Construction method and application of genome-wide methylation high-throughput sequencing library and

A whole-genome and sequencing library technology, applied in the field of constructing whole-genome methylation high-throughput sequencing libraries and kits for constructing whole-genome methylation high-throughput sequencing libraries, can solve problems that need to be improved

Active Publication Date: 2013-05-08
TIANJIN MEDICAL LAB BGI
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

[0003] However, current studies of genome-wide DNA methylation still need to be improved

Method used

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  • Construction method and application of genome-wide methylation high-throughput sequencing library and
  • Construction method and application of genome-wide methylation high-throughput sequencing library and
  • Construction method and application of genome-wide methylation high-throughput sequencing library and

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Embodiment 1

[0055] 1. Experimental process

[0056] 1. Use Msp I and ApeK I to digest genomic DNA:

[0057] 1.1 Msp I enzyme digestion: 100ng human mDC cell line whole genome DNA sample (mDC, mature dendritic cells) was digested with Msp I:

[0058] 1) Prepare the Msp I digestion reaction system in a 1.5ml centrifuge tube:

[0059]

[0060]The reaction system was reacted in a 37°C water bath for 7h. After the reaction was completed, the enzyme digestion reaction system was placed at 80° C. for 20 minutes to inactivate the restriction endonuclease Msp I.

[0061] 1.2 ApeK I digestion:

[0062] 1) Add 5 μL of ApeK I (NEB) (4,000 units / ml) directly to the restriction endonuclease-inactivated Msp I digestion reaction system

[0063] 2) The reaction system was subjected to enzyme digestion overnight (16-19h) in a water bath at 75°C.

[0064] 3) Add 1 μL of EDTA (1 mM) to the reaction system to inactivate the restriction endonuclease ApeK I.

[0065] 4) The DNA in the reaction system w...

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Abstract

The invention provides a construction method and application of a genome-wide methylation high-throughput sequencing library. The construction method of the genome-wide methylation high-throughput sequencing library comprises steps of: conducting digestion on a genome DNA with Msp I and a second restriction endonuclease; conducting end repair on DNA fragments; adding a basic group A on a 3'terminal of the DNA fragment subjected to end repair; connecting a DNA fragment with a cohesive end A to a methylation joint; conducting fragment selection on the connect products with the methylation joint, in order to obtain a target fragment; subjecting the target fragment to a bisulfite treatment, in order to convert unmethylated cytosine in the target fragment to uracil; subjecting the converted target fragment to PCR amplification; and separating and purifying the amplification products, wherein the amplification products form the genome-wide methylation high-throughput sequencing library. The construction method and application of the genome-wide methylation high-throughput sequencing library provided by the invention can conveniently and effectively construct the genome-wide methylation high-throughput sequencing library of the genome DNA sample.

Description

technical field [0001] The present invention relates to the field of biotechnology. Specifically, it relates to the detection technology of genome-wide methylation, especially the field of detection technology of whole-genome methylation of trace DNA. More specifically, the present invention provides a method for constructing a genome-wide methylation high-throughput sequencing library, a method for determining the methylation site of a genomic DNA sample, and a method for determining the methylation site of a genomic DNA sample. A set of methylation sites, a set of isolated restriction enzymes, and a kit for constructing genome-wide methylation high-throughput sequencing libraries. Background technique [0002] DNA methylation is the most deeply studied epigenetic mechanism. DNA methylation plays an important role in maintaining normal cell function, inhibiting the damage of genome integrity caused by parasitic DNA components, modifying chromatin structure, inactivating X ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C40B40/06C40B50/06C12Q1/68C12N15/10C12M1/34C12Q1/34
CPCC40B50/06C12Q1/68C12N15/1093C12N9/22
Inventor 高飞王君文夏渝东王俊
Owner TIANJIN MEDICAL LAB BGI
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