CN114807328A - Method for marking and identifying single-base resolution DNA - Google Patents

Abstract

The invention provides a method for marking and identifying DNA with single base resolution, which is based on N of DNA adenine ⁶ N of allyl or cytosine ⁴ Allyl labeling and chemical treatment to induce base mutation during DNA replication, and identifying the mutation site by nucleic acid sequencing to obtain 6aA or 4aC site. The method can be used for marking new DNA, can also be used for identifying 6mA or 4mC methyltransferase modification sites, and further can carry out specific marking on the 6mA or 4mC sites in the cells through the metabolic process of the cells, and the marking not only can replace the 6mA or 4mC sites in the cells in situ, but also can be positioned by means of mutation sequencing. Compared with the existing DNA marking and identifying technology, the method of the invention improves the precision of detecting the modified sites based on antibody immunoprecipitation and large-scale parallel sequencing method which are commonly adopted at present because the mutant sites can be accurate to the resolution of single baseAnd (3) the method is a direct single-base identification method.

Description

Method for marking and identifying single-base resolution DNA

Technical Field

The invention belongs to the field of gene sequencing, and particularly relates to a single-base resolution DNA marking and identifying method.

Background

DNA is composed of a combination of four bases, cytosine (C), thymine (T), guanine (G) and adenine (A). N is a radical of hydrogen ⁶ -methyladenine (6mA) and N ⁴ Methyl cytosine (4mC) is an important modified base on DNA, and has various biological functions in biological processes, such as regulation of gene expression and the like. The identification and sequencing method of the methyl modification sites are the precondition for researching the biological significance of the methyl modification sites. 6mA and 4mC are widely distributed in prokaryotic organisms and lower eukaryotic genomic DNA, and the methylation modification plays an important role in a bacterial self-protection mechanism and is used as an important mark of exogenous DNA in bacterial regions. Besides, the mammalian cell DNA is also found to be modified by 6mA and 4mC, and the current 6mA detection method is mainly based on an antibody immunoprecipitation sequencing technology, but the resolution is only limited to be within a range of 100-200 bases; the detection method of 4mC is mainly based on sulfite sequencing, but has low methylation modification content, incomplete oxidation reaction, low result reliability and indirect identification. The study of the source of methylation modifications, the sites of modifications and the corresponding biological functions in mammalian cell DNA remains controversial due to the low level of modifications and the lack of direct high resolution detection methods. In addition, in extreme environments, DNA can be damaged, triggering repair mechanisms within the cell. There is currently no direct high resolution identification of sites of injury and repair.

The invention hopes to utilize the recognizable modifying group, the modifying group is introduced into DNA to carry out iodine cyclization treatment, and then the site where the modifying group is located is recognized by means of mutation sequencing, thereby achieving the purpose of DNA marking. Therefore, methods for single base resolution labeling and identification of DNA are under development.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provide a method for marking and identifying DNA with single base resolution, which is used for directly identifying a DNA marking site with single base resolution by a mutation sequencing mode.

The invention adopts the following technical scheme:

a method of single base resolution DNA labeling and identification comprising the steps of:

(1) labeling of deoxyribonucleic acid (DNA) extracellularly with enzyme assistance or intracellularly by cellular self-metabolic processes:

the extracellular labeling process was as follows: adenosylmethionine transferases use the methionine analogue allyl-L-seleno/thiohomocysteine to generate the methyltransferase cofactor allyl-L-seleno/thioadenosylhomocysteine, or directly add allyl-L-seleno/thioadenosylhomocysteine, and methyltransferases use this cofactor to introduce an allyl group into a particular N of DNA ⁶ 6mA or N methyladenine ⁴ -methylcytosine at the 4mC site, forming N ⁶ -allyldeoxyadenosine 6aA or N ⁴ -allyldeoxycytidine 4 aC; or extracellular N-binding of N using active DNA polymerase/reverse transcriptase ⁶ Allyl deoxyadenosine triphosphate 6adATP, N ⁴ -random replication/reverse transcription of allyl deoxycytidine triphosphate 4adCTP into nascent DNA;

the labeling process inside the cell is as follows: cells use allyl-L-seleno/thiohomocysteine to introduce an allyl group to a specific 6mA or 4mC site of DNA by natural metabolism to form 6aA or 4 aC; the cells can also randomly introduce free 6aA or 4aC in the culture medium into the nascent DNA directly;

(2) enrichment of DNA containing 6aA or 4aC modifications: cutting the unmodified DNA sequence by using restriction endonuclease, and enriching the modified DNA sequence (the unmodified DNA sequence) by agarose gel electrophoresis; or enriching the 6aA or 4aC modified DNA by binding the 6aA or 4aC with an antibody by means of immunoprecipitation, eluting the DNA from the magnetic beads with a protease, and purifying the eluted DNA;

(3) iodine addition and circularization of DNA 6aA or 4 aC: n on DNA ⁶ -allyladenine or N ⁴ Allyl cytosine undergoes an iodine addition reaction, and then is induced to form a cyclized structure under an alkaline condition, so that base complementary pairing is shielded;

(4) extension and sequencing recognition of circularization-treated DNA: and (3) carrying out denaturation treatment on the DNA obtained in the step (3), adding DNA polymerase, extending the DNA containing a cyclization structure, introducing errors to a para-complementary base in the process that adenine or cytosine cyclized on the DNA generates a DNA complementary strand under the action of the DNA polymerase, and identifying a mutation site by a nucleic acid sequencing means so as to obtain a 6aA or 4aC site.

