CN103114139A - Multi-specific site DNA methylation electrochemical detection method based on ligase reaction - Google Patents
Multi-specific site DNA methylation electrochemical detection method based on ligase reaction Download PDFInfo
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Abstract
The invention discloses a multi-specific site DNA methylation electrochemical detection method based on a ligase reaction. According to the method, the direct electrochemical response of the specific immunoreaction and antibody-labeled enzyme on the titanium dioxide sol-gel film is utilized so that a novel reagent-free immunoassay method is developed, and an immunosensor is prepared. The antigenic molecules are fixed by utilizing a titanium dioxide sol-gel vapor deposition method, the competitive immunoassay method is utilized, and the concentration of the antigen to be tested is directly obtained by directly measuring the reduction of a direct electrochemical response signal which is reacted to the antibody-labeled enzyme on the surface of the immunosensor. According to the method, complicated gene sequencing steps are not required, the analysis system is simplified, the measurement cost is reduced, the method has high accuracy, repeatability and stability, and can be developed into a kit and contributes to market popularization.
Description
Technical field
The present invention relates to a kind of DNA methylation determination and analysis method, be specifically related to a kind of many specific sites DNA methylation electrochemical detection method based on the ligase enzyme reaction.
Background technology
DNA methylation is a kind of important gene epigenetic modification mode that extensively is present in higher organism, is also a kind of important expression and regulation mechanism of outer genetic system.DNA methylates and mainly occurs in the C of 5 '-CpG-3 ', generates 5-methylcytosine (5-mC).Studies show that in a large number, DNA methylation is in the situation that do not change the gene base sequence, by changing the methylation on CpG island, realizes regulate gene expression on transcriptional level, eliminate the potentially dangerous DNA sequence dna, regulate the functions such as growth course and various physiological responses.On the contrary, the abnormal genomic methylations such as DNA hypomethylation and CpG island hyper-methylation can cause not only that DNA replication dna postpones, the karyomit(e) compression, transcribe inhibition etc., and can affect susceptibility, genesis, the prognosis of disease and lapse to etc.In recent years find, the generation of the chronic disease that the genomic dna abnormal methylation is relevant to various diseases especially age is closely related.Because DNA methylation plays an important role in keeping normal cell function, genetic imprinting, fetal development and human tumor generation, set up accuracy DNA methylation detection method high, simple to operate, the early stage diagnosis and treatment of disease and community-based intervention etc. are had very important theory significance and practical value.
The detection of DNA methylation research can be divided into three levels: the whole methylation level of (1) genome detects, and (2) specific site methylation level detects, (3) newly the methylate searching in site.The detection method of conventional DNA methylation mainly relies on the susceptibility restriction endonuclease that methylates and at recognition site separately, the 5-mC susceptibility different with C is distinguished, or bisulfite changes the different information of sequence into to the information of methylating that methylates with the different DNA of making of chemically reactive of non-methylated cytosine comprise.method mainly comprises and methylating in conjunction with the albumen column chromatography, the MSssI methyltransgerase is analyzed and the monochloroacetaldehyde reaction method, methylation status of PTEN promoter (MSP), the analysis of the susceptibility that methylates single stranded conformational, heavy sodium bisulfite associating restriction endonuclease analysis (COBRA), the susceptibility that methylates spot analysis, the susceptibility that methylates denaturing gradient gel electrophoresis, responsive mononucleotide primer extension method (MS-SNuPE) methylates, PCR fluorescence sex change tracing analysis, denaturing high-performance liquid chromatography (DHPLC), fluoroscopic examination (MethyLight) methylates, the multi-link dependency probe amplification of methylation-specific (MS-MLPA), DNA methylation micro-array chip technology etc.Although methylation detecting method is greatly developed, the whole bag of tricks exists significantly restricted.The bisulfite method requires a large amount of cloning and sequencings, and process is loaded down with trivial details, expensive, processes simultaneously easily to cause false positive when incomplete; It is slightly complicated that the susceptibility that methylates mononucleotide primer deriving technology exists experimental procedure, if in the time of will detecting a plurality of site, needs a plurality of primers of design and exist radiocontamination and bisulfite to process incomplete problem; The susceptibility that methylates chain solution curve analytical technology etc. exists the too low shortcoming of sensitivity; The pcr amplification that relies on gene sequencing or multistep makes analytical procedure complicated, detects and takes length, and cost of determination is high; The use restriction enzyme can only obtain the methylation level of special restriction enzyme site, can not determine the situation that methylates in other site in sample DNA.Therefore, need to seek more simply, method fast.
