CN109022544A - A kind of kit and its method detecting DNA methylation transferase active - Google Patents
A kind of kit and its method detecting DNA methylation transferase active Download PDFInfo
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- CN109022544A CN109022544A CN201810759712.XA CN201810759712A CN109022544A CN 109022544 A CN109022544 A CN 109022544A CN 201810759712 A CN201810759712 A CN 201810759712A CN 109022544 A CN109022544 A CN 109022544A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
- C12Q1/44—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving esterase
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/48—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/90—Enzymes; Proenzymes
- G01N2333/91—Transferases (2.)
- G01N2333/91005—Transferases (2.) transferring one-carbon groups (2.1)
- G01N2333/91011—Methyltransferases (general) (2.1.1.)
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/90—Enzymes; Proenzymes
- G01N2333/914—Hydrolases (3)
- G01N2333/916—Hydrolases (3) acting on ester bonds (3.1), e.g. phosphatases (3.1.3), phospholipases C or phospholipases D (3.1.4)
- G01N2333/922—Ribonucleases (RNAses); Deoxyribonucleases (DNAses)
Abstract
The invention discloses a kind of methods of cycle signal amplification detection DNA methylation transferase active that join dependency is mediated based on monokaryon ribotide reparation, (1) the hair clip substrate that Dam MTase is acted on is added in excision reaction buffer solution and carries out cleavage reaction, the product of 24-nt will be generated；(2) above-mentioned cleaved products are added in amplified reaction buffer, carry out being connected by circulation dependence strand displacement amplification reaction, circulation generates the fluorescence signal of enhancing, passes through the activity of the strong and weak measurement Dam MTase of detection fluorescence signal；The present invention can be effectively suppressed independent of target and template/primer non-specific amplification, to substantially reduce background signal, the final index amplification for realizing signal, this method sensitivity and specificity with higher, Monitoring lower-cut is up to 4.8 × 10‑6U/mL。
Description
Technical field
The invention belongs to bioassay technique fields, and in particular to a kind of company mediated based on single ribonucleotide reparation
Connect the method for relying on cycle signal amplification detection DNA methylation transferase active.
Background technique
Genomic DNA methylation level occurs often on 4,5/ nitrogen of carbon of cytimidine (C) and 6, nitrogen of adenine (A), is
Most important epigenetic modification in genomic DNA.Adjusting genetic transcription, chromatin Structure, embryonic development and cell ageing
In play vital effect.Dnmt rna (MTase) is responsible for genomic DNA methylation level modification, can be catalyzed transfer
Methyl on adenosine-L-methionine (SAM) remains thin on adenine/cytosine residues into specific gene group DNA sequence dna
Stable methylation patterns intracellular.Dnmt rna (MTase) active exception can destroy normal DNA methylation patterns, most
The generation of induction kinds cancer eventually, including lung cancer, breast cancer, prostate cancer, colon cancer, liver cancer, kidney, cervix cancer, first shape
Gland cancer, retinoblastoma and hematological cancer.Therefore, as the potential source biomolecule marker and anticancer of early clinical diagnosis
The pharmacology target for the treatment of, exploitation can accurately, delicately detect dnmt rna (MTase) active method for carcinogenic
The research of mechanism and the discovery of anticancer drug are most important.
So far, a variety of methods for dnmt rna (MTase) detection have been developed, including have been based on putting
The gel electrophoresis of penetrating property label, enzyme-linked immunosorbent assay, high performance liquid chromatography (HPLC).But the detection sensitivity of these methods
It is low, time and effort consuming is operated, and need precision instrument, or even there is radiation hazradial bundle.In addition, some new methods include colorimetric method,
Electrochemical process, fluorescence method and chemoluminescence method have also been applied to the active detection of DNA MTase.Colorimetric method can pass through peroxide
Change hydrogen (H2O2) oxidation ABTS2-To generate coloured ABTS-, to realize the active visualization inspection of dnmt rna (MTase)
It surveys.Electrochemical process is by a series of nano materials (such as graphene (GO), silver nano-grain (AgNPs) and gold nano grain (AuNPs)
Deng) dnmt rna (MTase) active detection is used in conjunction with the restriction endonuclease of methyl-sensitive.Fluorescence
Method is using the fluorescence resonance energy transfer (FRET) based on graphene (GO) and quantum dot (QD) come effectively quantitatively DNA methyl shifts
The activity of enzyme (MTase).However the sensitivity of these methods need to be improved, and is related to cumbersome operation sequence, it is complicated
The modification of nano material synthesis and electrode surface.In order to improve detection sensitivity, signal amplification strategy is introduced into DNA first recently
In based transferase (MTase) activity test method.Typical example includes target circulation and core based on exonuclease III auxiliary
The fluorescence analysis of the cycle signal probe cutting of sour restriction endonuclease (such as Nt.BbvCI and Nt.Alwl) auxiliary, but it is detected
Limit can only achieve 0.01U/mL, 0.01U/mL and 0.04U/mL；And it is based on the chemiluminometry of rolling circle amplification (RCA),
This method instrument is simple but sensitivity is not high, and detection is limited to 1.29 × 10-4Furthermore U/mL boths refer to molecular beacon probe
The preparation of well-designed and complicated annular template, and also suffer from the highback as caused by non-specific digestion or non-specific amplification
Scape signal；It is worth noting that, conventional nucleic acid amplification technique (such as polymerase chain reaction (PCR), strand displacement amplification reaction
(SDA), rolling circle amplification reaction (RCA) and isothermal exponential amplification (EXPAR) reaction are normally based on archaeal dna polymerase or DNA polymerization
Enzyme and nicking enzyme are combined to produce the DNA fragmentation for being largely used to realize signal amplification, but they all inevitably by
Be attributable to high background signal, reason as caused by non-specific amplification: (1) some archaeal dna polymerases do not have outside calibration nucleic acid
For enzyme cutting activity to repair the deoxyribonucleotide of mispairing, this will lead to the non-specific segment of generation；(2) archaeal dna polymerase mediates
De novo formation and DNA double chain polymerization extend so that the recognition site of endonuclease is mixed at random, this will lead to non-spy
Anisotropic exponential amplification.In order to reduce high background signal, repair enzyme uracil-DNA glycosylase and endonuclease IV are introduced into
And cooperated with archaeal dna polymerase to start the nucleic acid amplification of uracil reparation mediation, but this amplification based on enzyme reparation
Reaction (ERA) needs two kinds of repair enzymes (i.e. uracil-DNA glycosylase and endonuclease IV) and is related to multistep cleavage reaction
(purine that the uracil base excision reaction that i.e. uracil-DNA glycosylase mediates is mediated with endonuclease IV-/without pyrimidine
(AP) site is cut), or even also needing can be well-designed with the DNA profiling of uracil mispairing.It would therefore be highly desirable to develop a kind of high
Sensitivity, high specific and simple dnmt rna (MTase) activity test method of design.
