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Single-base resolution localization analysis method for 5-carboxycytosine modification in DNA assisted by deaminase

A carboxylcytosine and analysis method technology, which is applied in the field of 5-carboxycytosine modified single base resolution localization analysis, can solve the problems of increasing purification steps, DNA loss and the like, achieves high deamination efficiency, is easy to operate, and is conducive to localization The effect of analysis

Active Publication Date: 2019-10-22
WUHAN UNIV
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Problems solved by technology

Because the M.SssI enzyme only methylates cytosines on CpG dinucleotides, other randomly distributed 5-caC sites cannot be detected; otherwise, NaBH 4 Treatment and M.SssI enzyme treatment will add additional purification steps, during which some DNA will be lost

Method used

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  • Single-base resolution localization analysis method for 5-carboxycytosine modification in DNA assisted by deaminase
  • Single-base resolution localization analysis method for 5-carboxycytosine modification in DNA assisted by deaminase
  • Single-base resolution localization analysis method for 5-carboxycytosine modification in DNA assisted by deaminase

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Experimental program
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Embodiment

[0045] Standard Synthetic DNA Analysis

[0046] Commercially synthesized DNA 100ng containing C, 5-mC, 5-hmC, 5-fC and 5-caC, respectively, was added with a certain amount of APOBEC3A protein (according to the actual activity of the APOBEC3A protein used to determine the most suitable deamination concentration ), 2 μL of 250 mM HEPES, add deionized water to make the reaction system 20 μL, and incubate in a 37°C water bath for 2 hours. This was followed by incubation for 10 minutes in a 90°C water bath.

[0047] The above reaction DNA was taken for polymerase chain amplification reaction. Reaction system: 2 μL of 10x amplification buffer, 1 μL of 10 μmol / L forward and reverse primers, 5 ng of template DNA, 10 U of taq DNA polymerase final concentration, add deionized water to make the system 20 μL. The annealing temperature of the amplification reaction is selected according to different regions; the amplification cycle time program: (1) denaturation at 95°C for 5min; (2) den...

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Abstract

The invention discloses a single-base resolution localization analysis method for 5-carboxycytosine modification in DNA assisted by deaminase. The cytosine deaminase APOBEC3A protein is used to efficiently deaminate normal cytosine, 5-methylcytosine, 5-hydroxymethylcytosine and 5-aldehyde cytosine in DNA, except 5-carboxycytosine. Then PCR amplification is carried out, followed by sequencing to obtain the site information of 5-carboxycytosine. This method has the advantages of high sensitivity, high selectivity and simple operation, and can be adopted to directly obtain the single base resolution location of 5-carboxycytosine in DNA without the treatment of a DNA sample with bisulfite.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a deaminase-assisted single-base resolution positioning analysis method for 5-carboxycytosine modification in DNA. Background technique [0002] In mammalian DNA, the 5-position of cytosine will undergo methylation modification under the action of DNA methyltransferase to form 5-methylcytosine (5-mC). 5-mC participates in many important biological processes, such as genome imprinting, gene expression regulation, etc., and is an important epigenetic modification. 5-methylcytosine can be further oxidized successively by TET proteins to 5-hydroxymethylcytosine (5-hmC), 5-formylcytosine (5-foC) and 5-carboxycytosine (5-caC) , forming a new modification. These newly discovered modifiers are closely related to a variety of important physiological functions, such as cell differentiation, cell reprogramming, nervous system development, the occurrence and development of diseases, and so on....

Claims

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Application Information

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IPC IPC(8): C12Q1/6869
CPCC12Q1/6869C12Q2521/539C12Q2531/113C12Q2535/101
Inventor 袁必锋李俏莹冯钰锜
Owner WUHAN UNIV
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