Electrochemical method has fast, and sensitivity is not affected by solution turbidity, and instrument is simple, easy to operate, and use cost is low, need not complicated pretreatment process, is easy to microminiaturized and realizes the characteristics such as Site Detection, has unique advantage in the DNA methylation context of detection.In recent years, the electrochemical detection method of DNA methylation has been obtained certain breakthrough, but from bibliographical information, the Electrochemical Detection of DNA methylation mainly concentrates on the detection of the whole methylation level of DNA, and the electrochemical method that methylation level detection and the novel site of specific site are sought rarely has report.
Summary of the invention
The object of the present invention is to provide a kind of many specific sites DNA methylation electrochemical detection method based on the ligase enzyme reaction.
The technical solution used in the present invention is:
A kind of polyspecific site DNA methylation detection method comprises the steps:
(1) according to one group of nucleic acid probe of flanking sequence design in each specific methylation site on target dna sequence, every group of nucleic acid probe is comprised of a separate probe and a bars probe, separate probe is modified with solid phase carrier, signal probe is modified with quantum dot, detects the quantum dot of modifying on the signal probe that uses in the different sites that methylate different;
(2) the target dna sample is processed with bisulfite, makes not methylated cytosine(Cyt) be converted into uridylic;
(3) separate probe and signal probe are added in target dna solution after step (2) is processed and hybridize;
(4) collect solid phase carrier, add the DNA ligase reaction solution to carry out ligation after cleaning, the reaction of unwinding after reaction finishes after separating end of chain (EOC), separates solid phase carrier while hot with reaction solution;
(5) collect solid phase carrier, utilize the electrochemical signals of the quantum dot that connects on the Voltammetry solid phase carrier after cleaning;
(6) determine methylated site and methylation according to ownership and the intensity of electrochemical signals.
The length of described separate probe is 35～45 bp, and the length of signal probe is 10～15 bp, to guarantee the selectivity of hybridization.
Described solid phase carrier is that magnetic microsphere, tetrafluoroethylene microballoon, gold plaque or finishing have carboxyl or amino micro materials.
The preparation method of separate probe is as follows: after solid phase carrier is used respectively 100 mM imidazole buffer solution and EDC/NHS solution-treated, add nucleic acid molecule, 35～45 ℃ of lower oscillatory reactions 1～3 hour, separate, use respectively 2 * SSC buffered soln (0.5% SDS) and 65 ℃ of high purity waters to clean, obtain separate probe.
The preparation method of signal probe is as follows: the quantum dot aqueous solution mixes with nucleic acid molecule, at N
2Protection is lower stirred 16～24 hours, dropwise added PBS(pH 7.4,0.24 M NaCl, 0.1% NaN
3) rear at PBS(pH 7.4,0.2 M NaCl) the middle dialysis 36～48 hours, the signal probe of centrifugal collection preparation.
The condition of the described hybridization of step (3) is: hybridized under 35～45 ℃ 1～3 hour.
Consisting of of step (4) described ligase enzyme reaction solution: 30 mM Tris-HCl, 4 mM MgCl
2, 10 mM (NH
4)
2SO
4, 0.005% BSA, 0.2 mM NAD and 0.5 U/L
EcoliDNA ligase.
The condition of ligation is: reacted under 35～40 ℃ 1～2 hour.
The condition of unwinding is: unwind under 85～95 ℃ 5～15 minutes.
The condition that step (5) is measured the Stripping Voltammetry curve is: glass-carbon electrode is in 300～500 μ g/L bismuth solution, and-0.9～-1.1 V deposits 10 minutes.
