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Simple and convenient high-throughput DNA methylation detection method

A detection method and high-throughput technology, applied in biochemical equipment and methods, microbial determination/inspection, etc., can solve problems such as high false positives and false negatives, affecting the quality of sequencing data and comparison accuracy, and complex operations.

Pending Publication Date: 2022-04-22
YEASEN BIOTECHNOLOGY (SHANGHAI) CO LTD
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this method has high false positives and false negatives, and the operation is more complicated
In addition, DNA methylation detection methods need to convert all unmethylated cytosines to thymines, which affects the quality of sequencing data and the accuracy of alignment

Method used

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  • Simple and convenient high-throughput DNA methylation detection method
  • Simple and convenient high-throughput DNA methylation detection method
  • Simple and convenient high-throughput DNA methylation detection method

Examples

Experimental program
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Effect test

Embodiment 1

[0030] Embodiment 1: Preparation of 5mC DNA standard

[0031] In this embodiment, we have constructed a DNA methylation standard (5mC DNA standard sequence such as figure 2 and shown in SEQ ID No.1), the specific reaction system is as follows:

[0032] Table 2

[0033]

[0034] After mixing, react according to the following procedure:

[0035] table 3

[0036]

[0037] After PCR, 40 μL Agencourt AMPure XP beads (Beckman, A63881) were added to mix well, and incubated at room temperature for 5 minutes. Put the PCR tube on the magnetic stand. After the solution is clarified, suck off the supernatant, add 200 μL of freshly prepared 80% ethanol and let it stand for 30 seconds, and then suck up the ethanol; add 200 μL of freshly prepared 80% ethanol and let it stand for 30 seconds, and then suck it up Ethanol, let stand at room temperature to dry for 3min. After adding 20 μL ddH2O to suspend the magnetic beads, let stand at room temperature for 5 min. Place the PCR tube...

Embodiment 2

[0038] Example 2: Effect of DMA-seq on DNA methylation detection.

[0039] In this example, we use the Hieff Ultima DNA Library PrepKit for Illumina has established the conversion process and detection method of DMA-seq to 5mC DNA standards (see the schematic diagram figure 1 and image 3 ), the specific process is as follows:

[0040] 1) DNA end repair and adapter ligation

[0041] Table 4

[0042] components Dosage 5mC DNA Standard 10ng Endprep Mix 10μL Add ddH2O to 60μL

[0043] 30°C for 10 minutes, 72°C for 20 minutes, and stored at 4°C.

[0044] table 5

[0045] components Dosage The above reaction system 60μL Ligation Enhancer 30 Fast T4 DNA Ligase 5 DNA Adapter 5 Total 100μL

[0046] React at 20°C for 15 minutes.

[0047] 2) Strong alkali high temperature treatment and acid neutralization

[0048] After the reaction, add 11ul 1M NaOH or KOH, and react at 60°C for 60min.

[00...

Embodiment 3

[0059] Example 3: Effect of NaOH treatment temperature on DMA-seq data in thermal alkali deamination reaction.

[0060] In this example, we verified the effect of different NaOH treatment temperatures on DMA-seq data in the thermal alkali deamination reaction, the specific process is as follows:

[0061] 1) DNA end repair and adapter ligation: the same as in Example 2.

[0062] 2) Strong alkali high temperature treatment and acid neutralization

[0063] After the reaction, add 11ul 1M NaOH, and react at 50-80°C for 60min.

[0064] After the reaction was completed, 11ul of 1M hydrochloric acid was added.

[0065] After the reaction, the DNA was recovered using magnetic beads.

[0066] 3) Library amplification and sequencing: the same as in Example 2.

[0067] The results of the sequencing data analysis are shown in Figure 5 , as the reaction temperature increases, both the positive rate and the false positive rate increase, among which 60-70°C is the best.

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Abstract

The invention provides a simple and convenient high-throughput DNA methylation detection method, which comprises the following steps: (1) carrying out oxidation treatment on a DNA sample by using strong base to oxidize cytosine in the DNA sample into uracil and oxidize methylated cytosine into thymine; (2) adding acid to neutralize strong base, and controlling the pH value of the reaction system within 6-8; and (3) carrying out library amplification and sequencing by adopting uracil-intolerant high-fidelity DNA polymerase. The invention develops a simple and convenient high-throughput DNA cytosine methylation detection technology which is named as DMA-seq. Compared with an existing DNA methylation detection kit, the DMA-seq has the advantages of being easy to operate, short in consumed time, small in DNA damage, good in data quality, high in methylation site targeting performance and the like, and is suitable for high-abundance DNA methylation site detection, especially in the early tumor screening direction.

Description

technical field [0001] The patent of the invention relates to a simple high-throughput DNA methylation detection method, which belongs to the field of biotechnology. Background technique [0002] DNA methylation is an important research direction in the field of epigenetics. Cytosine methylation (5mC) is the most common modified base on DNA, accounting for 1%-8% of all cytosines, some specific cytosine sites such as Dam methylation sites in bacteria or eukaryotic The methylation ratio of cytosine sites in CpG islands can be as high as nearly 100%, so 5mC is also called the "fifth base". DNA methylation is the main way for bacteria to identify and distinguish internal and external DNA, and it is also an identity marker for bacteria to identify themselves. In recent years, a large number of studies have shown that the abnormal level of DNA methylation is closely related to tumorigenesis, deterioration and cell carcinogenesis. Therefore, detection of cfDNA methylation in blo...

Claims

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Application Information

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IPC IPC(8): C12Q1/6858
CPCC12Q1/6858C12Q2531/113C12Q2521/101C12Q2535/101
Inventor 陈晶晶江翱卢瑶侯策王嫚曹振宋东亮
Owner YEASEN BIOTECHNOLOGY (SHANGHAI) CO LTD
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