Summary of the invention
For the technical problems in the prior art, an object of the present invention is to provide a kind of based on single ribose core
The kit for the join dependency cycle signal amplification detection DNA methylation transferase active that thuja acid reparation mediates.
The second object of the present invention is to providing a kind of application of mentioned reagent box in detection DNA methylation transferase active.
The third object of the present invention is to provide a kind of join dependency circulation letter mediated based on single ribonucleotide reparation
The method of number amplification detection DNA methylation transferase active, this method can effective unrelated non-of suppression target and template/primer
Specific amplification thus greatly reduces background signal, and has high sensitivity and specificity.
The fourth object of the present invention is that the detection method of above-mentioned DNA methylation transferase active is applied to from DNA methyl
Dam MTase is distinguished in transferase (MTase), screens Dam MTase in Dam MTase inhibitor and quantitative Escherichia coli
Activity.
To achieve the goals above, the present invention adopts the following technical scheme:
The present invention provides a kind of cycle signal amplification detection DNA that join dependency is mediated based on monokaryon ribotide reparation
The active kit of methylated transferase, which is characterized in that the kit includes hair clip substrate, linking probe LP1, connection spy
Needle LP2, signal probe；
The hair clip substrate is the palindromic sequence that there is 5'-G-A-T-C-3' in stem, there is the identification position of Dam MTase in ring portion
Point；
The LP1 is linear DNA, the terminal modified hydroxyl group of 3', LP1 it is upper one and only one with uracil base mispairing
Adenine base；The LP2 is the DNA of hairpin structure, and two complementary strands are contained in stem, and in sense strand, 3' is end modified
Phosphate group is to prevent the generation of polymerization reaction, and in antisense strand, phosphate group has been modified also to carry out subsequent company in the end 5'
It is reversed to answer, there is one section of sequence identical with LP1 Sequence in ring portion；The probe LP1 and probe LP2 can be with hair clip bottom
Object ring portion partial complementarity；
The signal probe is that both ends are modified with the DNA of FAM fluorescent reporter group and BHQ1 fluorescent quenching group, and include
One uracil ribonucleotide.
Preferably, the hair clip substrate bases sequence is as shown in SEQ ID NO.1, specifically: 5'-ACT TAT CAG
CTT AAG ATC CAC GAC AAA AAA GCA AGC AGG ATC TTA AGC TGA TAA GT-3', wherein underscore A
For the catalytic site of the Dam MTase in the hair clip substrate；
Preferably, the base sequence of the probe LP1 is as shown in SEQ ID NO.2, specifically: 5'-CCT GCT CTT
CTC TTC ACT CTC GTC CTG CTT GCT-3',
The base sequence of the probe LP2 as shown in SEQ ID NO.3, specifically: 5'-P-TTT TTG TCG TGG
ACC TGC TCT CCA CGA CA-P-3', wherein P represents the phosphate group of modification,
The base sequence of the signal probe as shown in SEQ ID NO.4, specifically: 5'- (FAM)TCC TGC TCU
TCT CTT(BHQ1) -3', wherein FAM is fluorescent reporter group, and BHQ1 is fluorescent quenching group, and italic U is uracil ribose core
Thuja acid.
It further include endonuclease DpnI, S adenosylmethionine (SAM), Dam MTase reaction in kit described above
Buffer, CutSmart buffer, dATPs, dUTPs, dGTPs, dCTPs, Taq DNA ligase, Bst archaeal dna polymerase,
RNase HII, Taq DNA ligase reaction buffer and ThermoPol reaction buffer.
The present invention also provides the application of kit described above in detection Dam MTase activity.
The present invention also provides a kind of cycle signal amplification detections that join dependency is mediated based on monokaryon ribotide reparation
The active method of Dam MTase, which comprises the following steps:
(1) the hair clip substrate that Dam MTase is acted on is added in excision reaction buffer solution and carries out cleavage reaction, will produced
The product of raw 24-nt；
(2) above-mentioned cleaved products are added in amplified reaction buffer, be connected by circulation that rely on strand displacement amplification anti-
It answers, circulation generates the fluorescence signal of enhancing, passes through the activity of the strong and weak measurement Dam MTase of detection fluorescence signal.
Preferably, in step (1), there be returning for 5'-G-A-T-C-3' in the hair clip substrate of the Dam MTase effect, stem
Literary sequence has the recognition site of a Dam MTase in ring portion；Preferably, the hair clip substrate bases sequence such as SEQ ID
Shown in NO.1, specifically: 5'-ACT TAT CAG CTT AAGATC CAC GAC AAA AAA GCA AGC AGG ATC
TTA AGC TGA TAA GT-3', wherein A is the catalytic site of Dam MTase in the hair clip substrate.
