is an important target for clinical testing. [0003] However, in addition to the V600E mutation in the BRAF gene, the K601E mutation also occurs. The V600E mutation is mainly related to papillary thyroid carcinoma (PTC), but the K601E mutation is thyroid follicular adenoma or follicular papillary thyroid carcinoma ( FVPTC). Clinical data confirms that, unlike the BRAF gene V600E mutation, th...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6886C12N15/11
CPCC12Q1/6886C12Q2600/118C12Q2600/156
Inventor 张宇清裴婷婷赵艳伟
Owner 上海润安医学科技有限公司