CN110093399A - A method of utilizing N6 generation methylation modifications of adenine in diphosphonic acid uracil deoxynucleotide detection nucleic acid - Google Patents
A method of utilizing N6 generation methylation modifications of adenine in diphosphonic acid uracil deoxynucleotide detection nucleic acid Download PDFInfo
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Abstract
The invention discloses a kind of methods that methylation modification occurs using adenine N6 in diphosphonic acid uracil deoxynucleotide detection nucleic acid.The detection method mainly consists of two parts.First part is that diphosphonic acid uracil deoxynucleotide (dUDP) is mixed genome sequence by extension to tested nucleic acid or control nucleic acid.Second part is to analyze extension using denaturing polyacrylamide gel as a result, obtaining the extension percentage of tested nucleic acid or control nucleic acid after data processing, is compared to realize recognition detection N6Methyl adenine and N1Methyl adenine.The method overcome existing detection method device requirements it is high, cumbersome the deficiencies of, high sensitivity, wide adaptation range are raw materials used simple and easy to get, easy to operate.
Description
Technical field
The invention belongs to molecular biology, nucleic acid chemistry and the fields epigenetics (Epigenetic), and in particular to one
N in kind genome6Methyl adenine (N6- methyladenine) recognition detection method.
Background technique
An important branch subject of the epigenetics as science of heredity, main research are drawn based on the change of non-genomic sequence
Heritable variation of the gene expression risen.Its mechanism of action specifically includes that DNA methylation, chromatin remodeling, non-coding RNA
Regulation, histone modification etc..Epigenetics is related with numerous vital functions, such as genomic imprinting, x-chromosome inactivation etc..
Simultaneously studies have shown that the hair of the diseases such as epigenetics and cancer, disease of immune system, several genetic baryencephalia diseases
Existence is in important association.
The epigenetics modification of DNA is broadly divided into two major classes, first is that C5 methylations are modified on cytimidine, referred to as
5-methylcytosine (5mC)；Second class is N6 generation methylation modifications on adenine, it is referred to as N by we6Methyl gland
Purine.Not only there is the differences of organism distributing position and content for both different epigenetic modifications, while their institutes
The biological function undertaken is also entirely different.For example, 5-methylcytosine is distributed mainly on mammal and other eukaryons are raw
In object, vital work is all played for the selective expression of gene, the stability of gene, ontogeny and the generation of disease
With.However in prokaryotes and some low eucaryotes, N6Methyl adenine then replaces 5-methylcytosine to become master
The epigenetics modification wanted, and execute its specific biological function.In the following period of time that the past is very long, due to detecting hand
The limitation of section, N6Methyl adenine-is directly considered in the DNA for being only distributed in bacterium.
With the development of detection technique, researcher has found N6Methyl adenine exists in some eucaryotes, together
When its potential epigenetics function be also gradually revealed.For example, it may be possible to important epigenetics information is also carried, and
And hereditary transmitting can be carried out in filial generation etc..
Different from DNA, it is known that RNA exists to be modified more than 100 kinds.And taking off with more epigenetic modification object functions
Show, it has been found that, in addition to hereditary information is transferred to protein from DNA, mRNA itself can also participate in many biological processes.
N6Methyl adenine is widely present in the mRNA and non-coding RNA of mammal, average to there is 3 on every mRNA
~5 N6Methyl adenine modified base.In addition, it has been discovered that N6The distribution of methyl adenine has certain spy
Sign.Firstly, their integrated distributions are near terminator codon and in the exon of interior minister, this illustrates N6Methyl adenine
It is likely to all play critically important effect in the translational control of mRNA and the alternative splicing of RNA.In addition, it has also been found that
N6Methyl adenine is widely present in 3 ' UTRs, and the N in these regions6Methyl adenine very likely influence itself with
The binding force of rna binding protein, and then influence a series of biological processes of RNA.Meanwhile there are also seminars to find in 3 ' UTRs
N6Methyl adenine is always distributed in front of the bond area miRNA just, and speculates N6Methyl adenine will affect miRNA and lure
The transcript degradation led and Translational repression.They also pass through analysis, and discovery contains N6The gene of methyl adenine much also with
RNA metabolism, intercellular signal transmitting and the nervous system disease are related.