The principle of the inventive method is as follows:
As shown in Figure 1, according to methylate flanking sequence design one group of nucleic acid probe (P3 and P4) of site B of the flanking sequence of the site A that methylates on the target dna chain one group of nucleic acid probe of design (P1 and P2), P1 and P4 difference mark CdS and PbS quantum dot are as signal probe (PbS-P1, CdS-P4), probe P2 and P3 are fixed on solid phase carrier (magnetic bead) together upward as carrier of separating (MB-P2P3).
As shown in Figure 2, at first the testing sample solution that contains the target dna chain is processed through bisulfite, methylated C deaminizating does not occur in the target dna chain be converted into U, and 5-mC remains unchanged.This solution to be measured and MB-P2P3 hybridization and magnetic resolution, the target dna chain is hybridized to solid phase carrier (MB) surface by selectivity, separates with the DNA fragmentation that coexists in solution.Further with this MB and PbS-P1 and CdS-P4 hybridization, PbS quantum dots and CdS are connected to the MB surface.Add
E. coli.The DNA ligase enzyme, this DNA ligase is had an effect in interstitial site (arrow indication position in Fig. 1), the misidentify hapto.When on object chain, the site methylates, corresponding adjacent two probe chains and object chain complete complementary, ligase enzyme connects into a new nucleic acid chains with these two probe chains.Otherwise when the site did not methylate, corresponding adjacent two probe chains and object chain formed single base mismatch, and ligase enzyme can not be had an effect.The gained magnetic bead heats in buffered soln and unwinds, magnetic resolution, and the mark quantum dot on the site that methylates is connected with magnetic bead by the DNA chain, the separation but not the mark quantum dot on the site that methylates is unwind.Add 1 M nitric acid in magnetic bead after unwind, utilize the electrochemical signals of the quantum dot that connects on the Voltammetry magnetic bead.Determine methylated site and methylation according to ownership and the intensity of electrical signal.
The invention has the beneficial effects as follows:
The method is utilized the Direct Electrochemistry response that the enzyme of specific immune response and traget antibody shows on the TiO 2 sol gel-film, it is novel without the reagent immune analysis method and prepared immunosensor to have developed.Utilize TiO 2 sol gel vapour deposition process immobilized antigen molecule, and utilize the competitive immunoassay method, directly obtain the concentration of determined antigen by the reduction of direct assaying reaction Direct Electrochemistry response signal of the enzyme of traget antibody to the immunosensor surface.The more existing immune analysis method of the method has the following advantages:
(1) stripping voltammetry has enrichment and process in leaching, and is highly sensitive, is a kind of electrochemical method of good detection trace metal ion concentration;
(2) bismuth film electrode replaces traditional mercury electrode, and the toxicity of bismuth metal own is less, environmental friendliness, and have the chemical property that compares favourably with mercury electrode, as large in the electromotive force window, highly sensitive, detectability is low etc.;
(3) need not to carry out loaded down with trivial details gene sequencing step, simplified analysis system, reduce cost of determination;
(4) need not the pcr amplification step, simplified the operation steps of analyzing, shortened analysis time, be applicable to on-line quick detection clinically;
(5) need not to use restriction enzyme, to specific site methylate research have universality, shortened analysis time, avoided the shortcoming of restriction enzyme;
(6) according to spike potential and the peak current of electrochemical signals, realize that how special methylating measure when the site,
(7) the method shows good accuracy, and is repeated and stable, can develop into test kit and to marketing.
Description of drawings
Fig. 1 is the complementary pairing graph of a relation of nucleic acid probe and target dna;
Fig. 2 is the many specific sites DNA methylation electrochemical detection method schematic diagram based on the ligase enzyme reaction;
Fig. 3 is the complementary pairing graph of a relation of embodiment 1 nucleic acid probe and target dna;
Fig. 4 is that hybridization temperature and hybridization time are on the impact (A. hybridization temperature, B. hybridization time) of the electrochemical gaging that methylates;
Fig. 5 is melting temperature(Tm) and the impact (A. melting temperature(Tm), B. unwind time) of time on the electrochemical gaging that methylates of unwinding;
Fig. 6 is bismuth ion concentration and the electrode pretreatment time impact (A. sedimentation potential, B. depositing time, bismuth ion concentration in C. solution, D. electrode pretreatment time) on the electrochemical gaging that methylates in sedimentation potential, depositing time, solution;
Fig. 7 is the methylate typical curve in site of embodiment 1 target dna chain difference.
