Reagent for detecting DNA methylation and application

A methylation and gene technology, applied in recombinant DNA technology, DNA/RNA fragments, determination/inspection of microorganisms, etc., can solve the problems of decreased expression of tumor suppressor genes, decreased methylation degree, and altered chromatin structure.

Pending Publication Date: 2021-07-16
SINGLERA GENOMICS (SHANGHAI) LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

When tumors occur, the degree of methylation of CpG sites in non-CpG island regions of tumor suppressor genes usually decreases, while

Method used

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  • Reagent for detecting DNA methylation and application
  • Reagent for detecting DNA methylation and application
  • Reagent for detecting DNA methylation and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0427] Example 1: Reduced Representation Bisulfite Sequencing (RRBS) screening of methylation sites for differences between benign and malignant thyroid nodules

[0428] 1) Sample preparation

[0429] DNA was extracted from 37 cases of thyroid cancer and 37 cases of benign thyroid nodules using QIAamp DNA Mini Kit (QIAGEN, product number: 51304); DNA concentration was detected by QubitTM dsDNA HS Assay Kit (Thermo, product number: Q32854); 1% agarose gel electrophoresis for quality inspection.

[0430] 2) MspI digestion

[0431] Prepare the reaction system as follows:

[0432]

[0433]

[0434] The reaction procedure is: 37°C for 2 hours, and store at 4°C.

[0435] 3) End repair and A addition

[0436] Prepare the reaction system as follows:

[0437] components Volume (μl) Digested DNA product 20.0 End Repair&A-Tailing Buffer 4.0 End Repair&A-Tailing Enzyme Mix 2.0 nuclease free water 14.0 total 40.0

[0438] The reaction...

Embodiment 2

[0481] Example 2, Methylation-specific PCR (Methylation-specific PCR, MSP) and quantitative methylation-specific PCR (Quantitative MSP, Q-MSP) to verify differential methylation sites

[0482] 1) Sample preparation

[0483] Use the QIAamp DNA Mini Kit (QIAGEN, Cat. No.: 51304) to extract DNA from tissues or plasma of 10 cases of thyroid cancer and 10 cases of benign thyroid nodules; use Qubit TM dsDNA HS Assay Kit (Thermo, catalog number: Q32854) was used to detect the concentration of DNA; 1% agarose gel electrophoresis was used for quality inspection.

[0484] 2) DNA conversion

[0485] Use MethylCode TM Bisulfite Conversion Kit (Thermo, Cat. No.: MECOV50) was used to perform bisulfite conversion on the DNA obtained in step 1, and unmethylated cytosine (cytosine, C) was converted into uracil (uracil, U); The cytosine does not change after conversion.

[0486] 3) PCR mixture preparation

[0487] Including PCR reaction solution, primer mix, probe mix, the preparation of...

Embodiment 3

[0498] Example 3, multiple pre-amplification methylation-specific PCR method (preAMP-MSP) for discrimination of benign and malignant thyroid nodules

[0499] 1) Sample preparation

[0500] Use QIAamp Circulating Nucleic Acid Kit (QIAGEN, catalog number: 55114) to extract cfDNA from the plasma of 20 cases of thyroid cancer and 20 cases of benign thyroid nodules; use Qubit TM dsDNA HS Assay Kit (Thermo, Cat. No.: Q32854) was used to detect the concentration of cfDNA; LabChip 3K Assay was used for quality inspection.

[0501] 2) DNA conversion

[0502] Use MethylCode TM Bisulfite Conversion Kit (Thermo, Cat. No.: MECOV50) performs bisulfite conversion on the cfDNA obtained in step 1, and unmethylated cytosine (cytosine, C) is converted into uracil (uracil, U); The cytosine does not change after conversion.

[0503] 3) Pre-amplification PCR reaction

[0504] The pre-amplification PCR mixture includes PCR reaction solution and primer mixture. The primer mix included RARG (S...

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Abstract

The invention discloses a method for identifying the property of a thyroid nodule. The method comprises the step of detecting the DNA methylation levels of regions selected from the following (1) and (2) in a sample: (1) fragments of one or more genes selected from the following: RARG, PRR15, DPYS, MCC, TBX15, TSHR, DAPK and CDH1, and (2) nucleic acid regions within the upstream and downstream 10Kb of the genes described in (1).

Description

technical field [0001] The invention belongs to the field of molecular auxiliary diagnosis, and specifically relates to the application in the screening of benign and malignant thyroid nodules. Background technique [0002] DNA methylation is a mechanism of epigenetics, a common epigenetic modification of eukaryotic cell genomes, and an important natural chemical modification of vertebrate DNA without changing the DNA sequence. , development and other aspects play an important role and are closely related to the occurrence and development of tumors. DNA methylation plays an important role in the body, and its effects include transcriptional repression, chromatin structure regulation, X chromosome inactivation, genome imprinting, etc. Abnormal DNA methylation can affect chromatin structure and the expression of oncogenes and tumor suppressor genes involved in tumorigenesis and progression. [0003] CpG dinucleotides are the most important target of DNA methylation in mammal...

Claims

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Application Information

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IPC IPC(8): C12Q1/6886C12N15/11
CPCC12Q1/6886C12Q2600/154C12N15/11
Inventor 刘蕊苏明扬何其晔
Owner SINGLERA GENOMICS (SHANGHAI) LTD
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