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Preparation method and application of reference substance with high tumor mutation load and microsatellite instability

A technology of microsatellite instability and mutation load, which is applied in the field of preparation of reference products for cancer cell detection, can solve problems such as the absence of MSI cell lines, and achieve good comparison and reference effects

Pending Publication Date: 2021-01-15
菁良科技(深圳)有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are currently no paired MSI cell lines and related reference products on the market, especially the combination of TMB and MSI in the same product

Method used

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  • Preparation method and application of reference substance with high tumor mutation load and microsatellite instability
  • Preparation method and application of reference substance with high tumor mutation load and microsatellite instability
  • Preparation method and application of reference substance with high tumor mutation load and microsatellite instability

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Embodiment Construction

[0029] The technical solutions of the present invention will be described below in conjunction with the accompanying drawings and embodiments.

[0030] The invention provides a method for preparing a high tumor mutation load and microsatellite instability reference product, which is based on a paired cell line with a high TMB value, knocking out part or all of the genes of the MMR system, and then extracting and purifying the knockout gene The DNA of the removed cells is finally diluted into a standard series of reference products according to the concentration gradient; including the following detailed steps:

[0031] Step S1, designing a specific sgRNA for the MMR system; the specific sgRNA targets the MSH2 gene and the PMS2 gene of the MMR system, and the detailed sequence is:

[0032] The sgRNA sequence targeting the MSH2 gene is:

[0033] Seq01: 5'-GCGCCGTATAGAAGTCGCCCCGG-3'

[0034] The sgRNA sequence targeting the PMS2 gene is:

[0035] Seq02: 5'-GTAAGTTGCACCAATCAGCT...

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Abstract

The invention aims to provide a preparation method of a reference substance with high tumor mutation load and microsatellite instability. The preparation method comprises the following steps of on thebasis of a paired cell line with high TMB value, knocking out partial or all genes of an MMR system, extracting and purifying the DNA of the gene-knocked-out cells, and finally diluting into a standard series reference substance according to the concentration gradient. The invention also relates to application of the reference substance in detection of tumor mutation load and microsatellite instability, the reference substance provided by the invention enables detection not to depend on a public database, TMB and MSI data can be obtained through one experiment, and better comparison and reference can be provided for detection of two biomarkers related to immunotherapy curative effects.

Description

technical field [0001] The invention relates to the technical field of preparation of cancer cell detection reference products, in particular to a preparation method of high tumor mutation load and microsatellite instability reference products. Background technique [0002] Tumor mutation burden (TMB) is a quantifiable biomarker used to reflect the number of mutations contained in tumor cells, usually measured by the number of mutations per million bases in the coding region of the tumor cell genome. Simply put, it is the number of mutations carried by tumor cells. The more non-synonymous mutations, the more abnormal proteins the tumor cells produced. The higher the tumor mutation load, the more neoantigens are produced, and there is a greater possibility of activating the immune system to recognize tumors. It has been confirmed in multiple cancers that patients with high TMB also have a higher overall survival rate. TMB is currently one of the most well-studied predictiv...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6886C12N15/87C12Q1/6806
CPCC12Q1/6886C12N15/87C07K14/47C12N9/22C12Q1/6806C12Q2600/156C12Q2600/166C12Q2545/113
Inventor 李菁华刘楚新林东旭于艳杰刘清波耿汇津陆春菊
Owner 菁良科技(深圳)有限公司