In transcript profile level, different types of mRNA has the N of different abundance6Methyl adenine modification.Moreover, N6Methyl
For adenine in different cell stages and different tissue samples, content is all different.Therefore, N6Methyl adenine very may be used
It can be metabolized in RNA and play critically important effect in growth and development.Currently, it has been found that its some critical functions,
Montage including mRNA, mRNA's goes out core, and the translation of mRNA accelerates the degradation of mRNA to reduce mRNA stability, influences to give birth to
The maintenance of object clock, the adjusting of cell cycle and the differentiation of mammalian embryonic stem cell.It can be seen that N6Methyl adenine
The important function for being difficult to substitute all is played during the various biological of mammal.
Gradually in-depth study shows the N in DNA and RNA6Methyl adenine has irreplaceable researching value, and
The convenient and efficient of its detection method is just more crucial to the influence of its functional characteristics, the mechanism of action etc. further studied.
Existing N6It includes ultra performance liquid chromatography-tandem mass spectrometry (Ultra- that methyl adenine, which predominantly detects method,
High Performance Liquid Chromatography-tandem Mass Spectrometry, UHPLC-MS/MS),
The methods of unicellular real-time sequencing (Single molecule real time sequencing, SMRT).Although these methods
Detection N can be reached6The purpose of methyl adenine, but they there are still some limitations.Such as unicellular real-time PCR sequencing PCR
It is difficult to N6Methyl adenine and N1The similar modified types of these structures of methyl adenine；And other methods complex steps
Or equipment is expensive.
Summary of the invention
In order to solve shortcomings and deficiencies of the existing technology, the object of the present invention is to provide one kind without using expensive instrument
Device, without tedious steps, it is environmental-friendly, practical and can efficiently, special and delicately N in recognition detection nucleic acid6Methyl gland is fast
The method of purine.
The present invention completes by following technical solution:
N is had using the identification of diphosphonic acid uracil deoxynucleotide the present invention provides a kind of6The nucleic acid of methyl adenine
Method, specifically include:
(1) raw material synthesized using diphosphonic acid uracil deoxynucleotide (dUDP) as DNA, close to suspected of N6Methyl
The primer that the site upstream of adenine is 17-20bp according to one length of template strand sequence design, in archaeal dna polymerase or reverse transcription
It is incubated for altogether under enzyme effect with nucleic acid and carries out extension, diphosphonic acid uracil deoxynucleotide (dUDP) is mixed into sequence, institute
Stating nucleic acid is suspected of containing N6The determined nucleic acid or control nucleic acid in the site of methyl adenine, the control nucleic acid are corresponding site
Without N6Methyl adenine, sequence and determined nucleic acid is close and other sites contain N6Methyl adenine level and determined nucleic acid phase
Same nucleic acid；
(2) step (1) extension products are analyzed by denaturing polyacrylamide gel, are extended through gel imager measurement
Concentration of substrate and not extended concentration of substrate obtain extending percentage, the extension percentage are as follows: extension percentage=prolonged
The concentration of substrate stretched/(extended concentration of substrate+concentration of substrate not being extended)；
(3) result judgement: if the extension percentage for extending percentage and being less than control nucleic acid of determined nucleic acid, that is, can recognize
With N6The nucleic acid of methyl adenine.
Preferably, 5 ' of primer described in step (1) terminal modified have FAM.
Preferably, step (1) archaeal dna polymerase is Bst archaeal dna polymerase.
Preferably, step (1) reverse transcriptase is M-MuLV reverse transcriptase.
Second aspect provides diphosphonic acid uracil deoxynucleotide in preparation identification with N6The nucleic acid of methyl adenine
Detection reagent in application.
The principle of technical solution of the present invention is: when using dUDP as synthesis material, using containing N6The nucleic acid of methyl adenine
The significance difference of the extension rate of chain and the nucleic acid chains containing unmodified adenine under archaeal dna polymerase or reverse transcriptase catalysis
Not, the accurate information of methylation decorating site is obtained.
