Single-base resolution positioning analysis method for N4-methylcytosine in deaminase-mediated DNA

A methylcytosine and analysis method technology, applied in the field of single-base resolution positioning analysis of N4-methylcytosine, to achieve the effects of shortened analysis time, mild reaction conditions, and small dosage

Pending Publication Date: 2022-03-04
WUHAN UNIV
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  • Abstract
  • Description
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  • Application Information

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Problems solved by technology

At present, there is no 4mC localization analysis method that does not rely on bisulfite treatment, high selectivity, and simple operation

Method used

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  • Single-base resolution positioning analysis method for N4-methylcytosine in deaminase-mediated DNA
  • Single-base resolution positioning analysis method for N4-methylcytosine in deaminase-mediated DNA
  • Single-base resolution positioning analysis method for N4-methylcytosine in deaminase-mediated DNA

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Experimental program
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Effect test

Embodiment 1

[0048] Embodiment 1. Utilize artificially synthesized 215bp DNA strands containing different modifications to distinguish cytosine, 5-methylcytosine and N 4 - Methylcytosine

[0049] In order to verify the accuracy of the method of the present invention, a 215bp DNA chain containing different modifications was first synthesized, reacted with A3A protein, and the deamination rate was verified by mass spectrometry and Sanger sequencing.

[0050] A. Synthetic method of 215bp DNA chain

[0051] In a 50 μL reaction system, add 0.5ng pUC19 DNA (Takara Biotechnology Co., Ltd.), 5 μL 10× reaction buffer, 2.5U Taq DNA polymerase (Takara), 2 μL 10 μM forward primer (sequence 5′-GAGTGAGTGAGGGAGGAAG -3'), 2 μL of 10 μM reverse primer (sequence 5'-CCACTCACAATTCCACACAACATAC-3'), 1 μL of dATP, dGTP, TTP and dCTP (all at a concentration of 2.5 mM). When synthesizing 5mC or 4mC modified DNA strands, just replace dCTP with 5mdCTP or 4mdCTP.

[0052] The polymerase chain reaction (PCR) progra...

Embodiment 2

[0062] Example 2. The deaminase-mediated 4mC single-base mapping method in DNA is used for the identification of the 4mC site in the whole genome of Deinococcus radiodurans

[0063] Deinococcus radiodurans is a specialized bacterium that is extremely resistant to the deadly effects of ionizing radiation, UV light, oxidation and desiccation. Studies have shown that 4mC plays an important role in maintaining the genome stability of Deinococcus radiodurans. Current methods for identifying 4mC are mainly based on bisulfite conversion or third-generation sequencing technologies, however, each of these technologies has its own shortcomings. For example, bisulfite conversion can cause a lot of degradation to DNA, and the accuracy of third-generation sequencing has always been a problem. Therefore, using the method established above, the 4mC modification at a specific site in the genomic DNA of Deinococcus radiodurans was identified.

[0064] In order to explore the distribution and...

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Abstract

The invention discloses a single base resolution positioning analysis method for N4-methylcytosine in deaminase mediated DNA, and belongs to the technical field of biology. According to the method disclosed by the invention, normal cytosine and 5-methylcytosine in DNA are subjected to deamination by utilizing cytosine deaminase A3A protein, then thymine is formed through subsequent polymerase chain reaction amplification, and a target analyte N4-methylcytosine can resist the deamination effect of cytosine deaminase A3A; therefore, cytosine is amplified in a subsequent polymerase chain reaction. In this way, only N4-methylcytosine modification can be read as cytosine in final sequencing. The method disclosed by the invention is high in selectivity and good in accuracy, and the single-base resolution map of N4-methylcytosine in DNA can be directly obtained without carrying out hydrosulfite treatment on a DNA sample.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a deaminase-mediated N 4 - A method for the single-base resolution localization analysis of methylcytosine. Background technique [0002] N 4 -Methylcytosine (4mC) is a DNA methylation modification, which mainly exists in the DNA of extreme thermophiles and some mesophilic bacteria, and has not been reported in eukaryotes, especially mammals. So far, a variety of bacterial DNA 4mC methyltransferases have been discovered, and these methyltransferases can combine with restriction enzymes to form sequence-specific restriction-modification systems. The 4mC base in host DNA plays a very important role in the recognition of restriction endonucleases for specific motifs and protection of the host genome. Although 4mC can be detected and quantified with high sensitivity by liquid chromatography, liquid chromatography-tandem mass spectrometry (LC-MS) and other techniques, their localizatio...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6806C12Q1/6869
CPCC12Q1/6806C12Q1/6869C12Q2527/101C12Q2521/539C12Q2531/113C12Q2537/164C12Q2535/101C12Q2535/122
Inventor 袁必锋熊军冯钰锜
Owner WUHAN UNIV
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