CN103589777A - DNA methylation detection probe, and detection method and detection kit thereof - Google Patents
DNA methylation detection probe, and detection method and detection kit thereof Download PDFInfo
- Publication number
- CN103589777A CN103589777A CN201210288700.6A CN201210288700A CN103589777A CN 103589777 A CN103589777 A CN 103589777A CN 201210288700 A CN201210288700 A CN 201210288700A CN 103589777 A CN103589777 A CN 103589777A
- Authority
- CN
- China
- Prior art keywords
- dna
- detection
- sequence
- detection probes
- methylation
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Classifications
-
- 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
- C12Q1/6813—Hybridisation assays
- C12Q1/6816—Hybridisation assays characterised by the detection means
- C12Q1/682—Signal amplification
-
- 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
- C12Q2521/00—Reaction characterised by the enzymatic activity
- C12Q2521/50—Other enzymatic activities
- C12Q2521/501—Ligase
-
- 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
- C12Q2523/00—Reactions characterised by treatment of reaction samples
- C12Q2523/10—Characterised by chemical treatment
- C12Q2523/125—Bisulfite(s)
Abstract
The invention relates to a DNA methylation detection probe, and a detection method and a detection kit thereof. The detection probe comprises a 3' end and a 5' end which are complementary to methylated DNA, and a specific sequence used for initiating an HRCA reaction in the middle. If DNA to be detected is the methylated DNA, the DNA to be detected can be completely complementarily paired with the detection probe, and the 3' end and the 5' end of detection probe are linked under the action of a subsequent DNA ligase to form annular DNA; and if the DNA to be detected is unmethylated DNA, the DNA to be detected cannot be completely complementarily paired with the detection probe, and no annular DNA is formed. The annular DNA can quantitatively analyze the methylation degree of the DNA to be detected through the subsequent HRCA reaction and signal detection, and the HRCA has a signal 10<9>-time amplification capability, so the detection ultrahigh-sensitivity requirement is guaranteed. By using the probe, there is no need to use an expensive fluorescently-labeled probe or carry out PCR amplification in the detection process, so the detection cost is substantially reduced. The detection method has no enzyme site requirements on the methylated DNA, so the method can be widely applied.
Description
Technical field
The present invention relates to biology field, particularly relate to a kind of detection probes, detection method and detection kit of DNA methylation.
Background technology
Along with completing of the Human Genome Project, one of next vital task is exactly to decipher genetic system, and human body cell is how to use genetic material to decide a certain specific gene when and where to be expressed in process of growth.DNA methylation can affect the genetic state of genetic expression because of it, has become an integral part in epigenetic system.Mammiferous DNA methylation occurs in CpG dinucleotide on cytosine(Cyt) mostly, and on the pyrimidine ring of cytosine(Cyt), No. 5 carbon locations add methyl.In normal cell, methylate and mainly occur in the genome area of repetition, comprise genetic elements and satellite deoxynucleotide, yet the CpG island relevant to gene promoter and exon normally there is no methylated.But the DNA methylation of mutagenicity can cause the Transcriptional Silencing of cancer suppressor gene on these regions, the DNA methylation of this mutagenicity can be used as the sign that various diseases comprises cancer.On CpG island, methylating of specific region may be relevant to the cancer of particular type.Therefore, in human genome, on arbitrary given position, the accurate quantitative analysis of DNA methylation is very important for the early treatment of human cancer.
Traditional is existing multiple for analyzing the detection method of single CpG position or short sequence DNA methylation.The PCR(MSP of methylation-specific) open the gate for methylation analysis by polymerase chain reaction (PCR), but because the analytical results of MSP is by observing the phenomenon of gel electrophoresis, caused MSP only can provide the qualitative analysis of experiment but not quantitative analysis.The methods such as the FRET (fluorescence resonance energy transfer) of the quantum dot of fluorescent mark Real-Time Monitoring PCR and methylation-specific (MSq-FRET) all need the PCR instrument of precise temperature control, and generally need to use the probe of luminescent dye molecule mark, have greatly increased the weight of experimental cost.Detection in conjunction with restrictive diges-tion analytical procedure (COBRA) specified rate of sulphite and sensitive DNA methylation provides another kind of selection, but the precondition of COBRA is the restriction enzyme site that will have the restrictive diges-tion enzyme of the susceptibility of methylating in analytic sample.Based on surface enhanced Raman spectroscopy method and mononucleotide amplification method, because lower sensitivity causes applying limited.