The advantages of the present invention
1. the present invention is not limited by detection limit and sequence length to be measured, testing result of the invention is imitated by the extension quantified
Fruit (percentage) analysis, when using dUDP as synthesis material, using containing N6The nucleic acid chains of methyl adenine with containing unmodified
Adenine nucleic acid chains archaeal dna polymerase or reverse transcriptase catalysis under extension rate marked difference, obtain methyl
Change the accurate information of decorating site.Simultaneously as the extension rate under archaeal dna polymerase or reverse transcriptase catalysis is exceedingly fast, not
Marked difference, therefore the resolution detected is not present in the extension time that modified sequence carries out under technical solution of the present invention
Rate can still be guaranteed that this advantage makes the present invention adapt to the nucleic acid chains pattern detection of greater depth range, improve it
Application value；
2. the present invention is that the pathogenesis of study of disease is provided convenience: current research has found the morbidity machine of certain cancers
System is related to the methylation modification of specific site in genome, and is limited to detection means, and the type of the modification that methylates can not be by
Accurately know.In present invention situation known to whole genome sequence and decorating site that may be present, further improve to gland
Adenine methyl type is probed into, to accurately understand the pathology cause of disease and specific aim exploitation specific medicament；
3. the present invention provides new approaches for the early diagnosis of the diseases such as cancer: the early detection of cancer has the inspection of blood item
Look into, Ag-Ab hybridization etc. modes, be limited in that design detection reagent it is with high costs, complex steps, interpretation of result interval
Time is long, and exists for the initial stage cancer detection effect of different times uncertain.The present invention is using common biochemical examination
Agent dUDP and Bst archaeal dna polymerase or M-MuLV reverse transcriptase can pass through healthy individuals and the comparison for suffering from cancer individual testing result
Confirm Testing index, and suffer from the unknown individual of cancer situation, therefore significant shortening reaction time as referring to detection, is sieved for cancer
Offer important reference is provided, while reducing detection and carrying out cost, mitigates burden on society, is had in medical testing agency wide
Application prospect.
Detailed description of the invention
Fig. 1 will be adenine or N in same site when being 63 DEG C6The DNA sequence dna of methyl adenine respectively with various concentration
Diphosphonic acid uracil deoxynucleotide (dUDP) Bst archaeal dna polymerase effect under altogether be incubated for treated extend percentage-
Time chart.
A is extension percentage-time plot of the DNA containing adenine, and B contains N6The extension hundred of the DNA of methyl adenine
Divide ratio-time plot.The concentration for being labeled as diphosphonic acid uracil deoxynucleotide (dUDP) of legend.
Fig. 2 will be adenine or N in same site when being 63 DEG C6The DNA sequence dna of methyl adenine respectively with 400 μM two
Modacrylic acyl of the phosphoric acid uracil deoxynucleotide (dUDP) after being incubated for different time altogether under Bst archaeal dna polymerase acts on
Amine gel analysis figure.(band above in electrophoretogram is to extend segment, and band below is the segment not extended, 6mA in legend
Represent N6Methyl adenine)
A is the DNA sequence dna for containing only adenine site, and band 1-14 corresponds to the result of different disposal time from left to right.B
To contain N6The DNA sequence dna in methyl adenine site, band 1-14 corresponds to the result of different disposal time from left to right.C be containing
There is the DNA sequence dna in adenine site and contains N6The DNA sequence dna of methyl adenine extends percentage-time graph.
Fig. 3 will be adenine or N in same site when being 42 DEG C6The RNA sequence of methyl adenine respectively with 100 μM two
Denaturation of the phosphoric acid uracil deoxynucleotide (dUDP) after being incubated for different time under RNA M-MuLV reverse transcriptase acts on altogether is poly-
Acrylamide gel analysis figure.(band above in electrophoretogram is to extend segment, and band below is the segment not extended, figure
6mA represents N in example6Methyl adenine)
A is the RNA sequence for containing only the site adenine (A), and band 1-14 corresponds to the knot of different disposal time from left to right
Fruit.B is to contain N6The RNA sequence in methyl adenine site, band 1-14 corresponds to the result of different disposal time from left to right.C
To contain only the RNA sequence in adenine site and containing N6The RNA sequence of methyl adenine extends percentage-time graph.
Specific embodiment
By following detailed description combination attached drawing it will be further appreciated that the features and advantages of the invention.Provided implementation
Example is only the explanation to the method for the present invention, remaining content without limiting the invention in any way announcement.Following embodiment institute
The nucleic acid sequence used such as the following 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' |
[embodiment 1] will be adenine or N in same site6The DNA sequence dna of methyl adenine respectively with various concentration
Diphosphonic acid uracil deoxynucleotide (dUDP) Bst archaeal dna polymerase effect under altogether be incubated for treated extend percentage-when
Between Relationship Comparison
1. 1 μ L 1 × ThermoPol buffer of pH 8.8 at 25 DEG C of configuration: 20mM Tris-HCl, 10mM ammonium sulfate,
10mM potassium chloride, 2mM magnesium sulfate, 0.1% Triton X-100.Be added 0.2 μ L double-stranded DNA DNA-17mer-A1 or
DNA-17mer-6mA1 makes its final concentration of 2 μM, and 0.1 μ L gradient concentration dUDP, 1 μ L Bst archaeal dna polymerase and primer is added
The concentration ratio of Primer, primer and double-stranded DNA is 3:5, uses ddH2O polishing volume is to 10 μ L.