Preferably, in step (1), the excision reaction buffer includes DNA methylation transferase to be measured, endonuclease
Enzyme DpnI, S adenosylmethionine (SAM), Dam MTase reaction buffer and CutSmart buffer.
Preferably, in step (1), cut off reaction condition be 37 DEG C of 2~2.5h of incubations, then 80 DEG C inactivate 20~
25min；Preferably, assigning the time is 2h, inactivates 20min.
Preferably, in step (2), the amplified reaction buffer includes linking probe LP1, linking probe LP2, signal spy
Needle, dATPs, dUTPs, dGTPs, dCTPs, Taq DNA ligase, Bst archaeal dna polymerase, RNase HII, Taq DNA connection
Enzyme reaction buffer solution and ThermoPol reaction buffer；
The LP1 is linear DNA, and there is hydroxyl group to modify at the end 3', LP1 it is upper one and only one with uracil base mistake
The adenine base matched；The LP2 is the DNA of hairpin structure, and two complementary strands are contained in stem, and in sense strand, 3' is repaired end
Phosphate group is adornd to prevent the generation of polymerization reaction, in antisense strand, it is subsequent to carry out that phosphate group has also been modified in the end 5'
Connection reaction, have one section of sequence identical with LP1 Sequence in ring portion；
The probe LP1 and probe LP2 can be with hair clip substrate ring portion partial complementarity；
Preferably, the base sequence of the probe LP1 is as shown in SEQ ID NO.2, specifically: 5'-CCT GCT CTT
CTC TTC ACT CTC GTC CTG CTT GCT-3',
The base sequence of the probe LP2 as shown in SEQ ID NO.3, specifically: 5'-P-TTT TTG TCG TGG
ACC TGC TCT CCA CGA CA-P-3', wherein P represents the phosphate group of modification,
The base sequence of the signal probe as shown in SEQ ID NO.4, specifically: 5'- (FAM)TCC TGC TCU
TCT CTT(BHQ1) -3', wherein FAM is fluorescent reporter group, and BHQ1 is fluorescent quenching group, and italic U is uracil ribose core
Thuja acid.
Preferably, in step (2), being connected by circulation and relying on the condition of strand displacement amplification reaction is 45 DEG C of 60~65min of incubation；
Preferably, incubation time 60min.
The present invention also provides detection methods described above to distinguish Dam MTase's in Dam MTase family member
Using the application in screening Dam MTase inhibitor and the application in quantitative Escherichia coli in the activity of Dam MTase.
The principle of the present invention are as follows:
The technical program is a kind of cycle signal amplification detection DNA that join dependency is mediated based on monokaryon ribotide reparation
The active new fluorescent method of methylated transferase.The present invention devises the hair clip substrate comprising stem and ring, stem's design
There is the palindromic sequence of 5'-G-A-T-C-3', can be used as the recognition site of Dam MTase, ring portion can be mutual with the part LP1 and LP2
It mends, causes connection reaction to form the template (LP1-LP2) of strand displacement amplification (SDA).At the end 3' there is hydroxyl (OH) group to repair
The linear LP1 of decorations and the partial complementarity of hair clip substrate, in addition, containing the single adenine with uracil (U) base mispairing on LP1
(A) base, and the SDA that the single uridylate reparation that can star RNase HII catalysis mediates.With hairpin structure
LP2 is also made of stem and ring portion, and two complementary strands (sense strand and antisense strand) is contained in stem, and in cochain, 3' is end modified
Phosphate group (PO4) to prevent the generation of polymerization reaction, in lower chain, 5' has also modified PO in end4Group is subsequent to carry out
Connection reaction, in ring portion, devises one section of sequence identical with LP1 Sequence, can be with amplified production (i.e. report spy
Needle) it is complementary, cause single being connected by circulation for uridylate reparation mediation and relies on SDA.Contain uracil ribonucleotide
Signal probe is the nucleotide sequence comprising 15 bases for having modified fluorescence (FAM) and quencher molecule (BHQ1).Amplification
The signal probe and report probes complementary of generation form double-stranded DNA, cause RNase HII to uridylate on double-stranded DNA
Excision, so as to cause the circulation digestion of signal probe, the final fluorescence signal for generating enhancing.
In the presence of Dam MTase, the methyl in SAM (S adenosylmethionine) is transferred to 5'-GATC- by Dam MTase
On adenine residue in 3' palindromic sequence, hair clip substrate is set to methylate, then, the hair clip substrate of methylation is by endonuclease
DpnI cutting generates the product of 24-nt；Linking probe 1 (LP1) and linking probe 2 (LP2) hybridize with the cleaved products of previous step
Connection forms the template (LP1-LP2) of SDA, in archaeal dna polymerase and four kinds of nucleotide (i.e. dATP, dGTP, dCTP and dUTP)
In the presence of, 24-nt cleaved products will carry out polymerization extension as primer, generate the stabilization that uracil (U) ribonucleotide is added
DNA duplex (dsDNA), uracil ribonucleotide will generate 5' phosphoric acid (PO by the direct specific excision of RNase HII4)
End and the end 3' hydroxyl (OH), the site with the end 3'-OH will become the new replication site of archaeal dna polymerase starting SDA, most
A large amount of report probe is generated eventually, and the report probe of release can be used as primer and hybridize with LP1-LP2 template, starts new polymerization and prolong
It stretches to form the new DNA duplex (dsDNA) with uridylate, subsequent RNase HII catalysis uridylate
Excision, induction generate the product of 30-nt；The 30-nt product of release will hybridize with free LP1 and LP2, causes connection and is formed newly
SDA template (LP1-LP2), the SDA of join dependency that starting monokaryon ribotide reparation mediates newly recycles, and passes through circulation
Connection, polymerization, cutting and release, multiple SDA reactions will be activated and generate a large amount of report probes；With uracil (U) ribose
The signal probe of nucleotide can hybridize with the report probe of generation to be combined, and being formed has uracil ribonucleotide mispairing
DNA duplex (dsDNA), RNase HII by the uracil ribonucleotide on the specific excision DNA duplex (dsDNA),
Signal probe is caused to be broken, to discharge fluorescence signal and report probe, the report probe of release again can be with other signal
Probe combines, and starts the hybridization of circulation, cutting and release, eventually leads to the fluorescence signal significantly increased.
