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DNA methylation marker screening kit and method

A methylation and kit technology, applied in the field of molecular biology, can solve the problems of low enrichment of CpG sites, large initial amount of DNA library construction, and low site reproducibility

Inactive Publication Date: 2019-10-15
SINGLERA GENOMICS (SHANGHAI) LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The disadvantage of this method is that the initial amount of DNA library construction is relatively large, the degree of enrichment of CpG sites in the promoter region and CpG island region is low (especially for FFPE samples with poor DNA quality), and the site duplication between different samples Not high

Method used

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  • DNA methylation marker screening kit and method
  • DNA methylation marker screening kit and method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0125] one. Magnetic bead purification of extracted DNA

[0126] 1. Take out the AMPure magnetic beads half an hour in advance and place them at room temperature. After vortexing the magnetic beads, add 50 microliters of magnetic beads to 50 microliters of DNA solution, and mix by pipetting. Let stand at room temperature for 5 minutes.

[0127] 2. Transfer to a magnetic stand until the liquid is completely clear.

[0128] 3. Keep on the magnetic stand, discard the supernatant, and wash the magnetic beads with 150 μl of freshly prepared 80% ethanol.

[0129] 4. Repeat step 3.

[0130] 5. Remove the ethanol completely and dry with the lid open for 5 minutes.

[0131] 6. Remove the sample from the magnetic stand, add 32 microliters of nuclease-free water, fully resuspend the magnetic beads with a pipette gun, and place at room temperature for 5 minutes.

[0132] 7. Transfer samples to a magnetic stand until completely clear.

[0133] 8. Transfer 30 µl of eluate to ...

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Abstract

The invention relates to DNA methylation marker screening kit and method. The method comprises the following steps: enzyme digestion, methylation linker connection, methylation treatment and index primer amplification; wherein restriction endonuclease insensitive to methylation is used for carrying out enzyme digestion on a desired DNA sequence, and an enzyme digestion product is obtained; the enzyme digestion product is directly connected with a linker with methylation modification without terminal repair and supplementation; the enzyme digestion product comprises an enzyme digestion productwith a cohesive end and an optional enzyme digestion product with a flat end. The invention also provides a corresponding linker sequence, an index primer sequence, a preferred reagent, a kit and related applications.

Description

technical field [0001] The invention relates to the technical field of molecular biology, in particular to a DNA methylation marker screening kit and method. Background technique [0002] DNA methylation mainly occurs in CpG islands and is an important epigenetic modification. DNA methylation is known to be involved in the process of gene transcription regulation and is associated with numerous biological processes, including the formation of cancer. Methods for screening DNA methylation markers related to biological processes include whole genome bisulfite sequencing (WGBS) and reduced methylation sequencing (RRBS). Although WGBS detection is comprehensive, the cost is high, and a large number of non-DNA methylated modified regions are sequenced. Compared with WGBS, RRBS can enrich CpG sites and greatly reduce the cost of sequencing. The traditional RRBS enriches CpG sites by enzymatic digestion, completes the ends, adds adapters through TA ligation, and then performs am...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C40B50/06C12Q1/6869C12N15/11
CPCC40B50/06C12Q1/6869C12Q2525/191C12Q2535/122C12Q2521/301
Inventor 刘蕊
Owner SINGLERA GENOMICS (SHANGHAI) LTD
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