Embodiment
The below detects as example take methylating of p53 tumor suppressor gene fragment, and the present invention is further illustrated, but be not limited to this.
One, preparation nucleic acid probe
1, the processing of target gene fragment
Process p53 tumor suppressor gene fragment with bisulfite, step is as follows:
According to bisulfite conversion reagent box explanation, appropriate bisulfite mixture with the water of 800 μ L stoning ribonuclease T.s at 60 ℃ of dissolve completes.Add 15 μ L DNA sample solutions (1 ng-2 μ g) in 200 μ L PCR pipes, the water of 5 μ L stoning ribonuclease T.s, 85 μ L bisulfite mixture solutions, 35 μ L DNA protection buffered soln.Cover PCR pipe lid, fully mix the bisulfite reaction solution, preserve under room temperature.95 ℃ of sex change 5 min in the PCR instrument, 60 ℃ of incubation 25 min continue at 95 ℃ of sex change 5 min, 60 ℃ of incubation 85 min, then at 95 ℃ of sex change 5 min, at 60 ℃ of incubation 175 min, spend the night 20 ℃ of preservations.
Gene fragment before processing is:
5’-CC CAC CAT GAG
CG C TGC TCA GAT AGC GAT GGT CTG GCC CCT CCT CAG CAT CTT ATC
CG A GTG GAA ACA-3’（SEQ ID NO:1）。
Gene fragment after processing is:
5’-UU UAU UAT GAG
CG U TGU TUA GAT AGU GAT GGT UTG GUU UUT UUT UAG UAT UTT ATU
CG A GTG GAA AUA-3’ （SEQ ID NO:2）。
2, designing nucleic acid probe
According to the base sequence (SEQ ID NO:2) of target gene after bisulfite is modified, corresponding nucleic probe (see figure 3) is designed for the line of delimitation in the site to methylate.
Nucleic acid probe 1(P1) with first site 5 ' terminal sequence (5 '-UU UAU UAT GAG C-3 ') complementation that methylates; Nucleic acid probe 2(P2) with first sequence (5 '-GU TGU TUA GAT AGU GAT GGT the UTG-3 ') complementation of site 3 ' end to target gene sequence mid point that methylate; Nucleic acid probe 3(P3) with methylate sequence (5 '-GUU UUT UUT UAG UAT UTT ATU C-3 ') complementation in site of target gene sequence mid point to the second; Nucleic acid probe 4(P4) be second to methylate site 3 ' end to target gene 3 ' terminal sequence (5 '-GA GTG GAA AUA-3 ') complementation.
Wherein, P2 and P3 be in when design, except with DNA sequence dna complementation to be measured, the 5 ' end of P2 and the 3 ' end of P3 have 6-8 bp sequence complementary, form the neck of crotch structure, the centre has 6-8 bp inertia sequence to form the triangular space to reduce the tension force of hybridizing.
Simultaneously, consider the DNA ligase reaction, 5 ' the end phosphate of P1 and P4 is modified, and the 3 ' terminal hydroxy group of P2 and P3 is modified.Article four, nucleic acid probe sequence is as follows:
P1 5’-P-GCTCATGGTGGG-C
6-SH-3’（SEQ ID NO:3）
P2 5’-NH
2-C
6-ACA CAC CAT GCA TGA CAA CAA CAA CAA CAC GCT ATC TGA GCA GC-OH-3’（SEQ ID NO:4）
P3 5’-P-GAT AAG ATG CTG AGG ACA ACA CAC ACA ACA CAT GCA TGA CAC AC-C
6-NH
2-3’（SEQ ID NO:5）
P4 5’-SH-C
6-TGTTTCCACTCG-OH-3’（SEQ ID NO:6）
3, quantum dot-labeled signal probe P1 and P4
P1 modifies with PbS quantum dots, and step is: the quantum dot of 1 mg/ml (PbS) aqueous solution mixes with 8 OD/ml nucleic acid probe P1, at N
2Protection is lower stirred 24 hours.Dropwise add PBS(pH 7.4,0.24 M NaCl, 0.1% NaN
3) rear at PBS(pH 7.4,0.2 M NaCl) the middle dialysis 48 hours.The PbS-P1 of centrifugal collection preparation adds PBS(pH 7.4,0.24 M NaCl, 0.1% NaN
3) be placed under 4 ℃ stand-by.
