A kind of micro-degradation genomic DNA methylation library sequencing method and its kit

A genome and methylation technology, applied in the field of genomics and molecular biology, can solve problems such as undetectable, achieve the effect of low initial amount, reduce preference, and improve reaction efficiency

Active Publication Date: 2021-05-04
SHENZHEN E GENE TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, it is impossible to detect genomic methylation library construction and sequencing of severely degraded trace samples such as puncture FFPE and cfDNA

Method used

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  • A kind of micro-degradation genomic DNA methylation library sequencing method and its kit
  • A kind of micro-degradation genomic DNA methylation library sequencing method and its kit
  • A kind of micro-degradation genomic DNA methylation library sequencing method and its kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Reagent configuration:

[0042] The composition of Repair Buffer includes MgCl 2 , NaCl, DTT, dATP, dTTP, dCTP, dGTP, Tris-HCl, and the concentrations are 10mM, 50mM, 5mM, 4mM, 1mM, 1mM, 1mM, 60mM respectively, wherein the pH value of Tris-HCl is 7.6;

[0043] Repair Enzymes are T4Polynucleotide Kinase, T4DNA Polymerase and Taqpolymerase, and the concentrations are 8U, 2U and 0.04U respectively;

[0044] Buffer A includes Tris-HCl, MgCl 2 , DTT, ATP, and the concentrations were 190mM, 27mM, 14mM, 7mM;

[0045] Buffer B includes PEG6000 and PEG8000, the concentration of PEG6000 is 30%, the concentration of PEG8000 is 20%

Embodiment 2

[0047] Severely degraded FFPE sample DNA methylation library construction

[0048] Genomic DNA was extracted using a commercial kit (Qiagen FFPE extraction kit). After extracting DNA from a puncture micro-tissue slide, methylation library construction was taken as an example. The DNA was eluted into 30ul of ultrapure water.

[0049] 1. Take 1uL samples respectively for Qubit HS dsDNA concentration determination and 2100 analyzer to evaluate the degree of sample degradation (such as figure 2 ), the results showed that the samples were distributed almost uniformly in the interval of 50bp-10kb, the samples were completely degraded, and the total amount of samples detected by Qubit was 23ng.

[0050] 2. DNA screening and fragmentation in different ranges: 19.5ul of Ampure XPBeads (Agencourt) was added to 30ul of DNA samples for 3 minutes, and the magnetic beads were adsorbed on the magnetic stand. After clarification, transfer the supernatant to a new EP tube, then add 30uL of A...

Embodiment 3

[0076] In this example, 5uL of DNA Repair Buffer, 2uL of Repair Enzymes and 13uL of ultrapure water were sequentially added to a 30uL DNA sample, mixed evenly and reacted on a PCR instrument according to the following process, 16°C, 40min; 75°C , 25min; 72°C, 25min; 37°C, 10min. After the reaction, add 11uL of Buffer A, 12uL of Buffer B, 1uL of the annealed joint, 3uL of water and 3uL of DNA ligase in the tube, mix well, and incubate at 24°C for 10min.

[0077] Other steps are with embodiment 2.

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Abstract

The invention discloses a method for building and sequencing a micro-degraded genomic DNA methylation library. The invention tests different library building technologies and optimizes the reaction process to establish a methylation library that can detect as little as 1ng of severely degraded genomic DNA. technology. By testing different enzyme combinations, the end repair of degraded DNA, the addition of A at the 3-end, and the repair of DNA double-strand gaps due to degradation were completed in the same reaction system; by optimizing the reaction system, the sequencing adapters were connected in the original reaction solution; After further testing the biotin labeling of the adapter, through the specific binding of biotin and streptomycin magnetic beads, the capture of the genome fragments of the ligated adapter before the subsequent sulfite treatment was realized, and the loss of the target fragment was reduced; in addition, the establishment of sulfite Salt reaction system and reaction time reduce DNA damage during processing, ensure methylation conversion rate, and realize methylation library construction and sequencing of micro-degraded samples.

Description

technical field [0001] The invention relates to the technical fields of genomics and molecular biology, in particular to a method for building a library and sequencing a micro-degraded genome DNA methylation. Background technique [0002] DNA methylation 5mC is a process in which the methyl group in the methyl donor S-adenoylmethionine (SAM) molecule is transferred to DNA cytosine mediated by methyltransferase (DNAmethyltransferase, MTase). In eukaryotes, DNA methylation mainly occurs at the 5-carbon position of the CpG dinucleotide cytosine, forming 5-methylcytosine (5mC). The methylation modification of DNA is involved in animal embryonic development, gene imprinting and X chromosome inactivation, and plays an important role in the regulation of gene expression. The disorder of DNA methylation modification provides new ideas for the exploration of abnormal embryonic development and the pathogenesis of cancer. In order to meet different scientific research needs, in the p...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6869C12Q1/6806C40B50/06
CPCC12Q1/6806C12Q1/6869C40B50/06C12Q2523/125C12Q2525/191C12Q2521/501
Inventor 王君文黄文蕊吴炜吉冠玉高飞
Owner SHENZHEN E GENE TECH
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