Summary of the invention
Based on this, be necessary to provide a kind of highly sensitive, DNA methylation detection probes and detection method that testing cost is low.
A kind of DNA methylation detection probes, comprise that its 5 ' end and 3 ' end are used for and the sequence of methylate DNA complementary pairing and the specific sequence reacting for causing using hyper-branched rolling circle amplification of middle portion, wherein, last base of detection probes 3 ' end is guanine, the methylated cytosine pairing at described guanine and methylate DNA middle part, and the sequence and the described methylated cytosine to 3 from middle part of methylate DNA of detection probes 3 ' end ' the sequence reverse complemental of end matches, the sequence and the deoxynucleotide to 5 that closes on from the described methylated cytosine in middle part of methylate DNA of detection probes 5 ' end ' the sequence reverse complemental of end matches.
In an embodiment, the DNA sequence dna of described detection probes is SEQ ID NO:1 therein.
The detection probes with SEQ ID NO:1 gene order can be applied in the detection field of human non-small cell lung cancer's clone genomic methylation degree.
This DNA methylation detection probes, comprise and can hold with the 3 ' end and 5 ' of methylate DNA complementation and the specific sequence reacting for causing using hyper-branched rolling circle amplification (HRCA) of middle portion, and last base pair of 3 ' end of detection probes is answered the methylated cytosine at methylate DNA middle part, can be specific with the methylated cytosine at methylate DNA middle part by the formation of three hydrogen bonds complementary pairing.If DNA to be measured is methylate DNA, it can match with detection probes complete complementary, and after complementary pairing, cause the 3 ' end and 5 ' of detection probes to hold close, under the effect of follow-up DNA ligase, 3 ' end and the 5 ' end of detection probes are connected to form cyclic DNA, thereby can not digested by DNA exonucleaseⅢ and I; But not methylated DNA is through bisulf iotate-treated, on cytosine(Cyt) ring, the amino of No. 4 positions is by carbonyl substituted, be that cytosine(Cyt) has been transformed into uridylic, yet uridylic can not with guanine complementary pairing, non-methylated DNA can not match with detection probes complete complementary, there is no the formation of cyclic DNA, thereby all DNA are digested by DNA exonucleaseⅢ and I.Cyclic DNA can be by follow-up HRCA reaction and the methylation of signal detection quantitative analysis DNA to be measured, because HRCA has superelevation amplification ability (10
9times signal amplifies), thus the hypersensitivity requirement detecting guaranteed.In addition, use this probe, do not need use expensive fluorescently-labeled probe or carry out pcr amplification in testing process, the cost of detection reduces greatly.
A detection method for DNA methylation, comprises the steps:, with bisulf iotate-treated DNA to be measured, to make unmethylated cytosine(Cyt) in described DNA to be measured change into uridylic; In the DNA to be measured after bisulf iotate-treated, add above-mentioned detection probes to carry out DNA fusion reaction; In the product obtaining to fusion reaction, add DNA ligase, carry out ligation; Use the using hyper-branched rolling circle amplification method amplification product that above step obtains and carry out spectral signal detection.
In an embodiment, described hydrosulphite is sodium bisulfite or Potassium hydrogen sulfite therein.
In an embodiment, be also included in the product obtaining after the backward fusion reaction of fusion reaction and add DNA Digestive system therein, digest the not step of the DNA sequence dna of complete complementary pairing.
Therein in an embodiment, described in to carry out that spectral signal detects be that the DNA product after using hyper-branched rolling circle amplification is mixed with SYBR Green I fluorescence dye, under fluorophotometer, detect the transmitted wave intensity of mixture.
By DNA to be measured and padlock probe are merged, if DNA to be measured is methylate DNA, can under the effect of DNA ligase, form cyclic DNA with padlock probe, but not methylated DNA can not with padlock probe complementary pairing; After increasing by HRCA, cyclic DNA is increased in a large number, thereby fluorescence intensity can be detected, do not have the sequence of complementary pairing can not be amplified, thereby use above-mentioned detection method can analyze comparatively easily DNA to be measured, whether methylate, and because HRCA has superelevation amplification ability, the sensitivity of detection is guaranteed, by spectroscopic analysis, also there is quantitative effect.In testing process, do not need to use expensive fluorescence labeling probe or carry out pcr amplification, the cost of detection reduces greatly.And this detection method does not have the requirement of restriction enzyme site to methylate DNA, during for the DNA methylation degree detecting of arbitrary given position, only need to change 3 ' and 5 ' terminal sequence of this detection probes, make itself and target dna sequence complementary, just can realize the detection of this position DNA methylation degree, being widely used property of the method is described.