In the above technical solution, further, in the step (1), the preparation method of allyl-L-seleno/thiohomocysteine comprises: under the protection of nitrogen, selenium powder Se and sodium borohydride NaBH are firstly mixed in a molar ratio of 1:1 ₄ Taking ethanol as a solvent as a raw material, heating and refluxing at 80 ℃ for 6-24 hours to prepare Na ₂ Se ₂ Heating and refluxing the compound and hydrobromide (compound 1) of (S) - (+) -2-amino-4-bromobutyric acid with a molar weight of one half to one third of selenium powder at 80 ℃ for 6-24 hours, stopping the reaction with acid, filtering to remove insoluble substances, washing off by-products with diethyl ether, adjusting the pH value to be neutral to obtain seleno-homocystine (compound 2), adding sodium borohydride with a molar weight of 1-2 times that of the selenium powder to reduce diselenide bonds, reacting the mixture with allyl bromide with a molar weight of 0.5-2 times that of the selenium powder at room temperature for 6-24 under an alkaline condition (sodium bicarbonate/sodium carbonate with a molar weight of 0.5-1.5 times that of the selenium powder/sulfur powder), and analyzing and purifying by High Performance Liquid Chromatography (HPLC) to obtain allyl-L-seleno-homocysteine (compound 3); sulfur S is used as raw material to prepare thiohomocystine (compound 4) and allyl-L-thiohomocysteine (compound 5).

Further, in the step (1), the preparation method of 6aA and 6adATP comprises the following steps: under the protection of argon or nitrogen, taking 6-chloropurine deoxynucleoside, allylamine hydrochloride and triethylamine in a molar ratio of 1: 1.5-3: 3-10 as raw materials, taking ethanol as a solvent, heating and refluxing at 80 ℃ for 3 hours, concentrating, and separating by high performance liquid chromatography to obtain 6aA (compound 7); adding completely dry trimethyl phosphate with the molar weight 5-50 times of 6-chloropurine deoxynucleoside and phosphorus oxychloride with the molar weight 6aA 1.1-1.3 times of the total molar weight, and stirring at 0-4 ℃ for 0.5-3 hours until a reaction solution is completely clear; then adding tributylamine pyrophosphate with the molar weight of 6aA 2-10 times, and stirring for 10-30 minutes at 0-4 ℃; then continuously stirring for 5-10 minutes at room temperature, and adding triethylammonium bicarbonate with the molar weight being at least 1 kilo time of 6aA to terminate the reaction; purification by HPLC analysis gave 6adATP (Compound 8).

Further, in the step (1), the preparation method of 4aC and 4adCTP comprises the following steps: mixing a mixture of 1: 0.14-0.17 of 2 ' -deoxyuridine and 4-dimethylaminopyridine as raw materials, acetic anhydride as a solvent, reacting at room temperature for 5-6 hours, concentrating, and purifying by a silica gel column to obtain 3', 5' -diacetyldeoxyuridine (compound 10); mixing 3', 5' -diacetyl deoxyuridine with the molar weight 1-1.3 times that of 2 ' -deoxyuridine and 1-hydrogen-tetrazole with the molar weight 1-1.3 times that of 2 ' -deoxyuridine, adding the mixture into a flask, removing water and oxygen, adding pyridine and 4-chlorophenyl dichlorophosphate with the molar weight 2-4 times that of 2 ' -deoxyuridine in an ice water bath under the protection of nitrogen or argon, reacting the mixture for 5-10 minutes in the ice water bath at room temperature for 5-7 hours, and adding saturated Na ₂ CO ₃ Quenching the solution, extracting with dichloromethane, washing with saturated saline, drying, evaporating and concentrating, and directly using the obtained solid for the next reaction; dissolving the solid obtained in the last step in acetonitrile, adding 1-1.5 times of potassium hydroxide and 1-1.5 times of allylamine hydrochloride into a flask in advance, sealing, sequentially adding water, acetonitrile, triethylamine and the product obtained in the last step dissolved in acetonitrile, reacting for 24 hours at room temperature, concentrating, and purifying by a silica gel column to obtain 3', 5' -diacetyl-N ⁴ -allyldeoxyuridine (compound 11); 3', 5' -diacetyl-N with the molar weight 1-2 times of that of 2 ' -deoxyuridine ⁴ Adding allyl deoxyuridine into methanol solution containing 2mol/L ammonia with a molar amount of at least 1 thousand times that of 2' -deoxyuridine, reacting at room temperature overnight, concentrating, and dryingTo give 4aC (Compound 12); under the protection of argon or nitrogen, adding completely dried trimethyl phosphate with the molar weight 10-50 times that of 2 '-deoxyuridine and phosphorus oxychloride with the molar weight 1.1-1.5 times that of 2' -deoxyuridine into the mixture, and reacting the mixture for 3-3.5 hours at the temperature of 0-4 ℃; then adding tributylamine pyrophosphate (which can be pre-dissolved in N, N '-dimethylformamide at the concentration of 0.6 g/mL) with the molar weight of 3-10 times of 2' -deoxyuridine, and stirring for 1-1.5 hours at the temperature of 0-4 ℃; then continuously stirring for 5-10 minutes at room temperature, and adding triethylammonium bicarbonate with the molar weight at least 1 thousand times of that of 2' -deoxyuridine to terminate the reaction; purification by HPLC analysis gave 4adCTP (Compound 13).

Further, in the step (1), the site-specific labeling of the DNA by using methyltransferase in the cell is as follows: 1-2. mu.g of DNA probe was added to a PCR tube, and ATP (10mM, 5. mu.L), allyl-L-selenocysteine (45mM, 2.5. mu.L), 10 XBuffer, Methionine Adenosyltransferase (MAT) (50. mu.M, 5. mu.L), 5' -Methylthioadenosylnucleosidase (MTN) (50-100. mu.M, 5. mu.L), 6mA or 4mC methyltransferase were added to the system in this order, and then water was added to make up the system to 50. mu.L, the reaction was carried out at 37 ℃ for 4 hours, and the reaction was carried out at 70 ℃ for 10 minutes. After the reaction, the DNA was purified.

