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Preparation and application of homologous recombination repair detection reference substance

A technology of homologous recombination and detection reference, which is applied in the field of preparation of cancer cell detection reference products, can solve the problems of self-evident importance and influence on the approval process

Pending Publication Date: 2021-01-12
菁良科技(深圳)有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In May 2020, the targeted anticancer drug Lynparza was approved by the US Food and Drug Administration for a new indication of prostate cancer. This approval makes the drug available for the treatment of ovarian cancer, breast cancer, pancreatic cancer and prostate cancer. It is bound to affect the approval process of the HRR companion diagnostic kit, and the reference material for homologous recombination repair is the main component of the approval and daily quality control, its importance is self-evident, and the reference material on the market is still blank

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  • Preparation and application of homologous recombination repair detection reference substance
  • Preparation and application of homologous recombination repair detection reference substance
  • Preparation and application of homologous recombination repair detection reference substance

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054]A homologous recombination repair detection reference product, the cell line used is the tumor cell line KBM-7, including 25 kinds of homologous recombination repair related genes with inactivating mutations, namely: ATM, ATR, BARD1, BLM, BRCA1 , BRCA2, BRIP1, CDK12, CHEK2, FANCA, FANCC, FANCD2, FANCE, FANCF, FANCI, FANCL, FANCM, NBN, PALB2, PPP2R2A, RAD51B, RAD51D, RAD50, RPA1, and RAD54L.

Embodiment 2

[0055] Example 2: A method for preparing a homologous recombination repair detection reference product (germline mutation)

[0056] Step S1: Perform gene editing on the selected tumor cell line. The target genes for gene editing include 25 genes related to homologous recombination repair, namely: ATM, ATR, BARD1, BLM, BRCA1, BRCA2, BRIP1, CDK12, CHEK2, FANCA , FANCC, FANCD2, FANCE, FANCF, FANCI, FANCL, FANCM, NBN, PALB2, PPP2R2A, RAD51B, RAD51D, RAD50, RPA1 and RAD54L, the detailed operation is as follows:

[0057] Step S11, design a specific gRNA for the target site of the target gene, construct a gRNA expression vector, design and synthesize single-stranded DNA, and use it as a template for gene editing and repair;

[0058] Step S12, co-transfecting the gRNA expression vector and the single-stranded DNA into the target cells;

[0059] Step S13, when the confluence of the cells in step S12 reaches 70%-90%, digest and collect the cells, count the cells accurately, and dilute ...

Embodiment 3

[0095] Example 3: A method for preparing a homologous recombination repair detection reference product (somatic mutation)

[0096] In this example, the tumor cell line KBM-7 is used for gene editing, gene and mutation site list, gDNA extraction method, probe list and ddPCR method as described in Example 2, because the frequency of somatic mutations is generally low , so the preparation of this reference product (single tube) was carried out by means of gDNA mixing.

[0097] Select 27 components of somatic mutation reference products in combination of all mutation sites, and the detailed site information has been verified by ddPCR as shown in the table below:

[0098]

[0099]

[0100] This combination forms a somatic mutation reference for genes involved in homologous recombination repair.

[0101] The homologous recombination repair detection reference product prepared above can be used in the detection of homologous recombination repair defects.

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Abstract

The invention aims to provide a preparation method of a homologous recombination repair detection reference substance, which comprises the following steps: carrying out gene editing on a tumor cell line, modifying 117 sites of 25 homologous recombination repair related genes to obtain embryonic system mutations of the homologous recombination repair related genes, and screening cells meeting the requirements through detection of a spectrophotometer and ddPCR (droplet digital PCR) to serve as the homologous recombination repair gene detection reference substance. The reference substance is derived from cytogenetic materials obtained after gene editing, is stable in source and has reproducible and stable performance; and in addition, the reference substance comprises a single mutation site and a combined mutation site, so that somatic mutation and embryonic system mutation can be detected, and the HRR detection range is comprehensively covered.

Description

technical field [0001] The invention relates to the technical field of preparation of a reference product for detection of cancer cells, in particular to a method for preparing a reference product for detection of homologous recombination repair and the application of the reference product. Background technique [0002] Human cells are suffering endogenous and exogenous damage every day, and DNA in cells can change and repair these damages through various mechanisms. Not only can normal cells use these repair pathways, but cancer cells can also repair damaged DNA in ever-proliferating cells. Homologous recombination repair (homologous recombination repair, HRR) and PARP (poly (ADP-ribose) polymerase) are two important mechanisms of DNA damage repair, in which HRR is involved in DNA double-strand damage repair, and PARP is responsible for DNA single-strand damage repair. When one of the two repair mechanisms is impaired in the repair process, the other mechanism can compensa...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C12Q1/6806C12Q1/6858
CPCC12Q1/6806C12Q1/6858C12Q2521/327C12Q2563/159C12Q2535/101C12Q2545/113
Inventor 李菁华梁达超史鹏燕林海久涂英美魏孝林于艳杰
Owner 菁良科技(深圳)有限公司
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