In addition, be also necessary to provide a kind of highly sensitive, test kit detecting for human non-small cell lung cancer that testing cost is low.
A detection kit, comprises detection probes, and the DNA sequence dna of described detection probes is SEQ ID NO:1.
In an embodiment, described test kit also comprises using hyper-branched rolling circle amplification primer therein, and the DNA sequence dna of the forward primer of described using hyper-branched rolling circle amplification and the DNA sequence dna of reverse primer are respectively SEQ ID NO:3 and SEQ ID NO:4.
In above-mentioned detection kit, detection probes has two specific sequences (sequence length is respectively 25 and 23) that can react with 3 ' end (13 bases) of the methylate DNA complementary pairing of human non-small cell lung cancer's clone (H157) and 5 ' end (21 bases) and for initiation HRCA, and last base of 3 ' end of detection probes is set to guanine, the methylated cytosine at the specific methylate DNA of energy middle part is complementary pairing by the formation of three hydrogen bonds; If this position DNA methylates in the cell of individuality to be measured, its can with detection probes complementary pairing, and it is close to cause the 3 ' end and 5 ' of detection probes to be held, under the effect of follow-up DNA ligase, the 3 ' end and 5 ' of detection probes is held and to be connected to form cyclic DNA; But not methylated DNA is through bisulf iotate-treated, on cytosine(Cyt) ring, the amino of No. 4 positions is by carbonyl substituted, be that cytosine(Cyt) has been transformed into uridylic, yet uridylic can not with guanine complementary pairing, from but not methylated DNA can not with detection probes complementary pairing, there is no the formation of cyclic DNA yet.Cyclic DNA reacts amplification by HRCA again, and under the existence of amplimer, extension and the strand displacement of primer constantly occur, and obtain the double-stranded DNA product that a large amount of length is different, and then these products can carry out spectrum detection by quantitative by fluorescence dye, goes out.Because HRCA has superelevation amplification ability, the sensitivity of detection is guaranteed, and by spectroscopic analysis, also has quantitative effect.In testing process, do not need to use expensive fluorescence labeling probe or carry out pcr amplification, the cost of detection reduces greatly.
Accompanying drawing explanation
Fig. 1 is the complete complementary pairing schematic diagram of methylate DNA and detection probes;
Fig. 2 is the part complementary pairing schematic diagram of non-methylated DNA and detection probes;
Fig. 3 is the overhaul flow chart of the DNA methylation of an embodiment;
Fig. 4 is the testing process schematic diagram of embodiment 1;
Fig. 5 is the electrophoresis proof diagram of ligation, the result figure that 10% polypropylene amine of sex change dyes by standard silver by gel electrophoresis, and runway M is DNA sign, road 1,2,3 represent respectively blank assay, 10nM is the connection product of methylate DNA and 10nM methylate DNA not;
Fig. 6 is for being the electrophoresis proof diagram of digestion product, and condition is identical with Fig. 5;
Fig. 7 is the condition optimizing of HRCA experiment in embodiment 1, the fluorescence intensity change figure that the not methylate DNA (solid line post) of the methylate DNA of 1nM (dotted line post) and 1nM obtains along with the variation of primer concentration;
Fig. 8 is the condition optimizing of HRCA experiment in embodiment 1, the fluorescence intensity change figure that the not methylate DNA (solid line post) of the methylate DNA of 1nM (dotted line post) and 1nM obtains along with the variation of dNTPs concentration;
Fig. 9 is the electrophoresis proof diagram of amplified reaction, use be the agarose gel electrophoresis method of SYBR Green I, runway with in Fig. 5, represent consistent;
Figure 10 is the fluoroscopic examination intensity map of the methylate DNA of different concns;
Figure 11 is the index linear graph of concentration and the fluorescence intensity of methylate DNA, linearity range: 1fmol/L is to 10pmol/L;
Figure 12 is the fluoroscopic examination result figure of different methylation content levels;
Figure 13 is the methylation content level that measures and the graph of a relation of the actual methylation content level of putting into;
Figure 14 is the H157(dotted line post of different concns) and H209(solid line post) genomic dna and the graph of a relation of its fluoroscopic examination value;
Figure 15 is 10nM methylate DNA (M) and the fluoroscopic examination value of methylate DNA (U) not.