P4 modifies with Quantum dots CdS, and preparation process is identical.
4, the common modified magnetic microballoon of nucleic acid probe P2 and P3
10 μ L magnetic microsphere solution clean twice with 100 mM imidazole buffer solution (pH 7.0), add 100 microlitre EDC/NHS solution reactions after 30 minutes, add 50 μ L 10 μ M P3 and P4 solution, 50 ℃ of lower oscillatory reactions 3 hours.After magnetic separates, clean 3 times with 2 * SSC buffered soln (0.5% SDS), 65 ℃ of high purity waters clean twice, are dissolved in PBS(pH 7.4,0.24 M NaCl, 0.1% NaN
3) in solution, be placed under 4 ℃ stand-by.
Described EDC/NHS solution is by 1:1(v/v by 1g/L EDC and 1g/L NHS) be mixed to get.
Two, condition determination optimization
1, hybridization temperature and hybridization time
In the DNA crossover process, hybridization temperature is during lower than 10 ~ 15 ° of C of molten chain temperature (Tm) of DNA, and complementary strand can form stable two strands, and mispairing is to reducing; If hybridization temperature is lower than 30 ° of C of Tm, between complementary strand, pairing reduces, and mispairing is to increasing, and hydrogen bonded weakens.General optimum temperuture theoretical calculation formula is T=25 ° of Tm – C.
In the DNA crossover process, hybridization temperature affects crossbreeding effect.Hybridization time is too short causes that in complementary strand, the mispairing situation increases, and the probe chain is the captured target chain well, makes signal false positive occur; The efficient of the long impact of hybridization time experiment and the real-time of detection method.
In the present embodiment, take the p53 tumor suppressor gene fragment of exhaustive methylation as research object, change respectively hybridization temperature and hybridization time, the analog value when selecting the maximum current response is as best hybridization conditions.
As shown in Fig. 4 A.Be respectively 10,25 at hybridization temperature, measure respectively the target p53 gene fragment of exhaustive methylation under 38 and 55 ° of C conditions.When hybridization temperature was 10 ° of C, because temperature is too low, the complementary base pairing reduced, and mispairing is to increasing, and a little less than the bonding force of hydrogen bond, so detection signal is less, is not suitable for the hybridization of DNA.Improve with hybridization temperature, detection signal obviously increases, and 38 ° of C reach maximum value.Continue rising hybridization temperature to 55 ° C, can cause melting chain, hybridization efficiency reduces, and signal descends.Therefore, select 38 ° of C as the final hybridization temperature of experiment.
Fig. 4 B has optimized the hybridization time under 38 ° of C.When hybridization time is 1 hour, detection signal is less.Extended hybridization time from 2 to 6 hours, detection signal increases and substantially reaches stable, show 2 h after hybridisation events tend towards stability.Therefore select 2 h to be the hybridization time after optimizing.
2, melting temperature(Tm) and unwinding the time
The time effects of the unwinding effect of unwinding.The time of unwinding is too short, unwinds not exclusively, causes false positive results; The overlong time of unwinding is in hot environment DNA and magnetic microsphere for a long time, causes the DNA splitting of chain to become single base, and perhaps magnetic microsphere sex change magnetic reduces, and loses a part of signal.
Fig. 5 A has studied the impact of melting temperature(Tm) on measuring take complete non-methylated target p53 gene fragment as object.The peak-to-peak signal that detects during 70 ° of C as seen from the figure is maximum, and in the time of can thinking 70 ° of C, temperature causes unwinding not exclusively not, can make detection false positive results occur.When melting temperature(Tm) is elevated to 90 ° of C from 70 ° of C, measured signal reduces rapidly.Temperature higher than 90 ° of C after, the measured signal value of tending towards stability, therefore selecting 90 ° of C is melting temperature(Tm).