Beneficial effects of the present invention:
1, low background signal:
The strand displacement polymerization of the excision of monokaryon ribotide and specific join dependency based on RNase HII catalysis extends can
To effectively prevent non-specific amplification, background signal is thus greatly reduced.
2, high sensitivity
The high specific of monokaryon ribotide excision based on RNase HII catalysis, is connected by circulation the efficient of the SDA of dependence
Rate amplification and the excision of monokaryon ribotide and specific join dependency strand displacement polymerase extend the non-specific amplification suppression mediated
The low background signal made and generated finally realizes the index amplification of signal, therefore the technical program may be implemented to Dam
The active highly sensitive detection of MTase.Detection is limited to 4.8 × 10-6The dynamic range of U/mL and 5 order of magnitude.
3, specificity is good
Specific join dependency strand displacement polymerization extends, and can prevent the non-specificity when target Dam MTase is not present
Template and primer dependent/non-dependent can be effectively suppressed in the monokaryon ribotide excision of amplification, ribalgilase (RNase HII) catalysis
DNA polymerization cause non-specific amplification, to generate higher signal-to-noise ratio.Therefore, the methylation of same family is turned
Enzyme such as M.CviPI MTase and M.SssI MTase are moved, they can neither cause the cutting of hair clip substrate, can not start monokaryon
The join dependency circulation SDA amplification that ribotide reparation mediates, therefore the technical program can detect with high specificity
DamMTase activity.
4, easy to operate
In invention, the monokaryon ribotide excision reaction of connection reaction and RNase HII catalysis is all under homogeneity mode
It carries out, is not related to any thermal cycle, washing and separating step, enormously simplifies experimental arrangement.
5, the present invention can be used for the screening of Dam MTase inhibitor, and can be also used for complex sample (such as cell cracking
Liquid) in the active quantitative detection of Dam MTase.
Detailed description of the invention
Fig. 1 is the cycle signal amplification detection DNA MTase of the join dependency mediated based on monokaryon ribotide reparation
Schematic diagram.
Fig. 2 (A) is with native polyacrylamide gel electrophoresis to the hair clip that Dam MTase is guided under different experimental conditions
The analysis of substrate cleavage reaction product.Swimming lane M is DNA marker (DNA marker), swimming lane 1 be Dam MTase and Dpn I not
In the presence of reaction product, swimming lane 2 is reaction product in the absence of Dpn I, and swimming lane 3 is in the absence of Dam MTase
Reaction product, swimming lane 4 be Dam MTase and Dpn I all in the presence of reaction product.Wherein SYBR Gold is as fluorescence
Indicator；
(B) it is situated between under different experimental conditions to based on the reparation of monokaryon ribotide with native polyacrylamide gel electrophoresis
The analysis for the SDA reaction product for being connected by circulation dependence led.Swimming lane M is DNA marke, and swimming lane 1 is not present in Dam MTase
Under reaction product, swimming lane 2 is reaction product in the presence of Dam MTase, and swimming lane 3 is the LP1 of synthesis, and swimming lane 4 is synthesis
LP2, swimming lane 5 be synthesis hair clip substrate.Silver staining kit dyes gel as fluorescence indicator；
(C) there are Dam MTase (curve 1) and there is no fluorescence intensities when Dam MTase (control, curve 2).
Fluorescence spectrum variation diagram of the Fig. 3 (A) in various concentration Dam MTase；(B) in Dam MTase concentration from 1 × 10-4
To the variation diagram of the fluorescence intensity of 40U/mL；Illustration indicates the linear pass between fluorescence intensity and the logarithm of Dam MTase concentration
System's figure.Error bar indicates the standard deviation of independent experiment three times.
Fig. 4 corresponds to reaction buffer, M.CviPI, the variation of M.SssI and Dam MTase fluorescence intensity.Error bar
Indicate the standard deviation tested three times.
Fig. 5 is the screening experiment of gentamicin inhibitor.
Fig. 6 is the active detection of Dam MTase in Escherichia coli.
Specific embodiment
It is noted that described further below be all exemplary, it is intended to provide further instruction to the application.Unless another
It indicates, all technical and scientific terms used herein has usual with the application person of an ordinary skill in the technical field
The identical meanings of understanding.
It should be noted that term used herein above is merely to describe specific embodiment, and be not intended to restricted root
According to the illustrative embodiments of the application.As used herein, unless the context clearly indicates otherwise, otherwise singular
Also it is intended to include plural form, additionally, it should be understood that, when in the present specification using term "comprising" and/or " packet
Include " when, indicate existing characteristics, step, operation, device, component and/or their combination.
As background technique is introduced, the sensitivity of DNA methylation transferase active method is detected also in the prior art
It is to be improved, and it is related to cumbersome operation sequence, but also high background signal can be generated, in order to which the technology for solving as above is asked
Topic, present applicant proposes a kind of cycle signal amplification detection DNA methyl that join dependency is mediated based on monokaryon ribotide reparation
Change the kit and method of transferase active.
