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Method for constructing target set for homologous recombination repair defect detection

A technology for homologous recombination and defect detection, applied in recombinant DNA technology, biochemical equipment and methods, and microbial assay/inspection, etc., can solve the problem of increasing the potential benefit population of PARP inhibitors, large amount of sequencing data, economical cost-effectiveness It can improve the economic efficiency of detection, expand the detection rate, and reduce the detection cost.

Active Publication Date: 2021-10-01
QIAGEN SUZHOU TRANSLATIONAL MEDICINE CO LTD
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] However, ovarian cancer patients with BRCA1 / 2 gene mutations only account for about 20%-30%, and on this basis, adding the assessment of genome instability can increase the proportion of homologous recombination-positive patients to 50%, which will greatly Increase the potential benefit population of PARP inhibitors, but there are still great difficulties in the assessment of genomic instability, mainly because genomic instability requires monitoring the entire genome to analyze and determine which regions have heterozygosity Instability events such as deletions or copy number amplifications
At present, some use whole genome sequencing (WGS) for corresponding detection and analysis, but the human genome has about 300 million bases, and only low-depth sequencing also requires a large amount of sequencing data, which will lead to high detection costs and economical extremely cost-effective

Method used

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  • Method for constructing target set for homologous recombination repair defect detection
  • Method for constructing target set for homologous recombination repair defect detection
  • Method for constructing target set for homologous recombination repair defect detection

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0104] In this example, DNA is extracted from ovarian cancer paraffin-embedded (FFPE) tumor tissue samples from a clinical cooperation unit, including the following steps:

[0105] (1) Dewaxing

[0106] 1) Add 1 mL of xylene to the wax tissue, cover the tube, vortex at speed 6 for 10 seconds, incubate at 50°C for 5 minutes, centrifuge at maximum speed for 2 minutes at room temperature, and carefully suck off the supernatant with a pipette tip ;

[0107] 2) Add 1 mL of absolute ethanol to the precipitate, vortex to mix, centrifuge at the maximum speed for 2 min at room temperature, and carefully suck off the supernatant with the tip of a pipette;

[0108] 3) Store at 56°C with the lid open until the ethanol evaporates completely;

[0109] (2) cracking

[0110] 1) Add 180 μL of buffer ATL to the product of step (1) to resuspend the pellet, add 20 μL of proteinase K, vortex and mix, and incubate at 56°C for 1 h;

[0111] 2) Shake and mix, and place at 90°C for 1 hour (wait for ...

Embodiment 2

[0122] In this example, the DNA extracted from the nucleic acid in Example 1 was used. After fragmentation, adapter ligation, two rounds of purification, PCR amplification and quality control testing were performed to construct a pre-library. The steps are as follows:

[0123] (1) Enzymatic reaction

[0124] Pre-mix 5 μL Fx buffer and 10 μL Fx Enzyme Mix, store on ice, mix with 35 μL sample DNA to form a 50 μL total system, pre-cool the PCR instrument to 4°C, and set the program: 32°C, 32 min, 65°C, 30 min, 4°C Hold, hot lid 70°C, transfer the product to ice after the PCR reaction;

[0125] (2) Joint connection

[0126] Pre-mix 15 μL of water, 20 μL of buffer and 10 μL of ligase, add 5 μL of adapter stock solution, and prepare a 100 μL total system with the product of the previous step, incubate in a PCR instrument at 20°C for 15 min, and close the heat cover;

[0127] (3) Purification after connection

[0128] 1) Take the AMPure XP beads out of the refrigerator at 4°C in a...

Embodiment 3

[0138] In this example, the pre-library constructed in Example 2 is used to capture the target fragment, and the steps are as follows:

[0139] (1) Add 8 μL blockers, 5 μL blocker solution and 5 μL probe composition of the present invention to the pooled pre-library (the molar concentration ratio of HRR gene sub-probe group A to SNP site sub-probe group B is 4:1) , placed in a vacuum concentrator and evaporated to dryness at 45°C. During this period, the streptavidin magnetic beads and the purified magnetic beads can be taken out and equilibrated at room temperature for 30 min. The specific design method of the sub-probe group A is as follows:

[0140] 1) According to the UCSC database, the genome corresponding sequences of the coding regions of 40 genes were obtained, and the repeated regions were marked according to the database. The repeated regions include SINE, LINE and LTR, etc., for these repeated regions, no probe design is performed;

[0141] 2) For the gene coding s...

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Abstract

The invention relates to a method for constructing a target set for homologous recombination repair defect detection. The target set comprises a homologous recombination repair (HRR) gene and SNP (Single Nucleotide Polymorphism) loci, the HRR gene comprises 40 genes represented by BRCA1 and BRCA2, and the SNP loci comprise 10913 SNP loci. The SNP loci capable of fully representing genome instability are screened and combined with the HRR gene to construct the target set, the probe composition is designed, HRR gene variation can be detected by utilizing the probe composition, meanwhile, genome instability is accurately evaluated, the positive detection rate of homologous recombination repair defects can be effectively increased. The detection cost is reduced.

Description

technical field [0001] The invention belongs to the technical field of molecular detection and high-throughput sequencing, and relates to a method for constructing a target set for homologous recombination repair defect detection. Background technique [0002] Ovarian cancer is a common malignant tumor of the female reproductive system. The traditional standard therapy is cytoreductive surgery combined with platinum-based chemotherapy. Although most patients can achieve clinical remission with standard therapy, the recurrence rate of ovarian cancer patients within three years is higher. [0003] In recent years, the emergence of polyglycoside diphosphate-ribose polymerase (PARP) inhibitors has changed the treatment pattern of ovarian cancer patients. At present, there are many PARP inhibitors on the market, which can significantly prolong the progression-free survival time of ovarian cancer patients. PARP The gene family includes 17 members, among which PARP1 and PARP2 main...

Claims

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Application Information

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IPC IPC(8): C12Q1/6886C12Q1/6827C12N15/11
CPCC12Q1/6886C12Q1/6827C12Q2600/156C12Q2525/173C12Q2535/122C12Q2525/191
Inventor 刘佳顾凯丽宋文惠韩竹青张亚飞
Owner QIAGEN SUZHOU TRANSLATIONAL MEDICINE CO LTD
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