Method for identifying base modification

A base, non-modification technology, applied in the field of identifying single-base methylation modification, can solve the problems of low repetition rate, no single-base precision method, and low resolution, so as to reduce false positives and save antibodies The effect of the enrichment step

Inactive Publication Date: 2019-07-19
SUN YAT SEN UNIV
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Problems solved by technology

However, the limitation of this method is that it can only confirm that the modification exists within a fragment of about 100 bases in length, but cannot determine the modified position of a single base
Subsequently, many laboratories improved on the basis of this method to reduce the demand for starting mRNA and improve the resolu

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Examples

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Example Embodiment

[0052] Example 1 RNA endonuclease MazF and ChpBK digest RNA oligonucleotides

[0053] The RNA oligonucleotide sequence synthesized in vitro was used for verification, and it was found that MazF can only cut the ACA sequence without methylation modification and cut at the 5'end of the ACA, but cannot cut the methylation modification. ACA sequence ( figure 1 ). We mix the RNA oligonucleotide sequences with and without methyl groups so that the proportions of modified sites are 0%, 20%, 40%, 60%, 80%, 100%. To simulate partial methylation in the body, and use MazF enzyme for digestion, it is found that the degree of digestion is proportional to the proportion of methylation contained in it ( figure 2 ), indicating that the enzyme can be used to estimate the ratio of N6-methyladenine. The MazF of the present invention comes from Baori Medical Biotechnology (Beijing) Co., Ltd., and its article number is 2415A. The restriction digestion system of RNA oligonucleotide sequence is as f...

Example Embodiment

[0059] Example 2 Process of building a small RNA library

[0060] Use as Figure 5 To establish a small RNA library, the specific operations are as follows:

[0061] 1) Demethylation treatment: use N6-methyladenine modified demethylase FTO to treat mRNA as a negative control. The mRNA must be heated in a PCR machine at 85°C for 5 minutes before the reaction, and then immediately placed on ice for 2 minutes to remove the secondary structure of the mRNA. The demethylation reaction system is as follows:

[0062] Final concentration or total mRNA ~200ng N6-methyladenine modified demethylase FTO2.5ug α-ketoglutarate (α-KG)300uM Ferrous ammonium sulfate (Fe(NH4)2(SO4)2)283uM Ascorbic acid (L-ascorbic acid) 2mM Tris-HCl buffer (pH 7.5) 50mM RNase inhibitor20U Enzyme-free water (RNase-free H2O) To 20ul

[0063] React at room temperature (25°C) for 3 hours, add 1ul 40mM EDTA to stop the reaction, or use RNA purification kit for purification.

[0064] 2) MazF digestion reaction: bef...

Example Embodiment

[0080] Example 3 High-throughput sequencing data analysis

[0081] use Image 6 The procedure for sequencing and analyzing the small RNA library obtained in Example 2 is as follows:

[0082] First, perform quality control on sequencing data, remove sequencing adapters, and retain remaining fragments larger than 15 nt sequencing short sequences (reads);

[0083] Use Hisat2 software to compare the sequencing data back to the reference genome to get its specific position on the genome.

[0084] Locate the ACA sequence that has not been digested by MazF from the alignment results. The specific method is that if an ACA sequence is at the beginning of sequencing reads, it is normally unmethylated A, because it is the site cut by the MazF enzyme; and when the ACA sequence appears in the middle of the reads, it is in parallel. Without being cut by the MazF enzyme, the first A is the methylation site. Since only reads above 15 nt are retained when the data is unlinked, the position of the id...

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Abstract

The present invention discloses a method for identifying a base modification. The method comprises the following steps: I, a sample with nucleic acids to be tested and a base to be identified are provided; II, a nuclease capable of specifically distinguishing between a modified and an unmodified base to be identified is utilized to conduct enzymatic cleavage of the sample with the nucleic acids tobe tested to obtain at least one nuclease-cleaved nucleic acid sequence; nuclease recognition sites comprise a base site to be identified, the nuclease performs cutting of the recognition sites to form a first end comprising the base site to be identified and a second end not comprising the base site to be identified; and III, the nucleic acid sequence after the enzymatic cleavage by the nucleasein the step II is analyzed and according to the nucleic acid sequence condition after the enzymatic cleavage by the nuclease, whether or not the modification site of the base exists in the sample with the nucleic acids to be tested is determined.

Description

[0001] Technical field: [0002] The present application relates to a method for identifying base modification, in particular to a method for identifying single base methylation modification. Background technique [0003] It is known that there are more than 100 different chemical modifications on RNA, among which N6-methyladenine modification is the most abundant chemical modification on eukaryotic mRNA, accounting for about 0.1%-0.4% of all adenines. N6-methyladenine modification affects the metabolic process of mRNA at various stages, such as the biosynthesis of lncRNA and microRNA, and affects various life activities including neural development, cell fate, immune response, DNA damage response and tumorigenesis. [0004] Since the N6-methyladenine modification is chemically very similar to normal adenine, it is difficult to identify it chemically. High-sensitivity mass spectrometry-liquid chromatography (LC-MS / MS) and antibody blotting (dot blot) developed in recent years...

Claims

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Application Information

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IPC IPC(8): C12Q1/6809C12Q1/6869
CPCC12Q1/6809C12Q1/6869C12Q2521/327C12Q2535/122
Inventor 骆观正张璋
Owner SUN YAT SEN UNIV
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