Be incubated for 5min at 2.63 DEG C, and be spaced different time sampling analyses, time interval with reaction lengthen with
The higher characterization result of discrimination is obtained, 45 μ L are added and stop buffer (95% formamide, 25mM EDTA, pH 8.0) with end
It only reacts, reaction system is immediately heated to 90 DEG C and keeps the temperature 10min, is cooled to 4 DEG C.
The separation of 3.20% denaturing polyacrylamide gel electrophoresis is completed extension and the segment of extension does not occur,
And characterized under gel imager, then obtain the ratio for completing the nucleic acid chains to be detected of extension.
As a result: the growth in reaction time can make the difference for extending percentage more obvious, and improve the specificity of reaction.
The optimization of reaction condition provides more suitable condition to subsequent detection, can be to sensitivity, standard required for detection method
The various aspects such as true property are improved.(Fig. 1)
[embodiment 2] the method for the present invention is applied to include N6The detection of the DNA sequence dna of methyl adenine
1. 1 μ L 1 × ThermoPol buffer of pH 8.8 at two groups 25 DEG C of configuration: 20mM Tris-HCl, 10mM sulfuric acid
Ammonium, 10mM potassium chloride, 2mM magnesium sulfate, 0.1% Triton X-100.It is each that 0.2 μ L double-stranded DNA DNA-17mer- is added
A1 or DNA-17mer-6mA1 makes its final concentration of 2 μM.It is added final concentration of 400 μM of dUDP, 1 μ L Bst archaeal dna polymerase,
And primer Primer, the concentration ratio of primer and double-stranded DNA is 3:5, uses ddH2O polishing volume is to 10 μ L.
2.63 being incubated for 5min at DEG C and being spaced different time sampling analyses, time interval lengthen to obtain with reaction
The higher characterization result of discrimination is obtained, 45 μ L are added and stop buffer (95% formamide, 25mM EDTA, pH 8.0) to terminate
Reaction, reaction system are immediately heated to 90 DEG C and keep the temperature 10min, are cooled to 4 DEG C.
The separation of 3.20% denaturing polyacrylamide gel electrophoresis extends with the segment not extended and in gel imager following table
Sign then determines and extends percentage.
As a result: for DNA sample, template sequence containing adenine extends high percentage in containing N6Methyl adenine
Template sequence extension percentage.(Fig. 2)
[embodiment 3] the method for the present invention is applied to include N6The detection of the RNA sequence of methyl adenine
1. 1 μ L 1 × M-MuLV RT buffer of pH 8.3 at 25 DEG C of configuration: 50mM Tris-HCl, 75mM potassium chloride,
Final concentration of 2 μM of the RNA-17mer-A1 or RNA-17mer-6mA1 of 0.2 μ L is added in 2mM magnesium chloride, 10nM dithiothreitol (DTT),
The concentration ratio of primer Primer, primer and double-stranded DNA is 3:5, the dUDP that 100 μM of final concentration, 1 μ L M-MuLV reverse transcriptase,
Use ddH2O polishing volume is to 10 μ L.
2. being incubated for 5min at 42 DEG C of control, it is spaced different period sampling analyses, when analysis is added 45 μ L and stops buffering
Liquid (95% formamide, 25mM EDTA, pH 8.0) is immediately heated to 90 DEG C to terminate reaction and keeps the temperature 10min, be cooled to 4
℃。
The segment that 3.20% denaturing polyacrylamide (PAGE) gel electrophoresis separation extends and do not extend, and in gel imaging
It is characterized under instrument, then determines and extend percentage.
As a result: for RNA sample, template sequence containing adenine extends high percentage in containing N6Methyl adenine
Template sequence extension percentage.(Fig. 3).
