Preparation method of gene mutation and fusion gene positive reference

A technology that combines genes and reference products, applied in botany equipment and methods, biochemical equipment and methods, genetic engineering, etc., can solve problems that affect diagnosis and treatment, errors, and product quality that cannot meet safety and effectiveness

Inactive Publication Date: 2018-11-02
辽宁琦润生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

If the setting of the reference product is unreasonable or has defects, it will cause major problems in the selection of raw materials for the entire product, the determination of the production process, and the composition of the reaction system, resulting in the quality of the product not being safe and effective for clinical use. It is easy to have wrong results in the process of clinical testing and affect clinical diagnosis and treatment

Method used

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  • Preparation method of gene mutation and fusion gene positive reference
  • Preparation method of gene mutation and fusion gene positive reference
  • Preparation method of gene mutation and fusion gene positive reference

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Use PCDH lentivirus as a vector to prepare BRAF V600E gene mutation positive reference products:

[0037] (1) First, call out the genomic DNA sequence of the corresponding mutation site of the gene to be detected from the Genbank of the NCBI website, such as: BRAF V600E gene mutation, check the BRAF genomic DNA sequence surrounding the V600E mutation site, and 下游约500bp复制其序列,序列如下:ATCTATTAGTCCCTTTCAGACCTCTGACCTTGCTCAGTGGTAGTTGAGATATAACTGAAGACTCTAAATTATATAACAATGAGGTGAGAAAAACATAATATTTCTCTTCCCTAAGTGCAGACTAAGATACTATCTGCAGCATCTTCATTCCAATGAAGAGCCTTTACTGCTCGCCCAGGAGTGCCAAGAGAATATCTGGGCCTACATTGCTAAAATCTAATGGGAAAGTTTTAGGTTCTCCTATAAACTTAGGAAA GCATCTCACCTCATCCTAAC ACATTTCAAGCCCCAAAAATCTTAAAAGCAGGTTATATAGGCTAAATAGAACTAATCATTGTTTTAGACATACTTATTGACTCTAAGAGGAAAGATGAAGTACTATGTTTTAAAAGAATATTATATTACAGAATTATAGAAATTAGATCTCTTACCTAAACTCTTCATAATGCTTGCTCTGATAGGAAAATGAGATCTACTGTTTTCTTACTTACCGTTATCAGTTCAAGTTGAAAG T GAAATCTCGATGGAGTGGGTCCCATCAGTTTGAACAGTTGTCTGGATCCATTTTGTGGATGGTAAGAATTGAGGCTATTTTCCA...

Embodiment 2

[0067] Use the pMXs mimetic virus as a vector to prepare a positive reference product for BRAF gene mutation:

[0068] The pMXs retrovirus was used as the vector to carry out the same construction, the upstream and downstream primer restriction sites of BRAF should be changed, and the upstream primer sequence is: GGAATTCGCATCTCACCTCATCCTAAC;,

[0069] The downstream primer sequence is: CCCAAGCTTTAGTAACTCAGCAGCATCTCAG,

[0070] Among them, GAATTC and AAGCTT are EcoRI and HindIII enzyme cutting sites respectively, and the carrier and primer are digested with these two same enzymes, and then purified, ligated, heat shock transformed, extracted and identified recombinant plasmids, and site-directed rapid mutation And the subsequent preparation of virus packaging and the establishment of stable cell lines are all the same as the above-mentioned steps in Example 1.

Embodiment 3

[0072] Use PCDH lentivirus as a carrier to prepare a gene mutation-positive reference product for combined mutation of two genes of Kras and BRAF:

[0073] (1) Call out the genomic DNA sequence of the corresponding mutation site of the Kras gene to be detected from the Genbank of the NCBI website, the sequence is as follows:

[0074] CCGCAGAACAGCAGTCTGGCTATTTAGATAGAACAACTTGATTTTAAGATAAAAGAACTGTCTATGTAGCATTTATGCATTTTCTTAA GCGTCGATGGAGGAGTTTG TAAATGAAGTACAGTTCATTACGATACACGTCTGCAGTCAACTGGAATTTTCATGATTGAATTTTGTAAGGTATTTTGAAATAATTTTTCATATAAAGGTGAGTTTGTATTAAAAGGTACTGGTGGAGTATTTGATAGTGTATTAACCTTATGTGTGACATGTTCTAATATAGTCACATTTTCATTTTTTATTATAAGGCCTGCTGAAAATGACTGAATATAAACTTGTGGGTAG GGTGGC GTAGGCAAGAGTGCCTTGACGATACAGCTAATTCAGAATCATTTTGTGGACGAATATGATCCAACAATAGAGGTAAATCTTGTTTTAATATGCATATTACTGGTGCAGGACCATTCTTTGATACAGATAAAGGTTTCTCTGACCATTTTCATGAGTACTTATCAAGATAATTATGCTGAAAGTTAAGTTATCTGAAATGTACCTTGGGTTTCAAGTTATATGTTGAACTTATAT G AGTATGTCAGGGTCCATG ATGTTCACTCTCTGTGCATTTTGATTGGAAGTGTATTTCAGA...

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Abstract

The invention aims to provides a preparation method of a gene mutation and fusion gene positive reference. The preparation method comprises the following steps: with a lentivirus or a retrovirus, usedfor integrating a foreign gene in a cell genome, as a vector, preparing a sequence meeting different gene mutations or fusion genes, and after the sequencing validation is error-free, embedding in the cell genome through the virus, screening out a monoclonal cell line with gene mutation or fusion gene from a drug, thereby obtaining the continual positive reference. The cell line is preferably 293T cell line, the mutation can be conducted at a single mutation site of a single gene, multiple site mutations of a single gene and multiple site mutations of multiple genes, and the fusion gene canbe a single fusion gene,and can also be multiple fusion genes connected in series. The preparation method has the advantages of being rapid, accurate, simple, low in cost, high in sensitivity, and capable of realizing mass production, and application and popularization in the markets are facilitated.

Description

technical field [0001] The invention belongs to the field of gene detection, and particularly provides a method for preparing a positive reference product for gene mutation and fusion gene. Background technique [0002] As we all know, genetic diagnosis is a field that has developed extremely rapidly in recent years. Genetic detection is required in the molecular diagnosis and targeted therapy of tumors, the determination of pathogens of infectious diseases, and the genotype (SNP) detection or analysis of individualized treatment of other diseases. technology, and therefore, there is an increasing clinical need for genetic testing. Currently widely used detection methods such as next-generation sequencing technology (Next-generation sequencing technology), amplification resistance mutation system (Amplification refractory mutation system, ARMS)-PCR, digital PCR (Digital PCR, dPCR) technology, etc., according to these technologies, are being Develop corresponding gene detect...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/867C12N5/10
CPCC12N15/86C12N2740/15043C12N2800/107C12Q1/6806C12Q2545/113
Inventor 高劲松张英杰魏潇魏奇
Owner 辽宁琦润生物科技有限公司
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