CN110093399A - A method of utilizing N6 generation methylation modifications of adenine in diphosphonic acid uracil deoxynucleotide detection nucleic acid - Google Patents

Abstract

The invention discloses a method for detecting the methylation modification at the N6 position of adenine in nucleic acid by using uridine diphosphate deoxynucleotide. The detection method mainly consists of two parts. The first part is the incorporation of uracil deoxynucleotide diphosphate (dUDP) into the genome sequence through the extension reaction of the test nucleic acid or the control nucleic acid. The second part is to use denatured polyacrylamide gel to analyze the extension reaction results. After data processing, the extension percentage of the tested nucleic acid or the control nucleic acid is obtained, and compared to realize the identification and detection of N ⁶ ‑methyl adenine and N ¹ ‑methyl adenine. The method overcomes the shortcomings of existing detection methods such as high equipment requirements and cumbersome operations, and has high sensitivity and wide application range. The raw materials used are simple and easy to obtain, and the operation is simple and convenient.

Description

A method for detecting the occurrence of adenine N6 position in nucleic acid by using uracil diphosphate deoxynucleotide Method for methylation modification

technical field

The invention belongs to the fields of molecular biology, nucleic acid chemistry and epigenetics, and specifically relates to a method for identifying and detecting N ^{6 -methyladenine (N 6 -methyladenine} ) in genome.

Background technique

As an important branch of genetics, epigenetics mainly studies the heritable changes in gene expression based on non-gene sequence changes. Its mechanism of action mainly includes: DNA methylation, chromatin remodeling, non-coding RNA regulation, histone modification, etc. Epigenetics is related to many life functions, such as genome imprinting, X-chromosome inactivation, etc. At the same time, studies have shown that epigenetics is important for the occurrence of diseases such as cancer, immune system diseases, and several inherited mental retardation diseases.

The epigenetic modification of DNA is mainly divided into two categories, one is the methylation modification at the C5 position on cytosine, which is called 5-methylcytosine (5mC); the second type is the methylation modification at the N6 position on adenine. Methylation modification, we call it N ⁶ -methyladenine. These two different epigenetic modifications not only have differences in the distribution position and content of organisms, but also have completely different biological functions. For example, 5-methylcytosine is mainly distributed in mammals and other eukaryotes, and plays a vital role in the selective expression of genes, gene stability, individual development and the occurrence of diseases. However, in prokaryotes and some lower eukaryotes, N ⁶ -methyladenine replaces 5-methylcytosine as the main epigenetic modification and performs its specific biological functions. For a long time in the past, due to the limitation of detection means, N ⁶ -methyladenine was considered to be only distributed in the DNA of bacteria.

With the development of detection technology, researchers have found that N ⁶ -methyladenine also exists in some eukaryotes, and its potential epigenetic functions have been gradually revealed. For example, it may also carry important epigenetic information and be able to carry out genetic transmission in offspring, etc.

Unlike DNA, more than 100 modifications are known to exist in RNA. With the revelation of the functions of more epigenetic modifiers, it has been found that, in addition to transmitting genetic information from DNA to proteins, mRNA itself also participates in many biological processes. N ⁶ -methyladenine widely exists in mammalian mRNA and non-coding RNA, and there are 3-5 N ⁶ -methyladenine modified bases on average in each mRNA. Furthermore, it has been found that the distribution of N ⁶ -methyladenine has certain characteristics. First, they are concentrated near the stop codon and in the long internal exon, which indicates that N ⁶ -methyladenine may play an important role in the translation regulation of mRNA and the selective splicing of RNA. In addition, people also found that N ⁶ -methyladenine widely exists in 3'UTRs, and N ⁶ -methyladenine in these regions is very likely to affect the binding force between itself and RNA-binding proteins, and then affect a part of RNA. series of biological processes. At the same time, another research group found that N ⁶ -methyladenine in 3'UTRs is always distributed just before the miRNA binding region, and speculated that N ⁶ -methyladenine would affect miRNA-induced transcript degradation and translation inhibition. Through analysis, they also found that many genes containing N ⁶ -methyladenine are also related to RNA metabolism, intercellular signal transmission, and nervous system diseases.