A kind of exemplary embodiment of the application provides and a kind of mediates join dependency based on monokaryon ribotide reparation
The kit of cycle signal amplification detection DNA methylation transferase active, which is characterized in that the kit includes hair clip bottom
Object, linking probe LP1, linking probe LP2, signal probe；
The hair clip substrate is the palindromic sequence that there is 5'-G-A-T-C-3' in stem, there is the knowledge of a Dam MTase in ring portion
Other site；
The LP1 is linear DNA, the terminal modified hydroxyl group of 3', LP1 it is upper one and only one with uracil base mispairing
Adenine base；The LP2 is the DNA of hairpin structure, and two complementary strands are contained in stem, and in sense strand, 3' is end modified
Phosphate group is to prevent the generation of polymerization reaction, and in antisense strand, phosphate group has been modified also to carry out subsequent company in the end 5'
It is reversed to answer, there is one section of sequence identical with LP1 Sequence in ring portion；The probe LP1 and probe LP2 can be with hair clip bottom
Object ring portion partial complementarity；
The signal probe is that both ends are modified with the DNA of FAM fluorescent reporter group and BHQ1 fluorescent quenching group, and include
One uracil ribonucleotide.
Preferably, the hair clip substrate bases sequence is as shown in SEQ ID NO.1, specifically: 5'-ACT TAT CAG
CTT AAG ATC CAC GAC AAA AAA GCA AGC AGG ATC TTA AGC TGA TAA GT-3', wherein underscore A
For the catalytic site of the Dam MTase in the hair clip substrate；
Preferably, the base sequence of the probe LP1 is as shown in SEQ ID NO.2, specifically: 5'-CCT GCT CTT
CTC TTC ACT CTC GTC CTG CTT GCT-3',
The base sequence of the probe LP2 as shown in SEQ ID NO.3, specifically: 5'-P-TTT TTG TCG TGG
ACC TGC TCT CCA CGA CA-P-3', wherein P represents the phosphate group of modification,
The base sequence of the signal probe as shown in SEQ ID NO.4, specifically: 5'- (FAM)TCC TGC TCU
TCT CTT(BHQ1) -3', wherein FAM is fluorescent reporter group, and BHQ1 is fluorescent quenching group, and italic U is uracil ribose core
Thuja acid.
It further include endonuclease DpnI, S adenosylmethionine (SAM), Dam MTase reaction in kit described above
Buffer, CutSmart buffer, dATPs, dUTPs, dGTPs, dCTPs, Taq DNA ligase, Bst archaeal dna polymerase,
RNase HII, Taq DNA ligase reaction buffer and ThermoPol reaction buffer.
The another embodiment of the application provides kit described above in detection Dam MTase activity
Using.
The another embodiment of the application provides a kind of following based on monokaryon ribotide reparation mediation join dependency
The active method of ring signal amplification detection Dam MTase, which comprises the following steps:
(1) the hair clip substrate that Dam MTase is acted on is added in excision reaction buffer solution and carries out cleavage reaction, will produced
The product of raw 24-nt；
(2) above-mentioned cleaved products are added in amplified reaction buffer, be connected by circulation that rely on strand displacement amplification anti-
It answers, circulation generates the fluorescence signal of enhancing, passes through the activity of the strong and weak measurement Dam MTase of detection fluorescence signal.
Preferably, in step (1), there be returning for 5'-G-A-T-C-3' in the hair clip substrate of the Dam MTase effect, stem
Literary sequence has the recognition site of a Dam MTase in ring portion；Preferably, the hair clip substrate bases sequence such as SEQ ID
Shown in NO.1, specifically: 5'-ACT TAT CAG CTT AAGATC CAC GAC AAA AAA GCA AGC AGG ATC
TTA AGC TGA TAA GT-3', wherein A is the catalytic site of the Dam MTase in the hair clip substrate.
Preferably, in step (1), the excision reaction buffer includes DNA methylation transferase to be measured, endonuclease
Enzyme DpnI, S adenosylmethionine (SAM), Dam MTase reaction buffer and CutSmart buffer.
Preferably, in step (1), cut off reaction condition be 37 DEG C of 2~2.5h of incubations, then 80 DEG C inactivate 20~
25min；Preferably, assigning the time is 2h, inactivates 20min.
Preferably, in step (2), the amplified reaction buffer includes linking probe LP1, linking probe LP2, signal spy
Needle, dATPs, dUTPs, dGTPs, dCTPs, Taq DNA ligase, Bst archaeal dna polymerase, RNase HII, Taq DNA connection
Enzyme reaction buffer solution and ThermoPol reaction buffer；
The LP1 is linear DNA, and there is hydroxyl group to modify at the end 3', LP1 it is upper one and only one with uracil base mistake
The adenine base matched；The LP2 is the DNA of hairpin structure, and two complementary strands are contained in stem, and in sense strand, 3' is repaired end
Phosphate group is adornd to prevent the generation of polymerization reaction, in antisense strand, it is subsequent to carry out that phosphate group has also been modified in the end 5'
Connection reaction, have one section of sequence identical with LP1 Sequence in ring portion；
The probe LP1 and probe LP2 can be with hair clip substrate ring portion partial complementarity；
Preferably, the base sequence of the probe LP1 is as shown in SEQ ID NO.2, specifically: 5'-CCT GCT CTT
CTC TTC ACT CTC GTC CTG CTT GCT-3',
The base sequence of the probe LP2 as shown in SEQ ID NO.3, specifically: 5'-P-TTT TTG TCG TGG
ACC TGC TCT CCA CGA CA-P-3', wherein P represents the phosphate group of modification,
The base sequence of the signal probe as shown in SEQ ID NO.4, specifically: 5'- (FAM)TCC TGC TCU
TCT CTT(BHQ1) -3', wherein FAM is fluorescent reporter group, and BHQ1 is fluorescent quenching group, and italic U is uracil ribose core
Thuja acid.
Preferably, in step (2), the condition for recycling strand displacement amplification reaction is 45 DEG C of 60~65min of incubation；Preferably,
Incubation time is 60min.