Further, in step (1), extracellular 6adATP, 4adCTP are replicated/reverse transcribed into nascent DNA using active DNA polymerase/reverse transcriptase:

reverse transcriptase is utilized to carry out reverse transcription on extracellular 6adATP and 4adCTP into nascent DNA, and the reverse transcriptase comprises the following specific steps: reacting 2-3. mu.g RNA with an equimolar ratio of primers at 70 ℃ for 5 minutes, and then adding an appropriate amount of RevertAId reverse transcriptase and Buffer, 0.5. mu.L RNase inhibitor, 2mM dNTPs (1.25. mu.L 8mM 4 addCTP, 1.25. mu.L 8mM dATP, 1.25. mu.L 8mM dTTP, 1.25. mu.L 8mM dGTP or 1.25. mu.L 8mM 6 addATP, 1.25. mu.L 8mM dCTP, 1.25. mu.L 8mM dTTP, 1.25. mu.L 8mM dGTP), reacting at 42 ℃ for 1 hour, and reacting at 70 ℃ for 15 minutes; then, 2. mu.L of RNase H, 2.5. mu.L of Buffer, 0.5. mu.L of RNase cocktail were added and reacted at 37 ℃ for 1 hour, and then 1. mu.L of EDTA was added to terminate the reaction, to obtain cDNA labeled with 6adATP or 4 adCTP.

The method is characterized in that active DNA polymerase is used for copying extracellular 6adATP and 4adCTP into nascent DNA, and specifically comprises the following steps: the PCR is performed by replacing dNTPs with appropriate amounts of 6adATP or 4adCTP, dATP, dCTP, dGTP, and dTTP according to the protocol of the polymerase, and DNA polymerases including but not limited to KOD-FX (TOYOBO), KOD-Plus-NEO (TOYOBO), KOD-FX-NEO (TOYOBO) can be used.

Further, in step (1), the cells randomly introduce free 6aA or 4aC in the culture medium directly into the nascent DNA: after the sample cells were cultured to a certain degree of fullness, 6aA or 4aC was added to the sample cells at a final concentration of 1mM, and gDNA was extracted from the sample cells after 4 days of culture.

Further, in the step (2), the method for enriching the DNA containing the 6aA or 4aC modification comprises the following steps: the unmodified DNA sequence is cleaved with restriction enzymes, and the uncleaved DNA (i.e., DNA with modifications) is enriched by agarose Gel electrophoresis and purified using a Kit, including but not limited to Gel Extraction Kit (QIAGEN).

Further, in the step (3), the method for iodine addition and cyclization comprises the following steps: dissolving 0.1-0.5M iodine simple substance in 0.2-1M potassium iodide to obtain a potassium iodide solution of iodine, reacting the potassium iodide solution of iodine with allyl on DNA 6aA or 4aC, and removing excessive iodine by using 0.1-0.5M sodium thiosulfate; adding 0.1-0.5M sodium carbonate, adjusting the pH value to 9-10, and inducing 1, N on DNA 6aA ⁶ 3, N in position or 4aC ⁴ The site forms a cyclic structure, thus shielding the normal hydrogen bonding pairing of adenine or cytosine.

Further, in the step (4), the method for extension and sequencing identification of the circularized DNA comprises the following steps: taking about 500ng of cDNA or double-stranded DNA, adding 2 mu L of primer, a proper amount of Buffer and DEPC water, reacting for 5 minutes at 100 ℃, and then carrying out gradient cooling to 25 ℃; adding dNTPs, DNA polymerase, Buffer and DEPC water into the system, and performing reaction according to the reaction conditions of the DNA polymeraseAnd (4) extending. DNA polymerases that can be used include, but are not limited to: coli DNA polymerase I Klenow Fragment (3 '-5' exo) ^- ) (NEB), Sulfolobus DNA polymerase IV (NEB), Human DNA polymerase eta, Therminator DNA polymerase (NEB), Bsu DNA polymerase (NEB), DNA polymerase I (NEB), E.coli DNA polymerase I Klenow Fragment (NEB), T4 DNA polymerase (NEB) and T7 DNA polymerase (NEB) and the like.

In step (4) above, the sequencing method after extension of the circularized DNA includes, but is not limited to, high-throughput sequencing for constructing a library, and low-throughput sequencing based on TA-cloning. Wherein, the PCR enzyme in TA-cloning method includes but is not limited to KOD-FX DNA polymerase, and the ligation of plasmid vector in TA-cloning method includes but is not limited to T vector; the Library construction method includes, but is not limited to, Library construction using the illumina Ultra II DNA Library Prep Kit.

In the step (1), the cells can be, but not limited to, host cells of general mammalian cells, mammalian cancer cells, mammalian stem cells, bacteria, viruses, and cells derived from various types of tissues and organs.

In the above steps (1) to (4), the nucleic acid purification step after each reaction may be carried out by a conventional purification method or a commercial purification kit. Methods include, but are not limited to: one or more of the techniques of silica gel membrane centrifugal column method, magnetic bead method, ethanol and isopropanol precipitation, etc. Purification kits include, but are not limited to: the AmpureXP bead is a new standard,

PCR purification Kit(Qiagen)，RNA Clean&Concentrator(Zymo)，DNA Clean&concentrator (zymo). The invention has the beneficial effects that:

(1) the single base resolution DNA marking and identifying method is based on the chemical marking and induced mutation of the deoxynucleic acid adenine or the deoxynucleic acid cytosine, the mutation site can be accurate to the resolution of a single base, the detection precision is improved, and the method is a direct method for high-throughput single base identification.

