Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

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
View PDF0 Cites 1 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

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 resolution of identified sites. However, there is no better method for single-base accuracy.
[0005] The method based on antibody enrichment not only has the problem of low resolution, but also has problems such as low repetition rate, complicated process, and inability to quantitatively calculate the ratio of N6-methyladenine modification.

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Method for identifying base modification
  • Method for identifying base modification
  • Method for identifying base modification

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Example 1 RNA endonucleases MazF and ChpBK cut RNA oligonucleotides

[0053] Using RNA oligonucleotide sequences synthesized in vitro for verification, it was found that MazF could only digest the ACA sequence without methylation modification, and cut at the 5' end of ACA, but could not digest the methylation modification ACA sequence ( figure 1 ). We mixed RNA oligonucleotide sequences with and without methyl groups so that the ratios of modification sites were 0%, 20%, 40%, 60%, 80%, 100%, respectively, using To simulate the situation of partial methylation in vivo, and use MazF enzyme to carry out enzyme digestion, it is found that the degree of enzyme digestion is directly proportional to the proportion of methylation contained in it ( figure 2 ), indicating that the ratio of N6-methyladenine can be estimated using this enzyme. The MazF of the present invention comes from Baoriyi Biotechnology (Beijing) Co., Ltd., and its product number is 2415A. The RNA oligon...

Embodiment 2

[0059] Example 2 Small RNA library construction process

[0060] use as Figure 5 The way to build a small RNA library, the specific operation is as follows:

[0061] 1) Demethylation treatment: mRNA was treated with N6-methyladenine modified demethylase FTO as a negative control. Before the reaction, the mRNA must be heated in a PCR machine at 85°C for 5 minutes, and then placed on ice for 2 minutes immediately 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 FTO 2.5ug α-Ketoglutarate (α-KG) 300uM Ammonium Ferrous Sulfate (Fe(NH4)2(SO4)2) 283uM Ascorbic acid (L-ascorbic acid) 2mM Tris-HCl buffer (pH7.5) 50mM RNase inhibitor 20U Enzyme-free water (RNase-free H2O) To 20ul

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

Embodiment 3

[0080] Example 3 High-throughput sequencing data analysis

[0081] use Figure 6 The flow process carries out sequencing analysis to the small RNA library that embodiment 2 obtains, and specific steps are as follows:

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

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

[0084] The ACA sequence not digested by MazF was located from the alignment results. The specific method is that if an ACA sequence is at the beginning of the sequencing reads, it is a normal 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 not Not cut by MazF enzyme, its first A is the methylation site. Since only reads above 15nt are retained when the data is de...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

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

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
IPC IPC(8): C12Q1/6809C12Q1/6869
CPCC12Q1/6809C12Q1/6869C12Q2521/327C12Q2535/122
Inventor 骆观正张璋
Owner SUN YAT SEN UNIV
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com