At the transcriptome level, different types of mRNAs have different abundances of N ⁶ -methyladenine modifications. Moreover, the content of N ⁶ -methyladenine is different in different cell stages and different tissue samples. Therefore, N ⁶ -methyladenine probably plays an important role in RNA metabolism and growth and development. At present, some important functions of it have been discovered, including splicing of mRNA, nuclear export of mRNA, translation of mRNA, accelerating mRNA degradation to reduce mRNA stability, affecting the maintenance of biological clock, regulation of cell cycle and mammalian embryonic stem cells. differentiation. It can be seen that N ⁶ -methyladenine plays an irreplaceable important role in various biological processes of mammals.

Gradually in-depth research shows that N ⁶ -methyladenine in DNA and RNA has irreplaceable research value, and the convenience and high efficiency of its detection method is more and more critical to further research on its functional characteristics and mechanism of action. .

Existing N ⁶ -methyladenine main detection methods include Ultra-High Performance Liquid Chromatography-tandem Mass Spectrometry (UHPLC-MS/MS), single cell real-time sequencing (Single molecule real time sequencing, SMRT) and other methods. Although these methods can achieve the purpose of detecting N ⁶ -methyladenine, they still have some limitations. For example, single-cell real-time sequencing methods are difficult to identify structurally similar modification types such as N ⁶ -methyladenine and N ¹ -methyladenine; while other methods have cumbersome steps or expensive equipment.

Contents of the invention

In order to solve the defects and deficiencies in the prior art, the purpose of the present invention is to provide a method that does not need to use expensive instruments, has no cumbersome steps, is environmentally friendly, has strong practicability, and can efficiently, specifically and sensitively identify and detect N ⁶ -formazan in nucleic acids. base adenine method.

The present invention is accomplished through the following technical solutions:

The present invention provides a method for identifying nucleic acid with N ⁶ -methyladenine by using uridine diphosphate deoxynucleotide, which specifically includes:

(1) Use uridine diphosphate (dUDP) as the raw material for DNA synthesis, and design a primer with a length of 17-20 bp according to the template strand sequence upstream of the site suspected to be N ⁶ -methyladenine, Extension reactions by incubation with nucleic acids suspected of containing N ⁶ -methyladenine under the action of DNA polymerase or reverse transcriptase to incorporate uracil deoxynucleotide diphosphate (dUDP) into the sequence The test nucleic acid or control nucleic acid at the site, the control nucleic acid is that the corresponding site does not contain N ⁶ -methyl adenine, the sequence is similar to the test nucleic acid, and the other sites contain N ⁶ -methyl adenine at the same level as the test Nucleic acid identical to nucleic acid;

(2) Step (1) The extension product is analyzed by denaturing polyacrylamide gel, and the concentration of the extended substrate and the concentration of the unextended substrate are measured by a gel imager to obtain the extension percentage, and the extension percentage is: extension Percent = extended substrate concentration/(extended substrate concentration+unextended substrate concentration);

(3) Result determination: if the extension percentage of the test nucleic acid is less than that of the control nucleic acid, the nucleic acid with N ⁶ -methyladenine can be identified.

Preferably, the 5' end of the primer described in step (1) is modified with FAM.

Preferably, the DNA polymerase in step (1) is Bst DNA polymerase.

Preferably, the reverse transcriptase in step (1) is M-MuLV reverse transcriptase.

The second aspect provides the application of uracil deoxynucleotide diphosphate in the preparation of detection reagents for recognizing nucleic acids with N ⁶ -methyladenine.

The principle of the technical solution of the present invention is: when dUDP is used as a synthetic raw material, the nucleic acid chain containing N ⁶ -methyladenine and the nucleic acid chain containing unmodified adenine are extended under the catalysis of DNA polymerase or reverse transcriptase Significant differences in reaction rates provide accurate information on methylation modification sites.