(2) The invention is realized for the first timeN of deoxyribonucleic acid (DNA) adenine in cell ⁶ N of allyl and cytosine ⁴ Allyl markers, which provide the possibility of subsequently identifying the 6mA or 4mC site by means of site mutations.

(3) The present invention provides two novel deoxynucleoside phosphates: n is a radical of ⁶ Allyl deoxyadenosine triphosphate (6adATP), N ⁴ Deoxycytidine allyltriphosphate (4adCTP), which can be incorporated into DNA directly by DNA polymerase or reverse transcriptase. Two novel deoxynucleosides are also provided: n is a radical of ⁶ Allyl deoxyadenosine (6aA) and N ⁴ Allyl deoxycytidine (4aC), which can be randomly introduced into nascent DNA by cells. The conventional synthetic method of modified DNA is usually a solid-phase synthetic method, which has complex operation and complicated purification process; the invention directly introduces the modified 6aA and 4aC into DNA by DNA polymerase or reverse transcriptase, has simple operation and purification process and good biocompatibility, and can be used for intracellular modification.

(4) Among the 9 th DNA polymerases used in the present invention, E.coli DNA polymerase I Klenow Fragment (3 '-5' exo) ^- ) And Sulfolobus DNA polymerase IV are generally used in the extension process of DNA, but the invention can be used for identifying the cyclization sites, thereby generating the phenomenon of mismatch, introducing mutation in the replication process and having better mutation effect.

(5) On the basis of mutation sequencing, the invention can be applied to various analysis methods based on gene sequencing, such as identification of methyltransferase modification sites, DNA damage site identification and the like.

Drawings

FIG. 1 is a schematic representation of the use of novel deoxynucleosides and deoxynucleoside phosphates for DNA labeling and identification;

FIG. 2 is N ⁶ Of allyl deoxyadenosine (6aA) ¹ H nuclear magnetic resonance spectroscopy;

FIG. 3 is N ⁶ Of allyl deoxyadenosine (6aA) ¹³ C nuclear magnetic resonance spectrum;

FIG. 4 is N ⁶ -high resolution mass spectrum of allyldeoxyadenosine (6 aA);

FIG. 5 is N ⁶ -allyl triphosphateHigh resolution mass spectrometry of deoxyadenosine (6 adATP);

FIG. 6 is N ⁴ Of allyl deoxycytidine (4aC) ¹ H nuclear magnetic resonance spectroscopy;

FIG. 7 is N ⁴ Of allyl deoxycytidine (4aC) ¹³ C nuclear magnetic resonance spectrum;

FIG. 8 is N ⁴ -high resolution mass spectrum of allyldeoxycytidine (4 aC);

FIG. 9 is N ⁴ -high resolution mass spectrum of deoxycytidine allyltriphosphate (4 adCTP);

FIG. 10 shows the results of DNA polymerase sequencing after replication of DNA with 6aA modification by iodine addition and induced circularization;

FIG. 11 is the result of separation by agarose gel electrophoresis in example 1;

FIG. 12 is the results of low throughput sequencing in example 1;

FIG. 13 is N in HEK293T cell DNA from example 2 ⁶ Mass spectrometry results of the allyladenine incorporation rate.

Detailed Description

To better illustrate the objects, aspects and advantages of the present invention, the present invention will be further described with reference to specific embodiments. They are not to be construed as limiting the scope of the invention.

FIG. 1 is a schematic diagram of the application of the novel deoxynucleoside and deoxynucleoside phosphate of the present invention in DNA labeling and identification.

FIGS. 2-4 are N prepared by the process of the present invention ⁶ -allyldeoxyadenosine (6aA) relative characterization data. FIG. 2 is N ⁶ Of allyl deoxyadenosine (6aA) ¹ H nuclear magnetic resonance spectrum: ¹ H NMR(DMSO-d ₆ 400MHz), δ (ppm) 8.35(s,1H),8.20(s,1H),8.02(s,1H),6.35(dd, J ═ 6.2,7.8Hz,1H),5.99-5.89(m,1H),5.32(d, J ═ 4.0Hz,1H),5.24(dd, J ═ 5.0,6.5Hz,1H),5.14(dd, J ═ 1.3,17.0Hz,1H),5.05(dd, J ═ 1.6,10.2Hz,1H),4.41(m,1H),4.11(s,2H),3.88(dd, J ═ 4.2, 6.7Hz,1H),3.65-3.60(m,1H), 3.55-3.7 (s,2H), 3.49 (dd, J ═ 8.2H), 8.8, 8.26H, 8(dd, 1H). FIG. 3 is N ⁶ Of allyl deoxyadenosine (6aA) ¹³ C nuclear magnetic resonance spectrum: ¹³ C NMR(100MHz,DMSO-d ₆ ) Delta (ppm) 154.38,152.27,148.18,139.42,135.60,119.60,114.94,87.98,83.92,70.94,61.86,40.10, 39.89. FIG. 4 is N ⁶ High resolution mass spectrum of allyldeoxyadenosine (6 aA): HRMS (ESI), M/z 292.1410([ M + H)] ⁺ ,calcd 292.1404)。

FIG. 5 is N ⁶ High resolution mass spectrum of allyldeoxyadenosine triphosphate (6 adATP): HRMS (ESI), M/z 530.0245([ M-H)] ^- ,calcd 530.0249)

