Primer combination, kit, model, construction method and detection method for detecting BRAF gene mutation

A technology of primer combination and kit, applied in biochemical equipment and methods, DNA/RNA fragments, recombinant DNA technology, etc., can solve the problems of inconvenient detection of BRAF gene-related site mutations, and achieve the effect of improving the detection level

Pending Publication Date: 2022-04-22
MYGENOSTICS (CHONGQING) GENE TECH CO LTD
View PDF0 Cites 1 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] In view of the above-mentioned deficiencies in the prior art, the problem to be solved in the present invention is: how to provide a primer combination, kit, model, constructi

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Primer combination, kit, model, construction method and detection method for detecting BRAF gene mutation
  • Primer combination, kit, model, construction method and detection method for detecting BRAF gene mutation
  • Primer combination, kit, model, construction method and detection method for detecting BRAF gene mutation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Specific primers for the 12th and 15th exons of the BRAF gene:

[0051]

[0052] Detection sites of the 12th and 15th exons of the BRAF gene:

[0053]

Embodiment 2

[0055] The experimental samples of this program are tested with samples of known clinical information, as follows

[0056] serial number sample number Gene Exon base amino acid mutation frequency 1 19CA25984 BRAF exon12 c.1458_1472del p.N486_T491delinsK 8％ 2 19CA25736 BRAF exon15 c.1799T>A p.V600E 3.54％

[0057] 1. Extraction of cell-free nucleic acid (cfDNA) from plasma samples

[0058] Plasma samples were extracted using the magnetic bead method large-volume free nucleic acid extraction kit (Tiangen, DP710-01) produced by Tiangen Biochemical Technology (Beijing) Co., Ltd., and Qubit detected the concentration of cfDNA after 1000 μl of plasma samples were extracted. The concentration range was 0.1ng / ul-50ng / ul range. After passing the test, it is recommended to perform PCR amplification immediately, or store directly at -20±5°C for no more than one month. During this period, repeated freezing and thawing should be avoide...

Embodiment 3

[0106] Study on the detection limit of the 12th and 15th exons of BRAF gene

[0107] The experimental samples of this scheme are tested with samples with known clinical information, such as the samples with known clinical information in Example 2.

[0108] serial number sample number Gene Exon base amino acid mutation frequency 1 19CA25984 BRAF exon12 c.1458_1472del p.N486_T491delinsK 8％ 2 19CA25736 BRAF exon15 c.1799T>A p.V600E 3.54％

[0109] 1. Extraction of cell-free nucleic acid (cfDNA) from plasma samples

[0110] Plasma samples were extracted using the magnetic bead method large-volume free nucleic acid extraction kit (Tiangen, DP710-01) produced by Tiangen Biochemical Technology (Beijing) Co., Ltd., and Qubit detected the concentration of cfDNA after 1000 μl of plasma samples were extracted. The concentration range was 0.1ng / ul-50ng / ul, it is recommended to perform PCR amplification immediately after the determinat...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

No PUM Login to view more

Abstract

The invention relates to the field of molecular biological detection, in particular to a primer combination, a kit, a model, a construction method and a detection method for detecting BRAF gene mutation. Comprising the following steps: extracting a cfDNA sample, configuring a reaction system for detecting BRAF gene mutation based on the extracted cfDNA sample, correspondingly performing PCR amplification, performing linker addition and PCR enrichment on an amplification product to prepare a sequencing library, performing quantification and quality control on the sequencing library, performing high-throughput sequencing by adopting a gene sequencer, and analyzing sequencing data by adopting biological information analysis software. According to the detection method, a free nucleic acid sample containing 0.09% of BRAF gene related site mutation can be detected for the 12th exon of the BRAF gene, and a free nucleic acid sample containing 0.05% of BRAF gene related site mutation can be detected for the 15th exon of the BRAF gene; the biological clinical detection level of BRAF gene related sites can be improved, and a foundation is laid for popularization and application.

Description

technical field [0001] The invention relates to the field of molecular biology detection, in particular to a primer combination, kit, model, construction method and detection method for detecting BRAF gene mutation. Background technique [0002] The clinical features and prognostic factors of Langerhans cell histiocytosis (LCH) in adults are unclear. However, recurrent somatic mutations of the BRAF V600E allele have been found in LCH, and most of the genome analysis comes from children. In newly diagnosed LCH tissue samples, the first-generation package plus the second-generation package gene exon mutation detection, 63.2 % of the children had BRAF gene mutations, and the mutation sites were mainly concentrated in the 12th and 15th exons. The p.V600E type accounted for 50.6%; the positive rate of non-p.V600E mutations was 12.6%, including: p.N486_T491delinsK, p.V600D(c.1799_1800delinsAT), p.N486_P490del, p.L485_P490delinsF, and three sporadic positive mutations (p.V600D(c....

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
IPC IPC(8): C12Q1/6883C12Q1/6869C12N15/11
CPCC12Q1/6883C12Q1/6869C12Q2600/156C12Q2531/113C12Q2525/191C12Q2535/122
Inventor 伍建姬晓雯武璇
Owner MYGENOSTICS (CHONGQING) GENE TECH CO LTD
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap