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Nucleotide sequence group for detecting BRAF gene mutation and application of nucleotide sequence group

A nucleotide sequence and gene technology, applied in the field of molecular biology, can solve the problems of complicated operation of sequencing method, high invasiveness of biopsy and high cost, and achieve intuitive data analysis and result interpretation, simple experimental operation and low detection cost. Effect

Pending Publication Date: 2020-09-01
成都华青精准医疗科技有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] In view of the defects in the prior art that the biopsy is invasive, the operation of the sequencing method is complicated, the cost is high and the cycle is long, and the sensitivity of the conventional fluorescent quantitative PCR detection method can only reach 1%, the technical problem to be solved by the present invention is to provide a method for improving BRAF Methods for Gene Mutation Detection Sensitivity

Method used

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  • Nucleotide sequence group for detecting BRAF gene mutation and application of nucleotide sequence group
  • Nucleotide sequence group for detecting BRAF gene mutation and application of nucleotide sequence group
  • Nucleotide sequence group for detecting BRAF gene mutation and application of nucleotide sequence group

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] Embodiment 1 Detection Sensitivity Experiment

[0056] The nucleotide sequence set used in this example is used to amplify the forward primer, reverse primer, blocking nucleotide sequence and probe of DNA containing the mutation site (the sequence was synthesized by Shanghai Sangon Bioengineering Co., Ltd.) , as shown in Table 1:

[0057] Table 1 Nucleotide sequence group

[0058]

[0059]

[0060] The fluorescent group of the probe is FAM, HEX, CY5, VIC, TET, JOE or ROX; the quenching group is BHQ-1, BHQ-2, TAMRA or MGB. Fluorescence signals were obtained by the probe method.

[0061] 1. Mutant cell line DNA

[0062] The mutant cell line DNA was purchased from Kebai Bio (Cat. No.: CBP10234), and the mutation frequency was quantified by NGS.

[0063] 2. Detection template processing for sensitivity experiments

[0064] In the detection template, the DNA mutation frequencies were 1%, 0.1%, 0.05%, and 0%, respectively.

[0065] The concentration of the positiv...

Embodiment 2

[0076] Embodiment 2 Detection Sensitivity Experiment

[0077] The nucleotide sequence group used in this example includes forward primers, reverse primers, blocking nucleotides and probes (the sequences were synthesized by Shanghai Sangon Bioengineering Co., Ltd.), as shown in Table 1:

[0078] Table 3 Nucleotide sequence group

[0079]

[0080] The fluorescent group of the probe is FAM, HEX, CY5, VIC, TET, JOE or ROX; the quenching group is BHQ-1, BHQ-2, TAMRA or MGB. Fluorescence signals were obtained by the probe method.

[0081] 1. Mutant cell line DNA

[0082] The mutant cell line DNA was purchased from Kebai Bio (Cat. No.: CBP10234), and the mutation frequency was quantified by NGS.

[0083] 2. Detection template processing for sensitivity experiments

[0084] In the detection template, the DNA mutation frequencies were 1%, 0.1%, 0.05%, and 0%, respectively.

[0085] The concentration of the positive reference product (that is, the mutant DNA containing the mutat...

Embodiment 3

[0095] Embodiment 3 Detection Sensitivity Experiment

[0096] The nucleotide sequence set used in this example includes a forward primer, a reverse primer and a probe (the sequence is synthesized by Shanghai Sangon Bioengineering Co., Ltd.) for introducing a mutation site, as shown in Table 1:

[0097] Table 5 Nucleotide sequence group

[0098]

[0099]

[0100] The fluorescent group of the probe is FAM, HEX, CY5, VIC, TET, JOE or ROX; the quenching group is BHQ-1, BHQ-2, TAMRA or MGB. Fluorescence signals were obtained by the probe method.

[0101] 1. Mutant cell line DNA

[0102] The mutant cell line DNA was purchased from Kebai Bio (Cat. No.: CBP10234), and the mutation frequency was quantified by NGS.

[0103] 2. Detection template processing for sensitivity experiments

[0104] In the detection template, the DNA mutation frequencies were 1%, 0.1%, 0.05%, and 0%, respectively.

[0105] The concentration of the positive reference product (that is, the mutant DNA ...

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Abstract

The invention discloses a nucleotide sequence group which comprises a forward primer and a reverse primer and is used for amplifying one part of a BRAF gene containing a mutation site, wherein the 3'end of the forward primer corresponds to a mutation site of the BRAF gene, the positions of 1-10 basic groups from the mutation site to the 5' end of the BRAF gene or the positions of 1-10 basic groups from the BRAF mutation site to the 3' end of the BRAF gene; or the 3' tail end of the reverse primer corresponds to the mutation site of the BRAF gene, the positions of 1-10 basic groups from the mutation site to the 5' end of the BRAF gene or the positions of 1-10 basic groups from the BRAF mutation site to the 3' end of the BRAF gene. By the adoption of the nucleotide sequence group and matching a fluorescent quantitative PCR detection method, the detection sensitivity of the BRAF mutant gene can be improved to 0.05%.

Description

technical field [0001] The invention relates to the field of molecular biology, in particular to a nucleotide sequence set for gene mutation detection and its application. Background technique [0002] BRAF is one of the most important human proto-oncogenes. Currently, more than 30 BRAF mutations have been detected, of which V600E accounts for more than 80%, which mainly occurs in melanoma, colon cancer and thyroid cancer. It is RAS-RAF-MEK- An important transduction factor in the ERK signal transduction pathway, it mainly functions through the serine-threonine protein kinase in the fibroin kinase pathway. The enzyme connects cell surface receptors and RAS proteins to nuclear transcription factors through MEK and ERK, and initiates a variety of factors to participate in the regulation of various biological events in cells, such as cell growth, differentiation and apoptosis. Mutations in this gene, the most common mutation being V600E, occur most frequently in melanoma, non-...

Claims

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Application Information

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IPC IPC(8): C12Q1/6886C12Q1/6851C12N15/11
CPCC12Q1/6886C12Q1/6851C12Q2600/156C12Q2600/106C12Q2531/113C12Q2525/186C12Q2545/101C12Q2563/107
Inventor 黎俊霞李超群王永成王旭谭英绮
Owner 成都华青精准医疗科技有限公司
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