FIGS. 6 to 8 show the obtained N ⁴ -data relating to the characterization of allyl deoxycytidine (4 aC). FIG. 6 is N ⁴ Of allyl deoxycytidine (4aC) ¹ H nuclear magnetic resonance spectrum: ¹ H NMR(400MHz,DMSO-d ₆ ) δ 7.84(t, J ═ 5.7Hz,1H),7.77(d, J ═ 7.4Hz,1H),6.16(dd, J ═ 7.5,5.9Hz,1H), 5.96-5.69 (m,2H), 5.23-5.07 (m,3H),4.97(t, J ═ 5.4Hz,1H),4.12(q, J ═ 5.2Hz,2H),3.90(d, J ═ 5.4Hz,2H),3.76(q, J ═ 3.7Hz,1H), 3.63-3.48 (m,2H),2.10(ddd, J ═ 13.1,6.0,3.2Hz,1H),1.93(ddd, J ═ 13.3,7.5,6.0, 1H). FIG. 7 is N ⁴ Of allyl deoxycytidine (4aC) ¹³ C nuclear magnetic resonance spectrum: ¹³ C NMR(101MHz,DMSO-d ₆ ) δ 163.15,155.04,139.96,134.79,115.49,94.53,87.11,84.79,70.36,61.31,54.03(d, J ═ 5.9Hz),41.89, 40.24. FIG. 8 is N ⁴ -high resolution mass spectrum of allyl deoxycytidine (4 aC): HRMS (ESI), M/z 268.1291([ M-H)] ^- ,calcd 268.1219)

FIG. 9 is N ⁴ -high resolution mass spectrum of deoxycytidine allyltriphosphate (4 adCTP): HRMS (ESI), M/z 506.0118([ M-H)] ^- ,calcd 506.0136)。

FIG. 10 shows the sequencing of TA clones by DNA polymerase after replication of DNA with 6aA modifications by iodine addition and induced circularization. The invention adopts E.coli DNA polymerase I Klenow Fragment (3 '-5' exo) ^- ) Nine DNA polymerases, such as Sulfolobus DNA polymerase IV, Human DNA polymerase eta, Therminator DNA polymerase, Bsu DNA polymerase, DNA polymerase I, E.coli DNA polymerase I Klenow Fragment, T4 DNA polymerase and T7 DNA polymerase, were used to replicate DNA that was subjected to iodine addition and induced circularization, and the mutation effect of each enzyme was tested. From the figure10 it is known that different DNA polymerases have different extension effects on the circularization site, E.coli DNA polymerase I Klenow Fragment (3 '-5' exo) ^- ) The ratio of the mismatches of Sulfolobus DNA polymerase IV and Human DNA polymerase eta at the cyclization site is higher, and the mutation effect is obvious.

The present invention is further illustrated below by the following examples of labeling of DNA with 6mA extracellularly and 6aA intracellularly.

Example 1 extracellular DNA N ⁶ Identification of the methyladenine (6mA) methylation modification site

1. DNA allyl labelling and extraction

(1) Preparing a DNA segment with the length of 202bp, and configuring a labeling reaction system according to table 1;

TABLE 1 DNA allyl labelling reaction System

TABLE 210X Reaction Buffer

(2) Reacting at constant temperature of 37 ℃ for more than 4 h;

(3) the reacted DNA was purified using a DNA Clean & Concentrator Kit (Zymo). Transferring 50 mu L of the reaction system to a 1.5mL centrifuge tube, adding 250 mu L of DNA Binding Buffer, and slightly shaking the centrifuge tube to mix the solution;

(4) transferring the mixed solution to Zymo-Spin ^TM Centrifuging a filter column/collection tube at 13000g for 30s, and discarding liquid in the collection tube;

(5) to Zymo-Spin ^TM Adding 200 mu L of DNA Wash Buffer into the centrifugal filter column, centrifuging for 30s at 13000g, and discarding the liquid in the collection pipe;

(6) finally, the filter column was transferred to a new collection tube, 15. mu.L of RNase-free water was added to the center of the filter in the centrifugal filter column, and the DNA was brought into full contact with the wells of the column, centrifuged at 13000g for 30s to obtain a DNA solution in the collection tube, and the DNA concentration was measured to be 200 ng/. mu.L by Bio Drop. The resulting DNA was stored at-20 ℃.

2. Containing N ⁶ Enrichment of DNA modified with-methyladenine

(1) Adding the DNA with allyl modification into an enzyme digestion system shown in the table 3, reacting for 1h at 37 ℃, and cutting the unmodified DNA by using MboI restriction enzyme;

TABLE 3 digestion system

(2) The cleavage reaction was subjected to agarose gel electrophoresis separation, and as shown in FIG. 11, the modified DNA was not cut, while the unmodified DNA was cut into shorter bands. The uncut DNA with modification was subjected to Gel-cutting purification using QIAquick Gel Extraction Kit (QIAGEN). Cutting the full-length DNA from the agarose gel, transferring the cut full-length DNA into a 1.5mL centrifuge tube, adding 300 mu L Buffer QG, and incubating at 50 ℃ for 10-20min until the gel block is completely dissolved;

(3) adding 100 mu L of isopropanol into the sample, shaking and uniformly mixing;

(4) transferring the mixed solution to a QIAquick spin centrifugal filter column/collection tube, centrifuging at 13000rpm for 1min, and discarding the liquid in the collection tube;

(5) adding 750 mu L of Buffer PE into a QIAquick spin centrifugal filter column, centrifuging at 13000rpm for 1min, and discarding the liquid in a collecting pipe;

(6) centrifuging the empty QIAquick spin centrifugal filter column/collection tube at 13000rpm for 1min, and discarding the liquid in the collection tube;

(7) finally, the filter column was transferred to a new collection tube, 25. mu.L of RNase-free water was added to the center of the filter membrane of the centrifugal filter column, and brought into full contact with the DNA in the well of the column, centrifuged at 13000rpm for 1min to obtain a DNA solution in the collection tube, and the DNA concentration was measured to be 200 ng/. mu.L by Bio Drop. The obtained DNA was used for the next reaction.

