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Nucleic acid methylation cytosine conversion method

A technology for methylated cytosine and nucleic acid, applied in the field of nucleic acid methylated cytosine conversion, can solve the problems that hinder the commercial application of TAPS technology, difficulty in automation, DNA loss, etc., and achieve small DNA damage, convenient automation, and high conversion rate high effect

Pending Publication Date: 2022-02-25
YEASEN BIOTECHNOLOGY (SHANGHAI) CO LTD
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This leads to severe DNA loss, complex operation, low conversion rate and difficulty in automation, which greatly hinders the commercial application of TAPS technology.

Method used

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  • Nucleic acid methylation cytosine conversion method
  • Nucleic acid methylation cytosine conversion method
  • Nucleic acid methylation cytosine conversion method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Embodiment 1: TAPS and eTAPS performance comparison

specific Embodiment approach

[0026] In this example, the conversion, DNA recovery and accuracy of TAPS and eTAPS were compared (see figure 1 ). The specific implementation is as follows:

[0027] 1) TAPS process:

[0028] In the first step, TET oxidation. Prepare the system according to the table below.

[0029] Table 1

[0030] components Dosage Methylated pUC19 DNA (Zymo Research, Cat#D5017) 10μL (10ng) 10× eTET1 Reaction Buffer 3μL 30× eTET1 auxiliary solution 1μL eTET1 (Yeasen, Cat#12219) 1μL H2O 15μL total 30μL

[0031] The 10× eTET1 reaction buffer included 500 mM tris, 10 mM α-ketoglutarate, 500 mM sodium chloride, and 2 mM dithiothreitol.

[0032] The 30× eTET1 supplement contains 3 mM ferrous ammonium sulfate and 60 mM vitamin C.

[0033] The second step is proteinase K treatment. Add 1 μL of 20 mg / mL proteinase K and react at 50°C for 0.5 h.

[0034] The third step is DNA recovery by magnetic bead method. Add 60 μL DNA Selected b...

Embodiment 2

[0057] Example 2: Application of eTAPS in the detection of RNA cytosine methylation.

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Abstract

The invention provides a nucleic acid methylation cytosine conversion method. The method is characterized by comprising the following steps: (1) carrying out TET enzyme oxidation treatment on a DNA or RNA sample; (2) adding pyridine borane into a reaction system for treatment; and (3) recovering DNA or RNA in the reaction system by magnetic beads. According to the present invention, the whole process of the nucleic acid methylation cytosine conversion method only comprises three steps such as TET enzyme oxidation, pyridine borane treatment and DNA magnetic bead recovery, such that the conversion efficiency of the TAPS is improved, the operation difficulty and the operation flow of the TAPS are simplified, and the nucleic acid methylation cytosine conversion method is named as Enhanced TET-assisted pyridine borane sequencing (eTAPS), the eTAPS has the advantages of high conversion rate, small DNA damage, simplicity in operation, convenience in automation and the like, and in addition, we find that the eTAPS also has efficient and sensitive detection efficiency in RNA cytosine methylation. Therefore, the eTAPS technology has an important value in nucleic acid methylation detection, especially in the fields of tumor early screening and disease diagnosis.

Description

technical field [0001] The patent of the invention relates to a nucleic acid methylation cytosine conversion 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 ratio of methylation modification at the cytosine site in the CpG island 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 ...

Claims

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Application Information

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IPC IPC(8): C12Q1/6858
CPCC12Q1/6858C12Q2527/125C12Q2521/537C12Q2535/122C12Q2563/143C12Q2563/149C12Q2521/131
Inventor 宋东亮江翱曹振陈晶晶刘倩侯策韦磊王嫚孙睿
Owner YEASEN BIOTECHNOLOGY (SHANGHAI) CO LTD
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