The another embodiment of the application provides detection method described above in Dam MTase family member
The application for distinguishing Dam MTase, the application in screening Dam MTase inhibitor and the Dam MTase in quantitative Escherichia coli
Activity in application.
It is described further below with reference to embodiment.
Embodiment 1
Dependence signal amplification: DNA methylation transferase (MTase) is connected by circulation based on what monokaryon ribotide reparation mediated
Detection be related to two continuous reaction steps.Firstly, with 1 × Tris-EDTA buffer (10 mMs every liter of trihydroxy methyl
Aminomethane (Tris), 1 mM every liter of ethylenediamine tetra-acetic acid (EDTA), pH 8.0) all oligonucleotide sequences of dilution with
Prepare stock solution.Then, by hair clip substrate and linking probe 2 (LP2) buffer (1.5 mMs every liter of magnesium chloride, 10
MM every liter three (methylol) aminomethane-hydrochloric acid (Tris-HCl), pH 8.0) in be diluted to 10 every liter of micromoles, and
It is incubated for 5 minutes, is then slowly cooled to room temperature to form hairpin structure under 95 degrees Celsius.Then, by 1 microlitre of hair clip substrate
(10 every liter of micromole) is added to 20 microlitres and includes Dam MTase, the SAM of 160 every liter of micromoles, and the Dpn I of 6 units, 2 is micro-
It is in the reaction system of the 10 × Dam MTase reaction buffer and 2 microlitres of 10 × CutSmart buffer that rise and Celsius 37
Degree is lower to be incubated for 2 hours, then inactivates 20 minutes under 80 degrees Celsius.Then 5 microlitres of above-mentioned reaction products are added to 20 microlitres of packets
Linking probe 1 (LP1) containing 500 every liter of nanomoles, the linking probe 2 (LP2) of 500 every liter of nanomoles, 500 every liter of nanomoles
DATPs, the dUTPs of 500 every liter of nanomoles, the dGTPs of 500 every liter of nanomoles, the dCTPs of 500 every liter of nanomoles, 900 receive and rub
Your every liter of signal probe, the Taq DNA ligase of 15 units, the Bst archaeal dna polymerase of 1 unit, 2 units
RNase HII, 2 microlitres of 10 × Taq DNA ligase reaction buffer and 2 microlitres of 10 × ThermoPol reaction buffer
Reaction system in, then be incubated for 60 minutes under 45 degrees Celsius.
Steady-state fluorescence measurement and gel electrophoresis analysis: 20 microlitres of reaction products are diluted to 60 microlitres with ultrapure water, then
Fluorescence spectrum is measured under the excitation wavelength of 492nm with Hitachi F-7000 sepectrophotofluorometer, and is by launch wavelength
Fluorescence intensity at 518nm is analyzed for data.In order to which that analyzes that cleavage reaction product and monokaryon ribotide reparation mediate follows
The SDA reaction product of ring join dependency.(1) under room temperature 110V constant voltage, in 1 × tbe buffer liquid (9 every liter of nanomoles
It is solidifying to carry out 12% non-denaturing polyacrylamide in 0.2 mM of every liter of EDTA, pH 7.9 for Tris-HCl, 9 mMs of every liter of boric acid
Gel electrophoresis (PAGE) analysis, using SYBR Gold as fluorescence indicator, electrophoresis time is 50 minutes.(2) in 110V, 1 × TBE
It is carried out in buffer (9 mMs of every liter of Tris-HCl, 9 mMs of every liter of boric acid, 0.2 mM of every liter of EDTA, pH 7.9)
12% native polyacrylamide gel electrophoresis (PAGE) analysis, after electrophoresis 45 minutes, gel is dyed with silver staining kit, is used in combination
ChemiDoc MP imaging system (Hercules, California, U.S.A) analysis.
Experimental principle (such as Fig. 1):
This programme relates generally to three continuous reaction steps: (1) cutting of the hair clip substrate of Dam MTase guidance, (2)
Based on the strand displacement amplification reaction (SDA) for being connected by circulation dependence that monokaryon ribotide reparation mediates, (3) ribonuclease H II
The circulation of (RNase HII) catalysis signal probe is cut to generate the fluorescence signal of enhancing.In the first step, Dam MTase will
Methyl in SAM (S adenosylmethionine) is transferred on the adenine residue in 5'-GATC-3' palindromic sequence, makes hair clip substrate
Methylation.Then, the hair clip substrate of methylation is cut the product for generating 24-nt by endonuclease DpnI.In second step, connection
Probe 1 (LP1) and linking probe 2 (LP2) hybridize the template (LP1-LP2) that connection forms SDA with the cleaved products of previous step.?
In the presence of archaeal dna polymerase and four kinds of nucleotide (i.e. dATP, dGTP, dCTP and dUTP), 24-nt cleaved products will be used as primer
Polymerization extension is carried out, the stabilized DNA duplex (dsDNA) that uracil (U) ribonucleotide is added is generated.Uridine diphosphate riboside
Acid will generate 5' phosphoric acid (PO by the direct specific excision of RNase HII4) end and the end 3' hydroxyl (OH).With the end 3'-OH
The site at end will become the new replication site of archaeal dna polymerase starting SDA, final to generate a large amount of report probe.The report of release
Probe can be used as primer and hybridize with LP1-LP2 template, starts new polymerization and extends to form the new DNA with uridylate
Duplex (dsDNA).The excision of subsequent RNase HII catalysis uridylate, induction generate the product of 30-nt.Release
30-nt product will hybridize with free LP1 and LP2, cause the template (LP1-LP2) that connection forms new SDA, starting monokaryon sugar
The SDA for the join dependency that nucleotide reparation mediates newly is recycled.By the connection of circulation, polymerization, cutting and release, multiple SDA are anti-
It should will be activated and generate a large amount of report probes.In the third step, the signal probe with uracil (U) ribonucleotide can be with
It is combined with the hybridization of the report probe of generation, forms the DNA duplex (dsDNA) with uracil ribonucleotide mispairing.RNase
Uracil ribonucleotide on the specific excision DNA duplex (dsDNA) is caused signal probe to be broken, to release by HII
Put fluorescence signal and report probe.The report probe of release can start the hybridization of circulation in conjunction with other signal probe again,
Cutting and release, eventually lead to the fluorescence signal significantly increased.