3. N of DNA ⁶ Iodine addition reaction and cyclization treatment of-allyladenine

(1) mu.L of the enriched DNA (200 ng/. mu.L) was transferred to a PCR tube, diluted to 26. mu.L with RNase-free water, added with 4. mu.L of a 0.125M iodine solution (dissolved in 0.25M potassium iodide) which became brown, and treated at 37 ℃ for 30 minutes;

(2) the brown solution was transferred to a new PCR tube, 4 μ L of 0.2M sodium thiosulfate was added until the solution was colorless, 6 μ L of 0.1M sodium carbonate (pH 9.5) was added, and treated at 37 ℃ for 30 minutes;

(3) mu.L of the above solution was mixed with 100. mu.L of glacial ethanol and 1. mu.L of glycogen, and then the mixture was blown and mixed, after overnight precipitation, the mixture was centrifuged at 4 ℃ at 15000rpm for 45 minutes, the precipitate was washed with 880. mu.L of 80% ethanol, centrifuged at 4 ℃ at 15000rpm for 15 minutes, the supernatant was removed again, air-dried for 3 minutes, and RNA was dissolved with 10. mu.L of RNase-free water to give a concentration of 100 ng/. mu.L.

4. Circularization treatment and extension of the resulting DNA fragment with DNA polymerase, and PCR

(1) Transferring the DNA obtained after the cyclization treatment into a PCR tube, adding 1 mu L of extension primer, complementing the volume to 15 mu L by RNase-free water, and reducing the temperature from 100 ℃ to 60 ℃ in a gradient manner at the speed of 1 ℃/s in a PCR instrument to combine the primer and the template;

(2) preparing a DNA polymerase extension system according to a reaction system shown in the table 4, reacting at 37 ℃ for 1h, and then heating at 75 ℃ for 20 min;

TABLE 4 DNA polymerase extension System

(3) 10. mu.L of the extension reaction solution was added to the PCR reaction system shown in Table 5;

TABLE 5 PCR reaction System

TABLE 6 DNA template sequences and PCR primer sequences

(4) The system is blown, beaten and mixed evenly, and then the running program in a PCR instrument is shown in the table 7;

TABLE 7 PCR amplification running program

(5) Purifying the double-stranded DNA fragment obtained above: adding 100 mu L of AMPure XP beads which are restored to the room temperature in advance into a reaction system, blowing and beating the mixture uniformly for 6 to 10 times, then incubating the mixture for 15 minutes at the room temperature, separating the mixture by using a magnetic frame, discarding the supernatant, washing the beads twice by using 80 percent ethanol, standing the mixture for 30 seconds each time for 5 to 10 minutes to volatilize the ethanol, eluting a double-stranded DNA fragment by using 10 mu L of resuspension buffer, incubating the mixture for 2 minutes at the room temperature, then separating the mixture by using the magnetic frame, and taking 1 mu L of the supernatant for the next reaction;

5 performing low-flux sequencing on the DNA fragment obtained after PCR by using TA cloning technology, and verifying mutation sites

(1) 1. mu.L of the above DNA product was subjected to T Vector ligation using pUCM-T Vector (Sangon Biotech, B522213) and reacted at 16 ℃ for 6 hours, and the pUCM-T Vector ligation system is shown in Table 8;

TABLE 8 pUCm-T Vector ligation System

(2) Thawing 100 μ L DH5 α competent cells on ice for 5min, suspending the cells uniformly, adding 5 μ L of the above connecting solution, beating gently, mixing well, and standing on ice for 25 min;

(3) performing heat shock in 42 ℃ water bath for 45 seconds, then placing on ice for 2 minutes, adding 700 mu L of SOC culture medium, and performing shake culture at 37 ℃ and 220rpm for 1 hour;

(4) centrifuging at 5000rpm for 1min, sucking out 600 μ L of supernatant with a pipette tip, and suspending the cells with the rest of the culture medium;

(5) preparing 50mL LB plate containing 50. mu.L IPTG (100mM), 100. mu. L X-gal (20mg/mL), 50. mu.L 1000 Xampicillin, 15mL each, and uniformly spreading the bacterial suspension on the LB plate;

(6) firstly, culturing the plate at 37 ℃ for 1 hour, then culturing the plate in an inverted mode for 16 hours, screening white single colony extraction plasmids in a blue-white spot, and carrying out Sanger sequencing to obtain a low-pass sequencing result;

(7) the results of the low-throughput sequencing are shown in FIG. 12, and three DNA polymerases, Klenow Fragment (3'→ 5' exo) ^- ) Sulfolobus DNA polymerase IV and Human DNA polymerase eta both mutate or delete at the A site of GATC and do not mutate or delete in the unclycled and unmodified DNA templates. Thus, it was confirmed that the modification site of DNA methylation was A in GATC.