Embodiment
Below in conjunction with drawings and the specific embodiments, DNA methylation detection probes, detection method and detection kit are further detailed.
The DNA methylation detection probes of one embodiment comprises the specific sequence reacting for causing using hyper-branched rolling circle amplification for the sequence with methylate DNA complementary pairing and middle portion of 5 ' end and 3 ' end, wherein, last base of detection probes 3 ' end is guanine, methylated cytosine pairing with methylate DNA middle part, and the sequence of detection probes 3 ' end and methylate DNA from middle part methylated cytosine to 3 ' the sequence reverse complemental of end matches, deoxynucleotide to another 5 ' the sequence reverse complemental of holding that closes on from middle part methylated cytosine of the sequence of detection probes 5 ' end and methylate DNA matches.
Last base pair of this DNA methylation detection probes 3 ' end is answered the methylated cytosine at methylate DNA middle part, can be specific with the methylated cytosine at methylate DNA middle part by the formation of three hydrogen bonds complementary pairing.As shown in Figure 1, if DNA to be measured is methylate DNA, it can match with detection probes complete complementary, and after complementary pairing, cause the 3 ' end and 5 ' of detection probes to hold close, under the effect of follow-up DNA ligase, 3 ' end and the 5 ' end of detection probes are connected to form cyclic DNA, can digested liquid digestion.As shown in Figure 2, non-methylated DNA is through bisulf iotate-treated, on cytosine(Cyt) ring, the amino of No. 4 positions is by carbonyl substituted, be that cytosine(Cyt) has been transformed into uridylic, yet uridylic can not with guanine complementary pairing, non-methylated DNA can not match with detection probes complete complementary, there is no the formation of cyclic DNA, thereby all DNA is digested by DNA exonucleaseⅢ and I.
Cyclic DNA can be by follow-up HRCA reaction and the methylation of signal detection quantitative analysis DNA to be measured, because HRCA has superelevation amplification ability (10
9times signal amplifies), thus the hypersensitivity requirement detecting guaranteed.And this detection probes can merge with numerous methylated DNA, compatible good.In addition, use this probe, do not need use expensive fluorescently-labeled probe or carry out pcr amplification in testing process, the cost of detection reduces greatly.
In addition, present embodiment also provides a kind of detection method of DNA methylation, as shown in Figure 3, comprises the steps:
Step S310, with bisulf iotate-treated DNA to be measured, makes unmethylated cytosine(Cyt) in DNA to be measured change into uridylic.
Hydrosulphite is sodium bisulfite or Potassium hydrogen sulfite etc.
Unmethylated cytosine(Cyt) is transformed uridylic by hydrosulphite, and methylated cytosine can not be converted.
Step S320 adds the detection probes of above-mentioned introduction to carry out DNA fusion reaction in the DNA to be measured after bisulf iotate-treated.
Padlock probe (Padlock probe) is a kind of longer single stranded oligonucleotide fragment, and two ends and target are adjoined complementation, and one section of middle catenation sequence, on not impact of detected result, can be used as the binding site of universal primer.Thereby after padlock probe and DNA merge, the two ends of probe are adjoined approaching, under the effect of follow-up DNA ligase, can couple together formation cyclic DNA.In the present embodiment, padlock probe is above-mentioned DNA methylation detection probes.
Step S330 adds DNA ligase in the product obtaining after fusion reaction, carries out ligation.If DNA to be measured is methylate DNA, can under the effect of DNA ligase, form cyclic DNA with padlock probe; But not methylated DNA can not with padlock probe complete complementary pairing, there is no the formation of cyclic DNA.
Step S340 adds DNA Digestive system in the product obtaining through digestion process, digests the not DNA sequence dna of complete complementary pairing, thereby obtains the cyclic DNA of purifying.Carrying out digestion step is mainly the amplification causing in order to reduce disconnected DNA, to reduce subsequent detection false positive or the interference of other DNA to experimental result.Be appreciated that in other embodiments, this step can be omitted.Step S350, is used the using hyper-branched rolling circle amplification method amplification product that above step obtains and carries out spectral signal detection.
