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Probe set, system and detection method for detecting unstable sites of next-generation sequencing microsatellite and application of probe set

A technology of microsatellite instability and microsatellite sites, which is applied in the determination/testing of microorganisms, biochemical equipment and methods, recombinant DNA technology, etc., and can solve problems such as complex operation process, loss of heterozygosity, and peak size change

Pending Publication Date: 2022-02-25
GENEIS TECH BEIJING CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0010] This method is currently the methodological "gold standard" for detecting MSI, but the operation process is complicated and expensive, and the following problems may be encountered in the judgment of the results, such as too strong or too little fluorescence, non-specific peaks, and insignificant Peak size changes, loss of heterozygosity, etc.

Method used

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  • Probe set, system and detection method for detecting unstable sites of next-generation sequencing microsatellite and application of probe set
  • Probe set, system and detection method for detecting unstable sites of next-generation sequencing microsatellite and application of probe set
  • Probe set, system and detection method for detecting unstable sites of next-generation sequencing microsatellite and application of probe set

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0093] 1. Pre-hybridization library construction

[0094] 1) Take 200ng of DNA, and use CovarisM220 to fragment the DNA.

[0095] 2) Use ABclonal's Rapid DNA Lib Prep Kit for nucleotide library construction: including end repair, adapter ligation, library enrichment and other steps.

[0096] 3) After purifying the nucleotide library using Agencourt AMpure XP magnetic beads, use Qubit4.0 and Agilent 2100 capillary electrophoresis for quality control.

[0097] 2. Probe hybridization capture

[0098] 1) Probe design

[0099] In this embodiment, the wild-type probes and flanking probes ( The probe sequence is shown in Table 1).

[0100] Table 1

[0101]

[0102] The table below shows the 138 MSI sites and the corresponding probe design regions.

[0103] Table 2

[0104]

[0105]

[0106]

[0107]

[0108]

[0109]

[0110]

[0111] 2) Library hybrid capture

[0112] Mix 500ng of the prepared pre-hybridization library with human cot-1DNA and library ...

Embodiment 2

[0121] 1. Library construction before hybridization of 36 samples

[0122] 1) Take 200ng of DNA, and use CovarisM220 to fragment the DNA.

[0123]2) Use ABclonal's Rapid DNA Lib Prep Kit for nucleotide library construction: including end repair, adapter ligation, library enrichment and other steps.

[0124] 3) After purifying the nucleotide library using Agencourt AMpure XP magnetic beads, use Qubit4.0 and Agilent 2100 capillary electrophoresis for quality control.

[0125] 2. Hybridization and capture of flanking probes alone and wild type + flanking probes for 36 samples

[0126] 1) Library hybrid capture

[0127] Mix 500ng of the prepared pre-hybridization library with human cot-1DNA and library blocking reagent, evaporate to dryness at 45°C using a vacuum filter pump, then redissolve in the hybridization solution, incubate at room temperature for 10 minutes, and then put on a PCR instrument, 95°C, 5 minutes Afterwards, add the mixed probes, and then hybridize at 65°C fo...

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Abstract

The invention discloses a probe set, a system and a detection method for detecting unstable sites of a next-generation sequencing microsatellite and application of the probe set. The method comprises the following steps: selecting at least one microsatellite site as a target; designing a probe set for the target, wherein the probe set comprises a flanking probe for the at least one microsatellite site, the flanking probe covers a flanking sequence of the at least one microsatellite site sequence; and capturing the target sequence by using the probe set. Compared with a conventional wild-type probe, the flank probe can effectively avoid a GC abnormal area and can effectively make up for lost effective sequencing reads, so that the coverage of microsatellite loci is remarkably improved. In addition, the unique probe design can greatly improve the capture efficiency of the target spot, and the specificity and GC uniformity of the probe are improved. Therefore, the probe set, the system and the detection method provided by the invention have relatively high detection value and clinical value and wide application prospect in cancer-related microsatellite detection.

Description

technical field [0001] The invention relates to the field of gene detection, in particular to a probe group, a system, a detection method and an application thereof for detection of microsatellite instability sites in second-generation sequencing. Background technique [0002] Microsatellite instability (Microsatellite Instability, MSI) refers to the phenomenon that the length of microsatellite (MS) sequence changes due to insertion or deletion mutations during DNA replication. MSI occurs in colorectal cancer, endometrial cancer, gastric cancer, hepatocellular carcinoma, breast cancer and other solid tumors. [0003] Clinical studies have confirmed that MSI is closely related to the prognosis of colorectal cancer. MSI-H colorectal cancer patients have a significant survival advantage compared with MSS patients, with worse clinical manifestations but better prognosis. Studies have confirmed that for patients with stage II / III colorectal cancer, the overall survival and dise...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6886C12Q1/6869C12N15/11
CPCC12Q1/6886C12Q1/6869C12Q2535/122C12Q2525/151
Inventor 孙雪赵颖王伟伟杨洲张吉娜田埂
Owner GENEIS TECH BEIJING CO LTD
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