EXAMPLE 2 with N ⁶ -allyldeoxyadenosine 6 aA-labeled HEK293T DNA

1. Culture of HEK293T cells, allyl labelling and gDNA extraction

(1) To normal cell culture medium was added 10% Fetal Bovine Serum (FBS), 1% 100 Xpenicillin-streptomycin diabody, N to a final concentration of 1mM ⁶ -allyldeoxyadenosine, HEK293T cells were cultured in this medium for 0,1,2,3,4 days;

(2) using Quick-DNA ^TM Microprep Kit (ZYMO) extracts cellular genomic DNA. Removing the culture medium by suction, suspending the cells by Phosphate Buffered Saline (PBS), transferring the cell suspension into a 1.5mL centrifuge tube, centrifuging at 4 ℃ and 2000rpm for 5min, and removing the supernatant;

(3) adding 400 mu L of Genomic lysine Buffer into a centrifuge tube, shaking and uniformly mixing, and cracking at room temperature for 5-10 min;

(4) transferring the mixed solution to Zymo-Spin ^TM Centrifuging IC centrifugal filter column/collection tube at 10000g for 1min, and discarding the collected tubeA liquid;

(5) to Zymo-Spin ^TM Adding 200 mu L of DNA Pre-Wash Buffer into an IC centrifugal filter column/collection tube, centrifuging for 1min at 10000g, and discarding liquid in the collection tube;

(6) to Zymo-Spin ^TM Adding 500 mu L g-DNA Wash Buffer into the IC centrifugal filter column/collection tube, centrifuging for 1min at 10000g, and discarding the liquid in the collection tube;

(7) finally, the filter column was transferred to a new collection tube, 50. mu.L of RNase-free water was added to the center of the filter membrane of the centrifugal filter column, and the filter membrane was brought into full contact with the DNA in the well of the column, centrifuged at 13000g for 1min to obtain a DNA solution in the collection tube, and the DNA concentration was measured to be 200 ng/. mu.L by Bio Drop. The resulting DNA was stored at-20 ℃.

2. DNA enzymatic hydrolysis and quantitation by mass spectrometry

(1) Taking 1-4 μ g of genome DNA, diluting to 26 μ L with RNase-free water, heating and denaturing at 100 deg.C for 3min, and standing on ice for 2 min;

(2) mu.L of 100mM NH pH 5.3 was added ₄ OAc and 1. mu.L of nucleic P1 (1U/. mu.L, WaKo USA), enzyme hydrolysis overnight at 42 ℃;

(3) 3.4. mu.L of NH was added to the above reaction system ₄ HCO ₃ (1M), then 1. mu.L of phosphodiesterase I (0.001U, Sigma-Aldrich) was added and incubated at 37 ℃ for 2 h;

(4) 1U of alkaline phosphatase (Sigma-Aldrich) was added to the above reaction system, and incubated at 37 ℃ for 2 h;

(5) finally, 50. mu.L of water was added for dilution, and the DNA was quantitatively analyzed for N by mass spectrometry ⁶ -content of allyladenine;

(6) the mass spectrum results are shown in FIG. 13, which illustrates N ⁶ Allyl adenine was successfully introduced into the cellular DNA, enabling the labeling of the newly generated DNA.

Claims (8)

1. A method for single base resolution DNA labeling and identification comprising the steps of:

(1) DNA is labeled extracellularly with enzyme assistance or intracellularly through the process of cellular self-metabolism:

the extracellular labeling process was as follows: adenosylmethionine transferases use the methionine analogue allyl-L-seleno/thiohomocysteine to generate the methyltransferase cofactor allyl-L-seleno/thioadenosylhomocysteine, or directly add allyl-L-seleno/thioadenosylhomocysteine, and methyltransferases use this cofactor to introduce an allyl group into a particular N of DNA ⁶ 6mA or N methyladenine ⁴ -methylcytosine at the 4mC site, forming N ⁶ -allyldeoxyadenosine 6aA or N ⁴ -allyldeoxycytidine 4 aC; or extracellular N-binding of N using active DNA polymerase/reverse transcriptase ⁶ Allyl deoxyadenosine triphosphate 6adATP, N ⁴ -random replication/reverse transcription of allyl deoxycytidine triphosphate 4adCTP into nascent DNA;

the labeling process inside the cell is as follows: cells use allyl-L-seleno/thiohomocysteine to introduce an allyl group to a specific 6mA or 4mC site of DNA by natural metabolism to form 6aA or 4 aC; or directly randomly introducing free 6aA or 4aC in the culture medium into the nascent DNA;

(2) enrichment of DNA containing 6aA or 4aC modifications: cutting off the unmodified DNA sequence by using restriction endonuclease, and enriching the modified DNA sequence by an agarose gel electrophoresis method; enriching 6aA or 4aC modified DNA by means of an immunoprecipitation method, eluting the DNA from magnetic beads by using protease, and purifying the eluted DNA;

(3) iodine addition and circularization of DNA 6aA or 4 aC: n on DNA ⁶ -allyladenine or N ⁴ Allyl cytosine undergoes an iodine addition reaction, and then is induced to form a cyclized structure under an alkaline condition, so that base complementary pairing is shielded;

(4) extension and sequencing recognition of circularization-treated DNA: and (3) carrying out denaturation treatment on the DNA obtained in the step (3), adding DNA polymerase, extending the DNA containing a cyclization structure, introducing errors to a para-complementary base in the process that adenine or cytosine cyclized on the DNA generates a DNA complementary strand under the action of the DNA polymerase, and identifying a mutation site by a nucleic acid sequencing means so as to obtain a 6aA or 4aC site.

2. The method for single-base resolution DNA labeling and identification according to claim 1, wherein in step (1), 6aA is prepared by: under the protection of argon or nitrogen, taking 6-chloropurine deoxynucleoside, allylamine hydrochloride and triethylamine in a molar ratio of 1: 1.5-3: 3-10 as raw materials, taking ethanol as a solvent, and taking 80 parts of ethanol as a solvent ^O C, heating and refluxing for 3 hours, concentrating, and separating by high performance liquid chromatography to obtain 6 aA.