Advantages and beneficial effects of the present invention

1. The present invention is not limited by the detection limit and the length of the sequence to be tested. The detection result of the present invention is obtained by analyzing the ^quantified extension effect (percentage). Accurate information on the methylation modification site can be obtained by the significant difference in the extension reaction rate of the nucleic acid chain containing purine and the nucleic acid chain containing unmodified adenine under the catalysis of DNA polymerase or reverse transcriptase. At the same time, since the extension reaction rate catalyzed by DNA polymerase or reverse transcriptase is extremely fast, there is no significant difference in the extension reaction time of the unmodified sequence under the technical solution of the present invention, so the detection resolution can still be guaranteed. This advantage enables the present invention to adapt to the detection of nucleic acid chain samples in a relatively large length range, improving its application value;

2. The present invention provides convenience for studying the pathogenesis of diseases: current research has found that the pathogenesis of some cancers involves methylation modification at specific sites in the genome, and is limited by detection means, the type of methylation modification cannot be accurately known. In the present invention, when the whole genome sequence and possible modification sites are known, the exploration of adenine methylation type is further improved, so as to accurately understand the pathological etiology and develop specific drugs;

3. The present invention provides a new idea for the early diagnosis of cancer and other diseases: the early detection of cancer has blood item inspection, antigen-antibody hybridization and other methods, the limitations of which are the high cost of designing detection reagents, cumbersome steps, and the interval time between results analysis Long, and there is uncertainty about the effect of early cancer detection in different periods. The present invention uses the common biochemical reagents dUDP and Bst DNA polymerase or M-MuLV reverse transcriptase to confirm the detection index by comparing the detection results of healthy individuals and cancer-stricken individuals, and use this as a reference to detect individuals with unknown cancer conditions. Therefore, the response time is significantly shortened, providing an important reference for cancer screening, while reducing the cost of testing and reducing social burdens, and has broad application prospects in medical testing institutions.

Description of drawings

Figure 1 shows the co-incubation of DNA sequences containing adenine or N ⁶ -methyladenine at the same site with different concentrations of uracil deoxynucleotide diphosphate (dUDP) under the action of Bst DNA polymerase at 63°C Percent elongation after treatment versus time plot.

A is the percentage elongation-time curve of DNA containing adenine, and B is the percentage elongation-time curve of DNA containing N ⁶ -methyladenine. Legend labeling is the concentration of uracil deoxyribonucleotide diphosphate (dUDP).

Figure 2 is the co-incubation of the DNA sequence of adenine or N ⁶ -methyladenine at the same site with 400 μM uridine deoxynucleotide diphosphate (dUDP) under the action of Bst DNA polymerase for different times at 63°C The final denaturing polyacrylamide gel analysis chart. (The upper band in the electropherogram is an extended fragment, and the lower band is an unextended fragment. In the legend, 6mA represents N ⁶ -methyladenine)

A is the DNA sequence containing only adenine sites, and bands 1-14 from left to right correspond to the results of different treatment times. B is the DNA sequence containing the N ⁶ -methyladenine site, and bands 1-14 from left to right correspond to the results of different treatment times. C is the extension percentage-time curve of the DNA sequence containing adenine site and the DNA sequence containing N ⁶ -methyladenine.

Figure 3 shows that the RNA sequences containing adenine or N ⁶ -methyladenine at the same site were co-sequenced with 100 μM uracil deoxynucleotide diphosphate (dUDP) under the action of RNA M-MuLV reverse transcriptase at 42°C. Denaturing polyacrylamide gel analysis pictures after incubation for different times. (The upper band in the electropherogram is an extended fragment, and the lower band is an unextended fragment. In the legend, 6mA represents N ⁶ -methyladenine)

A is the RNA sequence containing only the adenine (A) site, and bands 1-14 from left to right correspond to the results of different treatment times. B is the RNA sequence containing the N ⁶ -methyladenine site, and bands 1-14 from left to right correspond to the results of different treatment times. C is the elongation percentage-time curve of the RNA sequence containing only adenine site and the RNA sequence containing N ⁶ -methyladenine.