Cyclic DNA can be by follow-up HRCA reaction and the methylation of signal detection quantitative analysis DNA to be measured, because HRCA has superelevation amplification ability (10
9times signal amplifies), thus the hypersensitivity requirement detecting guaranteed.
In the present embodiment, carrying out signal detection is that the DNA product after using hyper-branched rolling circle amplification is mixed with SYBR Green I fluorescence dye, detects the transmitted wave intensity of mixture under fluorophotometer.
Whether above-mentioned detection method can be analyzed comparatively easily DNA to be measured and methylate, and because HRCA has superelevation amplification ability, the sensitivity of detection is guaranteed, and by spectroscopic analysis, also has quantitative effect.In testing process, do not need to use expensive fluorescence labeling probe or carry out pcr amplification, the cost of detection reduces greatly.
Further, present embodiment also provides a kind of human non-small cell lung cancer's detection kit, and it comprises detection probes, and the DNA sequence dna of detection probes is SEQ ID NO:1.This probe is the methylate DNA of SEQ ID NO:2 for detection of DNA sequence dna in mankind's non-small cell.
In addition, this detection kit also comprises using hyper-branched rolling circle amplification primer, and the DNA sequence dna of using hyper-branched rolling circle amplification primer is respectively SEQ ID NO:3 and the SEQ ID NO:4 in sequence table.
Detection probes in this detection kit has two specific sequences (sequence length is respectively 25 and 23) that can react with 3 ' end (13 bases) of the methylate DNA complementary pairing of human non-small cell lung cancer's clone (H157) and 5 ' end (21 bases) and for initiation HRCA, and last base of 3 ' end of detection probes is set to guanine, the methylated cytosine at the specific methylate DNA of energy middle part is complementary pairing by the formation of three hydrogen bonds; If this position DNA methylates in the cell of individuality to be measured, its can with detection probes complementary pairing, and it is close to cause the 3 ' end and 5 ' of detection probes to be held, under the effect of follow-up DNA ligase, the 3 ' end and 5 ' of detection probes is held and to be connected to form cyclic DNA; But not methylated DNA is through bisulf iotate-treated, on cytosine(Cyt) ring, the amino of No. 4 positions is by carbonyl substituted, be that cytosine(Cyt) has been transformed into uridylic, yet uridylic can not with guanine complementary pairing, from but not methylated DNA can not with detection probes complete complementary pairing, there is no the formation of cyclic DNA yet.
Below in conjunction with specific embodiment, verify the feasibility of above-mentioned detection probes and detection method
H157(human non-small cell lung cancer clone) and H209(mankind's small cell lung cancer cell system) methylate DNA of clone detects, as shown in Figure 4.
1, DNA extraction and digestion
1.1 cultivate respectively H157 and two kinds of clones of H209.In normal H157 clone, p16 promoter region is high methylation, and these regions in normal H209 clone be do not have methylated.
Culture condition: two kinds of clones are placed in respectively to the DMEM nutrient solution that contains 10% bovine serum albumin, are placed in the 37 ℃ of incubators that contain 5% carbonic acid gas that add wet treatment and cultivate.
1.2 extract DNA
With DNA extraction test kit, extract respectively the genomic dna of above-mentioned two kinds of clones, and adopt spectrophotometer to detect this DNA extraction solution in the absorption value of 260 nanometers, converse DNA concentration.
Two kinds of restrictive diges-tion enzymes of 1.3 use Pst I and BstE II are processed respectively the DNA of extraction, take digested genomic dna as shorter base fragment, obtain DNA to be measured.
In other embodiments, in clone, this step of the extraction of DNA can be omitted, and adopts the mode of directly buying to obtain methylated DNA and unmethylated DNA.
2, sodium bisulfite is processed DNA to be measured, makes unmethylated cytosine(Cyt) in described DNA to be measured change into uridylic.
Treatment condition are: in the sodium hydroxide solution that the concentration that is added to 20 μ L volumes in 1 μ g DNA to be measured is 0.35mol/L, 37 ℃ of reactions are after 20 minutes, by certain volume sodium sulfite solution and Resorcinol add in above-mentioned solution and make its final concentration be respectively 3.2mol/L and 0.5mmol/L, 50 ℃ of reaction 16-18 hour, then make solution cross desalting column, reclaim DNA; To the sodium hydroxide solution that adds certain volume in the DNA reclaiming, making its final concentration in the solution of 50 μ L is 0.3mol/L, and 37 ℃ of reactions 15 minutes, then neutralize this solution completely with Ammonium Acetate, finally in ethanol, precipitate the dry DNA powder that obtains.The DNA powder obtaining is dissolved in ultrapure water, in-20 ℃, saves backup.