Sequence table
<110>Wuhan University
<120>a kind of that methylation modification occurs using adenine N6 in diphosphonic acid uracil deoxynucleotide detection nucleic acid
Method
<160> 5
<170> SIPOSequenceListing 1.0
<210> 1
<211> 17
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 1
aatgccacat gctgcac 17
<210> 2
<211> 17
<212> DNA
<213>artificial sequence (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 (Artificial Sequence)
<400> 3
aaugccacau gcugcac 17
<210> 4
<211> 17
<212> RNA
<213>artificial sequence (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 (Artificial Sequence)
<220>
<221> misc_feature
<222> (1)..(1)
<223> 5'-fam
<400> 5
gtgcagcatg tggcat 16
Claims (5)
1. a kind of have N using the identification of diphosphonic acid uracil deoxynucleotide6The method of the nucleic acid of methyl adenine, feature exist
In specifically including:
(1) raw material synthesized using diphosphonic acid uracil deoxynucleotide (dUDP) as DNA, close to suspected of N6Methyl adenine
Site upstream be 17-20bp according to one length of template strand sequence design primer, acted in archaeal dna polymerase or reverse transcriptase
Lower and nucleic acid is incubated for altogether carries out extension, and diphosphonic acid uracil deoxynucleotide (dUDP) is mixed sequence, the nucleic acid
For suspected of containing N6The determined nucleic acid or control nucleic acid in the site of methyl adenine, the control nucleic acid are that corresponding site is free of N6-
Methyl adenine, sequence and determined nucleic acid is close and other sites contain N6Methyl adenine level core identical with determined nucleic acid
Acid；
(2) extension products of step (1) are analyzed by denaturing polyacrylamide gel, through the extended bottom of gel imager measurement
Object concentration and not extended concentration of substrate obtain extending percentage, the extension percentage are as follows: extension percentage=be extended
Concentration of substrate/(extended concentration of substrate+not extended concentration of substrate)；
(3) result judgement: if the extension percentage for extending percentage and being less than control nucleic acid of determined nucleic acid, that is, can recognize and have
N6The nucleic acid of methyl adenine.
2. the method according to claim 1, wherein 5 ' of primer described in step (1) terminal modified have FAM.
3. the method according to claim 1, wherein step (1) archaeal dna polymerase is BstDNA polymerase.
4. the method according to claim 1, wherein step (1) reverse transcriptase is M-MuLV reverse transcriptase.
5. diphosphonic acid uracil deoxynucleotide has N in preparation identification6Answering in the detection reagent of the nucleic acid of methyl adenine
With.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102399863A (en) * | 2010-09-16 | 2012-04-04 | 上海迦美生物科技有限公司 | Methylated DNA detection method based on fragment length analysis |
| CN103392008A (en) * | 2010-09-07 | 2013-11-13 | 加利福尼亚大学董事会 | Control of DNA movement in a nanopore at one nucleotide precision by a processive enzyme |
| CN104262434A (en) * | 2014-10-28 | 2015-01-07 | 武汉大学 | Chemical demethylation method of N<6>-methyladenosine in RNA |
| CN104350067A (en) * | 2012-04-10 | 2015-02-11 | 牛津纳米孔技术公司 | Mutant lysenin pores |
| CN107109489A (en) * | 2014-10-17 | 2017-08-29 | 牛津纳米孔技术公司 | Nano-pore RNA characterizing methods |
-
2019
- 2019-04-22 CN CN201910325528.9A patent/CN110093399A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103392008A (en) * | 2010-09-07 | 2013-11-13 | 加利福尼亚大学董事会 | Control of DNA movement in a nanopore at one nucleotide precision by a processive enzyme |
| CN102399863A (en) * | 2010-09-16 | 2012-04-04 | 上海迦美生物科技有限公司 | Methylated DNA detection method based on fragment length analysis |
| CN104350067A (en) * | 2012-04-10 | 2015-02-11 | 牛津纳米孔技术公司 | Mutant lysenin pores |
| CN107109489A (en) * | 2014-10-17 | 2017-08-29 | 牛津纳米孔技术公司 | Nano-pore RNA characterizing methods |
| CN104262434A (en) * | 2014-10-28 | 2015-01-07 | 武汉大学 | Chemical demethylation method of N<6>-methyladenosine in RNA |
Non-Patent Citations (5)
| Title |
|---|
| CAROLINE ROOST等: "Structure and Thermodynamics of N6-Methyladenosine in RNA: A Spring-Loaded Base Modification", 《JOURNAL OF THE AMERICAN CHEMICAL SOCIETY》 * |
| CASSANDRA R. BURKE等: "DNA synthesis from diphosphate substrates by DNA polymerases", 《PNAS》 * |
| CLAUDIO STASOLLA等: "Purine and pyrimidine nucleotide metabolism in higher plants", 《J. PLANT PHYSIOL.》 * |
| SHAORU WANG等: "N6-Methyladenine hinders RNA- and DNA-directed DNA synthesis: application in human rRNA methylation analysis of clinical specimens", 《CHEM. SCI.》 * |
| SHAORU WANG等: "The m6A methylation perturbs Hoogsteen pairing-guided incorporation of an oxidized nucleotide", 《CHEM. SCI.》 * |
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