Detailed ways

The features and advantages of the present invention can be further understood through the following detailed description in conjunction with the accompanying drawings. The examples provided are only illustrative of the method of the present invention and do not limit the rest of the present disclosure in any way. The nucleic acid sequences used in the following examples are shown in Table 1:

Table 1

name Sequence(from 5'to 3') DNA-17mer-A1 5'-AATGCCACATGCTGCAC-3' DNA-17mer-6mA1 5'-(N6-Me-dA)ATGCCACATGCTGCAC-3' RNA-17mer-A1 5'-(N1-Me-dA)ATGCCACATGCTGCAC-3' RNA-17mer-6mA1 5'-AAUGCCACAUGCUGCAC-3' Primer 5'-(N6-Me-A)AUGCCACAUGCUGCAC-3'

[Example 1] The DNA sequences containing adenine or N ⁶ -methyladenine at the same site were co-incubated with different concentrations of uracil deoxynucleotide diphosphate (dUDP) under the action of Bst DNA polymerase Post-elongation percentage-time relationship comparison

1. Prepare 1 μL of 1×ThermoPol buffer at pH 8.8 at 25° C.: 20 mM Tris-HCl, 10 mM ammonium sulfate, 10 mM potassium chloride, 2 mM magnesium sulfate, 0.1% polyethylene glycol octylphenyl ether. Add 0.2 μL double-stranded DNA DNA-17mer-A1 or DNA-17mer-6mA1 to make the final concentration 2 μM, add 0.1 μL gradient concentration dUDP, 1 μL Bst DNA polymerase, and primer Primer, the concentration ratio of primer and double-stranded DNA For 3:5, make up the volume to 10 μL with ddH ₂ O.

2. Incubate at 63°C for 5 minutes, and take samples for analysis at different intervals. The time interval increases with the progress of the reaction to obtain highly differentiated characterization results. Add 45 μL of stop buffer (95% formamide, 25mM EDTA, pH 8.0) to stop For the reaction, the reaction system was immediately heated to 90°C and kept for 10 minutes, then cooled to 4°C.

3. 20% denatured polyacrylamide gel electrophoresis to separate the fragments that have completed the extension reaction and the fragments that have not undergone the extension reaction, and characterize them under a gel imager, and then obtain the proportion of the nucleic acid chain to be detected that has completed the extension reaction.

Results: The increase of reaction time can make the difference of elongation percentage more obvious and improve the specificity of reaction. The optimization of reaction conditions provides more suitable conditions for subsequent detection, and can improve the sensitivity, accuracy and other aspects required by the detection method. (figure 1)

[Example 2] The method of the present invention is applied to the detection of DNA sequences containing N ⁶ -methyladenine

1. Prepare two sets of 1 μL 1×ThermoPol buffer solution at pH 8.8 at 25° C.: 20 mM Tris-HCl, 10 mM ammonium sulfate, 10 mM potassium chloride, 2 mM magnesium sulfate, 0.1% polyethylene glycol octylphenyl ether. 0.2 μL of double-stranded DNA DNA-17mer-A1 or DNA-17mer-6mA1 was added each to make a final concentration of 2 μM. Add dUDP at a final concentration of 400 μM, 1 μL of Bst DNA polymerase, and primer, the concentration ratio of primer and double-stranded DNA is 3:5, and make up the volume to 10 μL with ddH ₂ O.

2. Incubate at 63°C for 5 minutes and take samples for analysis at different intervals. The time interval increases with the progress of the reaction to obtain highly differentiated characterization results. Add 45 μL of stop buffer (95% formamide, 25mM EDTA, pH 8.0) to stop the reaction , the reaction system was immediately heated to 90°C and kept for 10 minutes, then cooled to 4°C.

3. 20% denaturing polyacrylamide gel electrophoresis to separate extended and unextended fragments and characterize them under a gel imager, and then determine the extension percentage.