3. The method for single-base resolution DNA labeling and identification according to claim 2, wherein in the step (1), the 6adATP is prepared by the following steps: adding completely dried trimethyl phosphate with the molar weight 5-50 times that of 6-chloropurine deoxynucleoside and phosphorus oxychloride with the molar weight 1.1-1.3 times that of 6aA into the prepared 6aA, and stirring for 0.5-3 hours at 0-4 ℃ until a reaction solution is completely clear; then adding tributylamine pyrophosphate with the molar weight of 6aA 2-10 times, and stirring for 10-30 minutes at 0-4 ℃; then continuously stirring for 5-10 minutes at room temperature, and adding triethylammonium bicarbonate with the molar weight being at least 1 kilo time of 6aA to terminate the reaction; purifying by high performance liquid chromatography to obtain 6 adATP.

4. The method for single-base resolution DNA labeling and identification according to claim 1, wherein in step (1), the 4aC is prepared by: mixing a mixture of 1: 0.14-0.17 of 2 ' -deoxyuridine and 4-dimethylaminopyridine serving as raw materials, acetic anhydride serving as a solvent, reacting at room temperature for 5-6 hours, concentrating, and purifying by a silica gel column to obtain 3', 5' -diacetyl deoxyuridine; mixing 3', 5' -diacetyl deoxyuridine with the molar weight 1-1.3 times that of 2 ' -deoxyuridine and 1-hydrogen-tetrazole with the molar weight 1-1.3 times that of 2 ' -deoxyuridine, adding the mixture into a flask, removing water and oxygen, adding pyridine and 4-chlorophenyl dichlorophosphate with the molar weight 2-4 times that of 2 ' -deoxyuridine in an ice water bath under the protection of nitrogen or argon, reacting the mixture for 5-10 minutes in the ice water bath at room temperature for 5-7 hours, and adding saturated Na ₂ CO ₃ Quenching the solution, extracting with dichloromethane, and then using saturated saltWashing with water, drying, evaporating and concentrating to obtain solid for the next reaction; dissolving the solid obtained in the last step in acetonitrile, adding 1-1.5 times of potassium hydroxide and 1-1.5 times of allylamine hydrochloride into a flask in advance, sealing, sequentially adding water, acetonitrile, triethylamine and the product obtained in the last step dissolved in acetonitrile, reacting for 24 hours at room temperature, concentrating, and purifying by a silica gel column to obtain 3', 5' -diacetyl-N ⁴ -allyldeoxyuridine; 3', 5' -diacetyl-N with the molar weight 1-2 times of that of 2 ' -deoxyuridine ⁴ Allyl deoxyuridine was added to a 2mol/L solution of ammonia in methanol in an amount of at least 1 thousand times the molar amount of 2' -deoxyuridine, reacted overnight at room temperature, concentrated and dried to give 4 aC.

5. The method for single-base resolution DNA labeling and identification of claim 4, wherein in step (1), 4adCTP is prepared by the following steps: adding completely dried trimethyl phosphate with the molar weight 10-50 times that of 2 '-deoxyuridine and phosphorus oxychloride with the molar weight 1.1-1.5 times that of 2' -deoxyuridine into the prepared 4aC under the protection of argon or nitrogen, and reacting for 3-3.5 hours at 0-4 ℃; adding tributylamine pyrophosphate with the molar weight 3-10 times that of 2' -deoxyuridine, and stirring for 1-1.5 hours at 0-4 ℃; then continuously stirring for 5-10 minutes at room temperature, and adding triethylammonium bicarbonate with the molar weight at least 1 thousand times of that of 2' -deoxyuridine to terminate the reaction; and (4) purifying by high performance liquid chromatography to obtain 4 adCTP.

6. The method for single-base resolution DNA labeling and identification of claim 1, wherein in step (2), the antibody used for binding 6aA is N ⁶ Antibodies to isopentenyl adenosine, 10 μ g of antibodies were used to enrich for 5-100 μ g of fragmented DNA.

7. The method for single-base resolution DNA labeling and identification according to claim 1, wherein the step (3) is specifically: i) denaturing the DNA fragment after iodine addition, heating at 95 deg.C for 10minStanding on ice for 5 min; ii) dissolving 0.1-0.5M iodine simple substance in 0.2-1M potassium iodide to obtain potassium iodide solution of iodine, and then enabling the potassium iodide solution of iodine and N on DNA 6aA ⁶ -allylic reaction of allyladenine followed by removal of excess iodine with 0.1-0.5M sodium thiosulfate; adding 0.1-0.5M sodium carbonate, adjusting the pH value to 9-10, and inducing N on DNA 6aA ⁶ 1, N of allyl adenine ⁶ N at position or 4aC ⁴ 3, N of allyl cytosine ⁴ The sites form a cyclic structure, thus shielding the normal hydrogen bond pairing.

8. The method for single-base resolution DNA labeling and identification according to claim 1, wherein the step (4) is specifically: i) extending the denatured DNA containing the circularized structure by using DNA polymerase; wherein the DNA polymerase is E.coli DNA polymerase I Klenow Fragment (3 '-5' exo) ^- ) Sulfolobus DNA polymerase IV, Human DNA polymerase eta, Therminator DNA polymerase, Bsu DNA polymerase, DNA polymerase I, E.coli DNA polymerase I Klenow Fragment, T4 DNA polymerase or T7 DNA polymerase; ii) adopting a DNA library preparation technology and a high-throughput sequencing means to identify the whole transcriptome mutation site, or adopting PCR and TA-cloning technology to identify and verify the mutation site on specific DNA.

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