Embodiment 2
(1) experimental verification of principle:
In order to verify whether Dam MTase can cut hair clip substrate in the presence of Dpn I, we use non denatured poly- third
Acrylamide gel electrophoresis simultaneously analyzes reaction product (Fig. 2A) using SYBR Gold as fluorescence indicator.In Dam MTase and
Dpn I all in the absence of (Fig. 2A, swimming lane 1), only Dam MTase (Fig. 2A, swimming lane 2) and only Dpn I (Fig. 2A swimming lane 3) are deposited
When, the band of only one 56-nt is observed, and (56nt) identical as the hair clip substrate size of synthesis shows do not have
Methylation and cleavage reaction occur.But in the presence of Dam MTase and Dpn I, observe the new band of a 24-nt
(Fig. 2A, swimming lane 4) shows that Dam MTase can trigger the methylation of hair clip substrate, the hair clip substrate then to methylate can again quilt
Dpn I is cut at the adenine site of methylation.
In order to verify the SDA reaction for being connected by circulation dependence mediated based on monokaryon ribotide reparation, we are with non denatured
Polyacrylamide gel electrophoresis simultaneously analyzes reaction product (Fig. 2 B) using silver staining kit as indicator.Dam MTase not
In the presence of (Fig. 2 B, swimming lane 1), only observe original LP1 (29nt, Fig. 2 B, swimming lane 4), LP2 (33nt, Fig. 2 B, swimming lane
3) with the band of hair clip substrate (56nt, Fig. 2 B, swimming lane 5), show there is no amplified reaction.In Dam MTase, there are situations
Under, it observes the characteristic bands (Fig. 2 B, swimming lane 2) of 30,24 and 15nt, corresponds respectively to the cleaved products of 30-nt, 24-nt
Cleaved products and 15-nt report probe, this shows that Dam MTase can cut hair clip substrate and be released in the presence of Dpn I
The cleaved products for putting 24-nt cause being connected by circulation for monokaryon ribotide reparation mediation consequently as primer and rely on SDA to produce
The cleaved products of raw 30-nt and the report probe (Fig. 2 B, swimming lane 2) of 15-nt.
Then we further monitor fluorescence signal (Fig. 2 C) at different conditions.It is right in the absence of Dam MTase
According to significant fluorescence signal (Fig. 2 C, curve 2) is not detected in group, show there is no amplified reaction in no Dam MTase
Occur.In the presence of Dam MTase, the fluorescence signal (Fig. 2 C, curve 1) significantly increased is observed, show that Dam MTase can be with
The signal amplification that induction monokaryon ribotide reparation mediated be connected by circulation dependence is to generate the glimmering of a large amount of report probes and enhancing
Optical signal.Gel electrophoresis (Fig. 2A and B) and fluorescence experiments (Fig. 2 C) are aware that hair clip substrate can be instructed by demonstrating Dam MTase
Cutting with start based on monokaryon ribotide reparation mediate the SDA for being connected by circulation dependence.It is worth noting that, being not present
DamMTase (Fig. 2 B, swimming lane 1) and there are DamMTase (Fig. 2 B, swimming lane 2) in the case where other miscellaneous bands are all not observed,
Show the high specific for being connected by circulation dependence SDA reaction that monokaryon ribotide reparation mediates, causes without non-specificity DNA piece
Section generates.
(2) sensitivity experiment:
In order to study the detection sensitivity of the technical program, it is dense that we determine difference Dam MTase at optimum conditions
The fluorescence intensity of degree.As shown in figure 3, as Dam MTase concentration is from 1 × 10-410U/mL is increased to, fluorescence intensity is with continuous
Increase, and reaches plateau in 40U/mL concentration.It is worth noting that, the logarithm of fluorescence intensity and Dam MTase concentration
Value is 1 × 10-4Good linear relationship (Fig. 3 B illustration) is shown within the scope of to 10U/mL.Corresponding equation is F=
201.7log10C+1205.1, related coefficient 0.9813, wherein F is fluorescence intensity level, and C is the concentration of Dam MTase.Detection
Limit is ultimately determined to 4.8 × 10-6U/mL。
(3) specificity experiments:
In order to study the specificity of the technical program, we use M.CviPI MTase and M.SssI MTase as yin
Property control.As shown in figure 4, under the same conditions, only can detecte high fluorescence signal in the presence of Dam MTase.With
By contrast, in the presence of M.CviPI MTase or M.SssI MTase, be observed without apparent fluorescence signal,
It is consistent with the control group for only including reaction buffer.The above result shows that this method can be very good to distinguish Dam MTase
With other DNA MTase family members, show that the technical program has specificity well.
(4) inhibitor screening is tested:
It can be used in the screening of inhibitor to study the technical program, gentamicin is used as the inhibition of Dam MTase
Agent.As shown in figure 5, the concentration with gentamicin increases to 50 every liter of micromoles from 0, the relative activity of Dam MTase with
Gradually decrease.Obtained 503nhibiting concentration is 12.21 every liter of micromoles, and is reported as obtained by fluorescence detection in the past
Numerical value be consistent.The above result shows that the technical solution can be used for the screening of Dam MTase inhibitor.