Fig. 5 B is the different impacts of time on measuring of unwinding under 90 ° of C melting temperature(Tm)s.Extend to 25 min from 5 min with the time of unwinding, measured signal descends rapidly, and the value of tending towards stability, and thinks and unwinds fully.Therefore, select 20 min to be unwinding the time after optimizing.
3, the stripping voltammetry condition is selected
Fig. 6 A study different sedimentation potentials to the impact of mensuration for take the target p53 gene fragment of exhaustive methylation as object.When sedimentation potential changed from-0.85 V to negative direction, measured signal increased sharply, and was tending towards ultimate value be defeated by-1.0 V in sedimentation potential after.Therefore, selecting the galvanic deposit current potential of optimization is-1.0 V.
Fig. 6 B is the affect situation of different depositing times on measured signal under-1.0 V sedimentation potentials.Extend to 450 s with depositing time from 50 s, peak current and depositing time are linear.After 450 s, the signal of Pb ion slightly descends, and the signal of Cd ion continues to increase, and reaches stationary value to 600 s.Therefore, selecting 600 s is best depositing time.
Fig. 6 C has studied the impact of bismuth ion concentration on the Stripping Voltammetry response in solution.When bismuth ion concentration is 0 ~ 200 μ g L
-1The time, the Cd peak current significantly increases, 200 ~ 600 μ g L
-1The time plateau, appear, greater than 600 μ g L
-1The time, peak current is downward trend.And for the peak current of Pb, when bismuth ion concentration is 0 ~ 200 μ g L
-1The time, peak current significantly increases, 200 ~ 600 μ g L
-1Shi Zengjia trend is slightly slow, greater than 600 μ g L
-1The time, peak current is downward trend.Need detect simultaneously Cd and Pb ionic concn in solution during detection, comprehensively both, select 400 μ g L
-1Concentration as bismuth ion in final solution.
Fig. 6 D is the optimization of scavenging period after determination of electrode.Because square wave voltammetry is a kind of scan method more fast, when therefore sweeping to positive potential from negative potential, residual metal to be measured and bismuth metal are arranged still on electrode, impact mensuration next time.Therefore, need to add a positive voltage and clean for some time on electrode, make the metal ion stripping completely on electrode, the reducing electrode of trying one's best is surperficial.Fig. 6 D is different scavenging period replications obtain for five times under+0.3 V voltage Cd and the response of Pb ion.When scavenging period is 15 s, repeatedly measure poor repeatability.Along with the growth of scavenging period, the repeatability of mensuration improves, and the scavenging period of experimental selection optimization is 75 s.
Three, the methylate typical curve in site of difference obtains
Get the magnetic microsphere that 10 μ L P2 and P3 nucleic acid probe modify and hybridized 2 hours under 38 ℃ with the object chain that methylates of series of standards concentration, then add respectively 10 μ L PbS-P1 with
CDS-P4 solution, under room temperature, hybridization is 1 hour.Collect magnetic microsphere, clean 2 times with ultrapure water, add 20 μ L enzyme reaction solutions (30 mM Tris-HCl, 4 mM MgCl
2, 10 mM (NH
4)
2SO
4, 0.005% BSA, 0.2 mM NAD and 0.5 U/L
EcoliDNA ligase), reaction 1 hour under 38 ℃.Reaction solution was unwind under 90 ℃ 10 minutes, collect magnetic microsphere, after while hot reaction solution being extracted out, use successively 1 * PBS solution (0.2% Tween 20) and ultrapure water to clean magnetic microsphere, add 100 μ L 1 M HNO
3Solution, glass-carbon electrode are in 400 μ g/L bismuth solution, and-1.1 V depositions 10 minutes obtain the Stripping Voltammetry curve.After replication three times, average.
