Construction method and application of genome-wide methylation high-throughput sequencing library and
A sequencing library and genome-wide technology, which is applied to the kit for constructing a genome-wide methylation high-throughput sequencing library and the field of constructing a genome-wide methylation high-throughput sequencing library, can solve problems that need to be improved
Active Publication Date: 2014-09-24
TIANJIN MEDICAL LAB BGI
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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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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]2) React the above reaction system in a water bath at 37° C. for 7 hours. 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 reac...
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Provided are a method for constructing a methylated high-throughput sequencing library for a whole genome and the use thereof. The method for constructing a methylated high-throughput sequencing library for a whole genome comprises: enzyme-digesting genome DNA with Msp I and a second restriction endonuclease; end-repairing the DNA fragments; adding base A at the 3' end of the DNA fragments that have been end-repaired; linking the DNA fragments with cohesive end A with a methylated linker; selecting fragments among the linking products with a methylated linker so as to obtain a target fragment; treating the target fragment with bisulfate so as to transform non-methylated cytosine in the target fragment to uracil; PCR amplifying the transformed target fragment; and isolating and purifying the amplification products. The amplification products constitute the methylated high-throughput sequencing library for the whole genome. The whole genome methylated high-throughput sequencing library of the genome DNA sample can be conveniently and effectively constructed using the method for constructing a methylated high-throughput sequencing library for a whole genome and the use thereof of the present invention.
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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IPC IPC(8): C40B40/06C40B50/06C12Q1/68C12N15/10C12M1/34C12Q1/34
CPCC12N15/1093
Inventor 高飞王君文夏渝东王俊
Owner TIANJIN MEDICAL LAB BGI