(5) Dam MTase Activity determination is tested in Escherichia coli:
It can be used for the active quantitative detection of Dam MTase, Escherichia coli in Escherichia coli to study the technical program
Cell GW5100 and JM110 are, respectively, used as the Dam MTase positive and negative sample.As shown in fig. 6, under the same conditions,
In GW5100 sample, high fluorescence signal can detecte.And in JM110 sample, it is observed without apparent fluorescence signal
It arrives, and it is consistent with the control group for only including lysis buffer.The above result shows that this method can be used for complex sample
The active quantitative detection of Dam MTase in (such as cell pyrolysis liquid).
Above-mentioned, although the foregoing specific embodiments of the present invention is described with reference to the accompanying drawings, not to invention protection scope
Limitation, those skilled in the art should understand that, based on the technical solutions of the present invention, those skilled in the art are not required to
It is still within the scope of the present invention to make the creative labor the various modifications or changes that can be made.
Claims (10)
1. a kind of mediate the cycle signal amplification detection DNA methylation of join dependency to shift enzyme activity based on monokaryon ribotide reparation
Property kit, which is characterized in that the kit include hair clip substrate, linking probe LP1, linking probe LP2, signal visit
Needle；
The hair clip substrate is the palindromic sequence that there is 5'-G-A-T-C-3' in stem, there is the identification position of a Dam MTase in ring portion
Point；
The LP1 is linear DNA, and the terminal modified hydroxyl group of 3', LP1 is upper, and one and only one is fast with the gland of uracil base mispairing
Purine base；The LP2 is the DNA of hairpin structure, and stem contains two complementary strands, in sense strand, the end modified phosphoric acid of 3'
Group is to prevent the generation of polymerization reaction, and in antisense strand, it is anti-to carry out subsequent connection that phosphate group has also been modified in the end 5'
It answers, there is one section of sequence identical with LP1 Sequence in ring portion；The probe LP1 and probe LP2 can be with hair clip substrate ring
Portion's partial complementarity；
The signal probe is modified with the DNA of FAM fluorescent reporter group and BHQ1 fluorescent quenching group for both ends, and includes one
Uracil ribonucleotide.
2. kit according to claim 1, which is characterized in that the hair clip substrate bases sequence such as SEQ ID NO.1
It is shown；The base sequence of the probe LP1 is as shown in SEQ ID NO.2, the base sequence of the probe LP2 such as SEQ ID
Shown in NO.3, the base sequence of the signal probe is as shown in SEQ ID NO.4.
3. kit according to claim 1, which is characterized in that further include endonuclease DpnI, S in the kit
Adenosylmethionine (SAM), Dam MTase reaction buffer, CutSmart buffer, dATPs, dUTPs, dGTPs, dCTPs,
Taq DNA ligase, Bst archaeal dna polymerase, RNase HII, Taq DNA ligase reaction buffer and ThermoPol reaction
Buffer.
4. application of any kit of claims 1 to 3 in detection Dam MTase activity.
5. a kind of active side of cycle signal amplification detection Dam MTase for mediating join dependency based on monokaryon ribotide reparation
Method, which comprises the following steps:
(1) it will be added in hair substrate that Dam MTase is acted in excision reaction buffer solution and carry out cleavage reaction, by hair clip bottom
The cutting of object ring portion generates the product of 24-nt；
(2) above-mentioned cleaved products are added in amplified reaction buffer, carry out circulation strand displacement amplification reaction, circulation generates increasing
Strong fluorescence signal passes through the activity of the strong and weak measurement Dam MTase of detection fluorescence signal.
6. according to the method described in claim 5, it is characterized in that, in step (1), the hair clip bottom of the Dam MTase effect
There are the palindromic sequence of 5'-G-A-T-C-3' in object, stem, there is the recognition site of a restriction endonuclease in ring portion；Preferably, the hair clip
Substrate bases sequence is as shown in SEQ ID NO.1.
7. according to the method described in claim 5, it is characterized in that, in step (1), the excision reaction buffer include to
Survey DNA methylation transferase, endonuclease DpnI, S adenosylmethionine (SAM), Dam MTase reaction buffer and
CutSmart buffer.
8. according to the method described in claim 5, it is characterized in that, the condition for cutting off reaction is 37 DEG C and is incubated for 2 in step (1)
~2.5h, then 80 DEG C of 20~25min of inactivation；Preferably, assigning the time is 2h, inactivates 20min.
9. according to the method described in claim 5, it is characterized in that, the amplified reaction buffer includes connection in step (2)
Probe LP1, linking probe LP2, signal probe, dATPs, dUTPs, dGTPs, dCTPs, Taq DNA ligase, Bst DNA are poly-
Synthase, RNase HII, Taq DNA ligase reaction buffer and ThermoPol reaction buffer；
The LP1 is linear DNA, and the end 3' has a hydroxyl group modification, LP1 it is upper one and only one with uracil base mispairing
Adenine base；The LP2 is the DNA of hairpin structure, and two complementary strands are contained in stem, and in sense strand, 3' is end modified
Phosphate group is to prevent the generation of polymerization reaction, and in antisense strand, phosphate group has been modified also to carry out subsequent company in the end 5'
It is reversed to answer, there is one section of sequence identical with LP1 Sequence in ring portion；The probe LP1 and probe LP2 can be with hair clip bottom
Object ring portion partial complementarity；
Preferably, the base sequence of the probe LP1 is as shown in SEQ ID NO.2, the base sequence such as SEQ of the probe LP2
Shown in ID NO.3, the base sequence of the signal probe is as shown in SEQ ID NO.4；
Preferably, in step (2), the condition for recycling strand displacement amplification reaction is 45 DEG C of 60~65min of incubation.
10. any detection method of claim 5~9 distinguishes answering for Dam MTase in Dam MTase family member
With；Application of any detection method of claim 5~9 in screening Dam MTase inhibitor；Claim 5~9 times
Application of the detection method described in one in quantitative Escherichia coli in the activity of Dam MTase.
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