Fig. 7 A is the response working curve of A methylated object chain concentration in site to the Pb ion that only methylate, in solution with the increase of object chain concentration, the response peak electric current of Pb ion increases gradually, response peak current value and object chain concentration present good linear relationship, linear equation is:
I pa(μ A)=-0.0505
c(nmol L
-1) – 1.096 (n=3, R=0.9663), linearity range and detection limit are 10 nmol L successively
-1～80 nmol L
-1With 230 pmol L
-1(S/N=3).
Fig. 7 B is the response working curve of B methylated object chain concentration in site to the Cd ion that only methylate.With the increase of object chain concentration in solution, the response peak electric current of Cd ion increases, and presents good linear relationship.Linear equation is:
I pa(μ A)=-0.0471
c(nmol L
-1) – 0.7533 (n=3, R=0.9637), linearity range and detection limit are 10 nmol L successively
-1～80 nmol L
-1With 300 pmol L
-1(S/N=3).
Fig. 7 C is the volt-ampere response relation of two methylated object chain concentration of sites while and Pb and Cd ion, shows the linear relationship identical with Fig. 7 B with Fig. 7 A, and explanation can detect the situation that methylates in two sites simultaneously.
Four, the methylated mensuration of p53 tumor suppressor gene fragment specific site
(1) p53 tumor suppressor gene fragment sample is processed with bisulfite:
The bisulfite mixture of getting test kit encapsulation with the water of 800 μ L stoning ribonuclease T.s at 60 ℃ of dissolve completes.Add 15 μ L DNA sample solutions (1 ng-2 μ g) in 200 μ L PCR pipes, the water of 5 μ L stoning ribonuclease T.s, 85 μ L bisulfite mixture solutions, 35 μ L DNA protection buffered soln.Cover the PCR lid, fully mix the bisulfite reaction solution, preserve under room temperature.95 ℃ of sex change 5 min in the PCR instrument, 60 ℃ of incubation 25 min continue at 95 ℃ of sex change 5 min, 60 ℃ of incubation 85 min, then at 95 ℃ of sex change 5 min, at 60 ℃ of incubation 175 min, spend the night 20 ℃ of preservations.
(2) get magnetic microsphere and the p53 tumor suppressor gene fragment sample that 10 μ L P2 and P3 nucleic acid probe modify and hybridized 2 hours under 38 ℃, then add respectively 10 μ L PbS-P1 with
CDS-P4 solution, under room temperature, hybridization is 1 hour.
(2) collect magnetic microsphere, clean 2 times with ultrapure water, add 20 μ L enzyme reaction solutions (30 mM Tris-HCl, 4 mM MgCl
2, 10 mM (NH
4)
2SO
4, 0.005% BSA, 0.2 mM NAD and 0.5 U/L
EcoliDNA ligase), reaction 1 hour under 38 ℃.Reaction solution was unwind under 90 ℃ 10 minutes.
(3) collect magnetic microsphere, after while hot reaction solution being extracted out, use successively 1 * PBS solution (0.2% Tween 20) and ultrapure water to clean magnetic microsphere, add 100 μ L 1 M HNO
3Solution, glass-carbon electrode are in 400 μ g/L bismuth solution, and-1.1 V depositions 10 minutes obtain the Stripping Voltammetry curve.
(4) utilize calibration curve method, calculate the methylated concentration of specific site in p53 tumor suppressor gene fragment sample.
＜110〉Zhongshan University
＜120〉a kind of many specific sites DNA methylation electrochemical detection method based on the ligase enzyme reaction
<130>
<160> 6
<170> PatentIn version 3.5
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Claims (10)
1. a polyspecific site DNA methylation detection method, comprise the steps:
(1) according to one group of nucleic acid probe of flanking sequence design in each specific methylation site on target dna sequence, every group of nucleic acid probe is comprised of a separate probe and a bars probe, separate probe is modified with solid phase carrier, signal probe is modified with quantum dot, detects the quantum dot of modifying on the signal probe that uses in the different sites that methylate different;
(2) the target dna sample is processed with bisulfite, makes not methylated cytosine(Cyt) be converted into uridylic;
(3) separate probe and signal probe are added in target dna solution after step (2) is processed and hybridize;
(4) collect solid phase carrier, add the DNA ligase reaction solution to carry out ligation after cleaning, the reaction of unwinding after reaction finishes after separating end of chain (EOC), separates solid phase carrier while hot with reaction solution;
(5) collect solid phase carrier, utilize the electrochemical signals of the quantum dot that connects on the Voltammetry solid phase carrier after cleaning;
(6) determine methylated site and methylation according to ownership and the intensity of electrochemical signals.