3, ligation
3.1 according to the sequences Design padlock probe of DNA to be measured
In the present embodiment, the sequence of DNA to be measured is as follows respectively:
Methylate DNA sequence is: GAG GGT GGG G
mcG GAC
mcG
mc GTG
mcGC T
mcGG
mcG GCT G(SEQ ID NO:2), wherein,
mc represents methylated cytosine(Cyt);
Methylate DNA sequence is not: GAG GGT GGG GCG GAC CGC GTG CGC TCGGCG GCT G(SEQ ID NO:5).
The sequence of the padlock probe of design: CAC GCG ATC CGC CCC ACC CTC ATT AGG TTACTG CGA TTA GCA CAA GCA CCA AGA GCA ACT ACA CGA ATT CCA ACCGCC GAA CG(SEQ ID NO:1).
In the present embodiment, in order to strengthen the specificity of detection, in the sequence of padlock probe, done special design: the overall length of padlock probe is 83 bases, there are 21 bases with 5 ' end of methylate DNA complementary pairing, 3 ' end has 13 bases, 49 bases of middle one-tenth loop section, the asymmetric sequential structure of this two ends complementary pairing, will be conducive to the combination of goal gene and padlock probe; Secondly, last base of 3 ' end of padlock probe is set to guanine, the methylated cytosine of specificity and methylate DNA is complementary pairing by the formation of three hydrogen bonds, and not methylate DNA by the processing of sodium bisulfite, on cytosine(Cyt) ring, the amino of No. 4 positions is by carbonyl substituted, be that cytosine(Cyt) has been transformed into uridylic, can not with guanine complementary pairing.
In other embodiments, padlock probe can adopt other rational designs, and for example the sequence length for complementary pairing of 5 ' end is not limited to 21bp, and the sequence length for complementary pairing of 3 ' end is also not limited to 13bp.As long as the design of padlock probe meets requirement of experiment.Guarantee that the sequence of padlock probe 5 ' and 3 ' end matches with 5 ' and 3 ' sequence reverse complemental of object methylate DNA respectively.
The condition of 3.2 ligations
The 2L of different concns DNA to be measured is mixed with the padlock probe of the 1mol/L of 2L, the mixed solution of preparation 20L, in mixed solution, contain: 20mmol/L Tris-HCl(pH 7.6) damping fluid, 25mmol/L Potassium ethanoate, 10mmol/L magnesium acetate, the niacinamide adenosine dinucleotides of 1mmol/L and 0.1%Triton X-100.95 ℃ are reacted 5 minutes, then add the Taq DNA ligase of 12 units, and 65 ℃ are reacted 60 minutes.
With after bisulf iotate-treated DNA to be measured, unmethylated cytosine(Cyt) changes into uridylic, and methylated cytosine(Cyt) does not change, in ligation, methylated DNA can with the two ends complete complementaries pairing of padlock probe, the 3 ' end and 5 ' of padlock probe is held close, under the effect of ligase enzyme, 3 ' end and the 5 ' end of padlock probe couple together, and form ring-type padlock probe.Unmethylated DNA is because sequence has had significant difference, can not match with padlock probe complete complementary, therefore 3 ' end and 5 ' is held and can not be connected enzyme and couple together, thereby methylate DNA and not methylate DNA can be distinguished thus, because the cyclisation of padlock probe connects high specificity, therefore this cyclisation that utilizes padlock probe connects for the method detecting that methylates and has high specific.
The checking of 3.3 ligation products
In order to prove the feasibility of the method, in the present embodiment, select electrophoresis experiment to verify, as shown in Figure 5, Fig. 5 is 10% polyacrylamide gel electrophoresis figure of the standard silver of the ligation product sex change of dying, runway M is DNA sign, and swimming lane 1,2,3 represents respectively the not connection product of methylate DNA and 10nM methylate DNA of blank assay, 10nM.Because linear padlock probe runs a good foot than ring-type padlock probe, the existence of electrophorogram proof ligation product, method is feasible.
