DNA methylation sequencing library and construction method and detection method thereof

A technology for constructing methods and sequencing libraries, applied in chemical libraries, biochemical equipment and methods, libraries, etc., can solve the problems of uncorrectable methylation information, data contamination, and distortion of methylation information, so as to avoid redundant sequencing data. extra effect

Pending Publication Date: 2022-02-11
竹石生物科技(苏州)有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This method does not use sulfite, which is easy to damage DNA. Therefore, this method has little damage to DNA structure, and will not cause DNA chain breakage, base loss, etc., but this method uses unmethylated dNTP to double Repairs missing ends of strands of DNA
For DNA with uneven double strands and sticky single-stranded ends, the repaired bases will also be read by the sequencer, so this method introduces new and different levels of methylation when repairing the missing ends, This distorts the methylation information
In addition, since it is difficult to know how many bases are missing in the original double-stranded DNA, these distorted methylation information cannot be corrected in subsequent bioinformatics analysis
Furthermore, the number of bases in the sequenced DNA sequence is more than the number of bases in the original DNA sequence, which will cause data pollution

Method used

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  • DNA methylation sequencing library and construction method and detection method thereof
  • DNA methylation sequencing library and construction method and detection method thereof
  • DNA methylation sequencing library and construction method and detection method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0091] The present embodiment provides a method for obtaining target double-stranded DNA, which comprises the following steps:

[0092] 1. Extract the DNA in the target sample, detect the concentration of DNA, and use electrophoresis to detect whether the molecular weight of the extracted DNA band belongs to the molecular weight of the target DNA. If so, proceed to the next step. If not, the DNA was re-extracted.

[0093] 2. Ultrasonic fragmentation of extracted DNA, which specifically includes: determining the amount of DNA loaded according to the concentration of DNA and electrophoretic bands. Generally, the initial amount of fragmentation is 150ng, and the total volume is 100μL. Place it in a fragmentation instrument for fragmentation. For fragmentation, the fragmented DNA is subjected to 2% agarose gel electrophoresis detection, and when the DNA fragment is between 200bp and 300bp, the DNA sequencing sample containing the target double-stranded DNA is obtained. The progr...

Embodiment 2

[0097] This embodiment provides a method for methylation protection of target double-stranded DNA, which includes the following steps:

[0098] 1. Add internal reference DNA:

[0099] Take 26 μL of the fragmented DNA sequencing sample obtained in Example 1, add 1 μL of fragmented λDNA and 1 μL of fragmented pUC19 DNA (containing 0.01-0.02ng), and mix to obtain a DNA mixed sample with a total volume of 28 μL.

[0100] In this example, the added pUC19 DNA is fully methylated, that is, all cytosine (C) on it is fully methylated, and the methylation rate of pUC19 is used to indicate the degree of methylation of cytosine in the experiment. protection is effective. If in the final analysis results, the methylation rate of pUC19 is greater than 98%, it can be considered as a successful methylation protection. Therefore, the added pUC19 DNA can be used as an internal control.

[0101] In this example, the added λDNA is completely unmethylated, that is, all the cytosines (C) thereon...

Embodiment 3

[0113] Embodiment provides a kind of method of DNA denaturation, it comprises the steps:

[0114] Take 16 μL of the purified DNA obtained in Example 2 and place it in a PCR reaction tube, add 4 μL of formamide, put it into a preheated PCR instrument, cover with a thermal lid, and incubate at 85°C for 10 minutes. After the incubation, remove it immediately. Store the reaction tubes on an ice box.

[0115] Wherein, formamide is used as a denaturant to denature purified double-stranded DNA into single-stranded DNA. In this application, the methylated cytosine is protected by an oxidation reaction, and then denatured into a single-chain conversion unmethylated cytosine, which is determined by the design of the enzymatic conversion method of NEB Company.

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Abstract

The invention discloses a DNA methylation sequencing library and a construction method and a detection method thereof. According to the method, when the sequencing library is established, the double-stranded DNA is not subjected to terminal repair, but is firstly denatured into the single-stranded DNA, and then the single-stranded DNA is used as a library establishment template and is combined with an enzymatic conversion method to establish the DNA methylation sequencing library, so that the phenomenon that a repaired basic group is read by a sequencer due to terminal repair can be avoided, and thus the sequencing data redundancy phenomenon caused by the method is avoided. In addition, the method does not adopt unmethylated dNTPs to carry out terminal repair, and a new methylation level is not introduced to the terminal, so that a distortion phenomenon of the measured methylation level is not caused.

Description

technical field [0001] The present application relates to the technical field of gene detection, in particular to a DNA methylation sequencing library and its construction method and detection method. Background technique [0002] DNA in living organisms includes four bases, namely adenine (A), guanine (G), thymine (T) and cytosine (C). Among them, part of cytosine (C) will be methylated to carry a methyl group. DNA methylation is the process by which DNA methyltransferases selectively add methyl groups to cytosine to form methylated cytosine. The level of DNA methylation is usually related to the induction or repression of gene expression. If the methylation of DNA is abnormal, it may be related to the occurrence and development of tumors. Therefore, sequencing specific genes to know their methylation levels is of great value for early screening of tumors. [0003] Currently, high-throughput sequencing platforms are used to sequence specific genes, which first requires t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6806C40B50/06C40B40/06C12Q1/6869
CPCC12Q1/6806C40B50/06C40B40/06C12Q1/6869C12Q2600/154C12Q2531/113C12Q2525/191C12Q2535/122
Inventor 陈澍宜
Owner 竹石生物科技(苏州)有限公司
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