2. method according to claim 1, is characterized in that, the length of described separate probe is 35～45 bp, and the length of signal probe is 10～15 bp.
3. method according to claim 1, is characterized in that, described solid phase carrier is that magnetic microsphere, tetrafluoroethylene microballoon, gold plaque or finishing have carboxyl or amino micro materials.
4. according to claim 1～3 described methods of any one, it is characterized in that, the preparation method of described separate probe is as follows: after solid phase carrier is used respectively 100 mM imidazole buffer solution and EDC/NHS solution-treated, add nucleic acid molecule, 35～45 ℃ of lower oscillatory reactions 1～3 hour, separate, use respectively 2 * SSC buffered soln (0.5% SDS) and 65 ℃ of high purity waters to clean, obtain separate probe.
5. method according to claim 1 and 2, is characterized in that, the preparation method of described signal probe is as follows: the quantum dot aqueous solution mixes with nucleic acid molecule, at N
2Protection is lower stirred 16～24 hours, dropwise added PBS(pH 7.4,0.24 M NaCl, 0.1% NaN
3) rear at PBS(pH 7.4,0.2 M NaCl) the middle dialysis 36～48 hours, the signal probe of centrifugal collection preparation.
6. method according to claim 1, is characterized in that, the condition of the described hybridization of step (3) is: hybridized under 35～45 ℃ 1～3 hour.
7. method according to claim 1, is characterized in that, the consisting of of step (4) described ligase enzyme reaction solution: 30 mM Tris-HCl, 4 mM MgCl
2, 10 mM (NH
4)
2SO
4, 0.005% BSA, 0.2 mM NAD and 0.5 U/L
EcoliDNA ligase.
8. method according to claim 1, is characterized in that, the condition of the described ligation of step (4) is: reacted under 35～40 ℃ 1～2 hour.
9. method according to claim 1, is characterized in that, the described condition of unwinding of step (4) is: unwind under 85～95 ℃ 5～15 minutes.
10. method according to claim 1, is characterized in that, the condition that step (5) is measured the Stripping Voltammetry curve is: glass-carbon electrode is in 300～500 μ g/L bismuth solution, and-0.9～-1.1 V deposits 10 minutes.
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| CN104062334A (en) * | 2014-07-11 | 2014-09-24 | 福州大学 | Quantitative analysis method aiming at DNA methylation monitoring |
| CN106556630A (en) * | 2016-10-31 | 2017-04-05 | 中山大学 | A kind of DNA methylation real-time detection method and its application |
| CN109136335A (en) * | 2018-09-06 | 2019-01-04 | 中国人民解放军陆军军医大学 | A kind of electrochemical analysis method of DNA methylation specific site |
| WO2020001416A1 (en) * | 2018-06-26 | 2020-01-02 | 深圳市圣必智科技开发有限公司 | Method for determining dna methylation level at specific site in biological sample and application thereof |
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Cited By (5)
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| CN104062334A (en) * | 2014-07-11 | 2014-09-24 | 福州大学 | Quantitative analysis method aiming at DNA methylation monitoring |
| CN106556630A (en) * | 2016-10-31 | 2017-04-05 | 中山大学 | A kind of DNA methylation real-time detection method and its application |
| CN106556630B (en) * | 2016-10-31 | 2019-04-16 | 中山大学 | A kind of DNA methylation real-time detection method and its application |
| WO2020001416A1 (en) * | 2018-06-26 | 2020-01-02 | 深圳市圣必智科技开发有限公司 | Method for determining dna methylation level at specific site in biological sample and application thereof |
| CN109136335A (en) * | 2018-09-06 | 2019-01-04 | 中国人民解放军陆军军医大学 | A kind of electrochemical analysis method of DNA methylation specific site |
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