Results: For DNA samples, the percentage extension of the template sequence containing adenine was higher than that of the template sequence containing N ⁶ -methyladenine. (figure 2)

[Example 3] The method of the present invention is applied to the detection of RNA sequences containing N ⁶ -methyladenine

1. Prepare 1 μL of 1×M-MuLV RT buffer at pH 8.3 at 25°C: 50 mM Tris-HCl, 75 mM potassium chloride, 2 mM magnesium chloride, 10 nM dithiothreitol, and add 0.2 μL of RNA-17mer at a final concentration of 2 μM -A1 or RNA-17mer-6mA1, primer Primer, the concentration ratio of primer and double-stranded DNA is 3:5, the final concentration is 100 μM dUDP, 1 μL M-MuLV reverse transcriptase, and the volume is made up to 10 μL with ddH ₂ O.

2. Incubate at 42°C for 5 minutes, take samples for analysis at different time intervals, add 45 μL of stop buffer (95% formamide, 25mM EDTA, pH 8.0) to stop the reaction, immediately heat to 90°C and keep warm for 10 minutes, cool to 4°C.

3. 20% denaturing polyacrylamide (PAGE) gel electrophoresis to separate extended and unextended fragments, and characterize them under a gel imager, and then determine the extension percentage.

Results: For RNA samples, the percent extension of the template sequence containing adenine was higher than that of the template sequence containing N ⁶ -methyladenine. (image 3).

sequence listing

<110> Wuhan University

<120> A method for detecting methylation at the N6 position of adenine in nucleic acid by using uridine diphosphate nucleotides

<160> 5

<170> SIPOSequenceListing 1.0

<210> 1

<211> 17

<212>DNA

<213> Artificial Sequence

<400> 1

aatgccacat gctgcac 17

<210> 2

<211> 17

<212>DNA

<213> Artificial Sequence

<220>

<221> modified_base

<222> (1)..(1)

<223> N6-Methyl

<400> 2

aatgccacat gctgcac 17

<210> 3

<211> 17

<212> RNA

<213> Artificial Sequence

<400> 3

aaugccacau gcugcac 17

<210> 4

<211> 17

<212> RNA

<213> Artificial Sequence

<220>

<221> misc_feature

<222> (1)..(1)

<223> N6-Methyl

<400> 4

aaugccacau gcugcac 17

<210> 5

<211> 16

<212>DNA

<213> Artificial Sequence

<220>

<221> misc_feature

<222> (1)..(1)

<223> 5'-fam

<400> 5

gtgcagcatg tggcat 16

Claims (5)

1. A method utilizing uridine diphosphate to identify a nucleic acid with N ⁶ -methyladenine, characterized in that it specifically comprises: (1) Use uridine diphosphate (dUDP) as the raw material for DNA synthesis, and design a primer with a length of 17-20 bp according to the template strand sequence upstream of the site suspected to be N ⁶ -methyladenine, Extension reactions by incubation with nucleic acids suspected of containing N ⁶ -methyladenine under the action of DNA polymerase or reverse transcriptase to incorporate uracil deoxynucleotide diphosphate (dUDP) into the sequence The test nucleic acid or control nucleic acid at the site, the control nucleic acid is that the corresponding site does not contain N ⁶ -methyl adenine, the sequence is similar to the test nucleic acid, and the other sites contain N ⁶ -methyl adenine at the same level as the test Nucleic acid identical to nucleic acid; (2) The extension product of step (1) is analyzed by a denaturing polyacrylamide gel, and the concentration of the extended substrate and the concentration of the unextended substrate are determined by a gel imager to obtain the extension percentage, which is: Extension percentage=extended substrate concentration/(extended substrate concentration+unextended substrate concentration); (3) Result determination: if the extension percentage of the test nucleic acid is less than that of the control nucleic acid, the nucleic acid with N ⁶ -methyladenine can be identified. 2. The method according to claim 1, characterized in that, the 5' end of the primer described in step (1) is modified with FAM. 3. The method according to claim 1, characterized in that the DNA polymerase in step (1) is BstDNA polymerase. 4. The method according to claim 1, characterized in that the reverse transcriptase in step (1) is M-MuLV reverse transcriptase. 5. The application of uracil deoxynucleotide diphosphate in the preparation of detection reagents for recognizing nucleic acids with N ⁶ -methyladenine.