4, digestion reaction
The condition of 4.1 digestion reactions
The solution of getting after 10L ligation mixes with 10L Digestive system, and 37 ℃ are reacted 2 hours, finally at 95 ℃ of 10 minutes inactivations.The DTT that contains 1mmol/L in 10L Digestive system, the magnesium chloride of 6.7mmol/L, DNA excision enzyme I and the exonucleaseⅢ of glycine-potassium hydroxide damping fluid ，10 unit of the pH 9.5 of 67mmol/L.
DNA excision enzyme I and exonucleaseⅢ can digest non-annularity DNA, but can not digest cyclic DNA, thereby obtain pure ring-type padlock probe.In the present embodiment, non-annularity DNA refers to methylate DNA, methylate DNA and the padlock probe of ligation does not occur not.
The checking of 4.2 digestion reaction products
In the present embodiment, selecting electrophoresis experiment to verify, is the denaturing polyacrylamide gel electrophoresis figure that digestion reaction product dyes by standard silver as shown in Figure 6, from Fig. 6, sees, all non-annularity DNA are digested clean ( runway 1 and 2 in Fig. 6) all, except ring-type padlock probe (in Fig. 6, runway 3).The existence of ring-type padlock probe has further proved the feasibility of ligation, simultaneously in order to reduce the generation of disconnected specific amplification in amplified reaction below, further improves specificity, and the digestion of excision enzyme is extremely important.
5, using hyper-branched rolling circle amplification reaction
5.1 according to two primers of padlock probe design
The condition of 5.2 using hyper-branched rolling circle amplification reactions
The digestion reaction product of 10L is mixed with 20L amplification solution, and 63 ℃ are reacted 1 hour.In amplification solution, contain: primer 1 and the primer 2 of 0.05 μ mol/L, the deoxynucleoside triphosphate mixed solution of 400 μ mol/L and the Bst archaeal dna polymerase of 8 units.
As Fig. 7, concentration reduction to 0.05 μ mol/L by 1 μ mol/L along with two primers, the gene that methylates increases gradually with the difference of the fluorescence intensity of the gene that do not methylate, therefore, the selected 0.05 μ mol/L of the present embodiment is the optimal concentration of two kinds of primers, because the primer of higher concentration, will cause dimerization and nonspecific amplification of primer self.In addition, as shown in Figure 8, the increase to 400 μ mol/L by 2 μ mol/L along with dNTPs concentration of substrate, the gene that methylates increases gradually with the difference of fluorescence intensity of the gene that do not methylate, so the present embodiment to select 400 μ mol/L be the optimum concn of dNTPs substrate.
The checking of 5.3 using hyper-branched rolling circle amplification reaction product
In the present embodiment, select electrophoresis experiment to verify, that using hyper-branched rolling circle amplification reaction product be take the agarose gel electrophoresis figure that SYBR Green I is dyestuff as shown in Figure 9, from Fig. 9, runway 3 can be seen, the gene that methylates can obtain a large amount of DNA products by cyclisation padlock probe and then amplification, and blank assay (in Fig. 9, runway 1) and not methylate DNA (in Fig. 9, runway 2) can not increase, thereby can not get product.
6, detect
6.1 testing conditions
The SYBR Green I (20 times) of the 30 reacted solution of μ L using hyper-branched rolling circle amplification and 1 μ L is mixed, add deionized water to 100 μ L.Incubated at room 10 minutes, detects this solution with fluorophotometer, fluoroscopic examination condition: excitation wavelength is 488 nanometers, and spectra collection scope is 500-650 nanometer, in 520 nanometers, measures its transmitted wave intensity afterwards.
6.2 detected result
The highly sensitive accurate quantitative analysis of DNA methylation is very important for the early treatment of cancer.In order to prove the high sensitivity of the method, the present embodiment has been studied the fluoroscopic examination result of the gene that methylates of different concns.As shown in figure 10, along with the increase of the mrna concentration that methylates, its fluoroscopic examination value also increases, and concentration and signal strength values exponent function relation, be logarithmic value and the signal strength values linear (as Figure 11) of concentration, and linear relationship covers 4 orders of magnitude, by 1fmol/L to 10pmol/L.Linear relation is: I
f=34.54+102.98log10C, wherein I
ffor fluorescence signal intensity value, C is the concentration (expense mole every liter) of gene of methylating.By this equation analysis margin value, add that the fluorescence intensity of 3 times of deviates obtains detecting 0.8 expense that is limited to mole every liter.This detects limit value and exceeds 8 orders of magnitude than with the colorimetric method of gold nano, high 3 orders of magnitude that obtain than the Raman enhanced spectrum with single base amplification.
In addition, the present embodiment is also mixed to get methylate gene and the gene that do not methylate artificial mixed solution with certain proportion, and its methylation is detected.As Figure 12, along with the increase of methylation, the fluorescence intensity level obtaining also and then increases.And as Figure 13 shows, detect the methylation and the actual methylation adding that obtain and almost coincide.And, the method can successfully detect in biased sample 0.01% the base that methylates, the resolving power obviously obtaining than following method is high: MALDI-MS mass spectrum (5%), fluorescence energy transfer based on quantum dot (1%), positively charged ion conjugated polymers ionogen method (1%), the PCR(0.1% of methylation specific), even can with MS-qFRET(0.01%) compare.
In the present embodiment, in order further to verify, actual sample is detected the reliability of above-mentioned detection method.By this method, investigated Lines H157 and small cell lung cancer cell is the situation that methylates on six CpG island in p16 promoter region fragment in H209.While detecting the actual sample of clone by this analysis method, the present embodiment had been processed genomic dna with restrictive diges-tion enzyme before experiment, and object is in order to prevent the secondary structures such as the superhelix of the formation of DNA in test subsequently or super cyclisation.As shown in figure 14, along with the increase of the amount of genomic dna, detect the fluorescence intensity that H157 obtains and increase thereupon, H209 remains unchanged, and the detection of H157 is limited to 2ng.Show thus the situation of DNA methylation in the detection of lung cancer clone of the detection method energy higher sensitivity in this patent.
The specific detection gene that methylates
Research in the early time shows, methylating of the cancer suppressor gene in p16 will cause kinds cancer.In order to prove the feasibility of the method, the present embodiment has synthesized section of DNA, and sequence is identical with one section of cancer gene in p16, and sequence is: GAG GGT GGG G
mcG GAC
mcG
mc GTG
mcGC T
mcG G
mand methylating of this segment DNA detected CG GCTG(SEQ ID NO:2).As shown in figure 15, the fluorescence intensity level being obtained by 10nM methylate DNA is 698.8 ± 25.3, higher 23 times than methylate DNA does not obtain by 10nM fluorescence intensity level (30.2 ± 5.5), illustrate the method can be successfully for the detection of methylate DNA.
The above embodiment has only expressed several embodiment of the present invention, and it describes comparatively concrete and detailed, but can not therefore be interpreted as the restriction to the scope of the claims of the present invention.It should be pointed out that for the person of ordinary skill of the art, without departing from the inventive concept of the premise, can also make some distortion and improvement, these all belong to protection scope of the present invention.Therefore, the protection domain of patent of the present invention should be as the criterion with claims.
Claims (9)
1. a DNA methylation detection probes, it is characterized in that, the specific sequence reacting for causing using hyper-branched rolling circle amplification for the sequence with methylate DNA complementary pairing and middle portion that comprises 5 ' end and 3 ' end, wherein, last base of detection probes 3 ' end is guanine, the methylated cytosine pairing at described guanine and methylate DNA middle part, and the sequence and the described methylated cytosine to 3 from middle part of methylate DNA of detection probes 3 ' end ' the sequence reverse complemental of end matches, the sequence and the deoxynucleotide to 5 that closes on from the described methylated cytosine in middle part of methylate DNA of detection probes 5 ' end ' the sequence reverse complemental of end matches.
2. DNA methylation detection probes according to claim 1, is characterized in that, the DNA sequence dna of described detection probes is SEQ ID NO:1.
3. the application of the detection probes that a DNA sequence dna is SEQ ID NO:1 in detecting human non-small cell lung cancer's clone genomic methylation degree.
4. a detection method for DNA methylation, is characterized in that, comprises the steps:
With bisulf iotate-treated DNA to be measured, make unmethylated cytosine(Cyt) in described DNA to be measured change into uridylic;
In the DNA to be measured after bisulf iotate-treated, add detection probes as claimed in claim 1 to carry out DNA fusion reaction;
In the product obtaining to fusion reaction, add DNA ligase, carry out ligation;
Use the using hyper-branched rolling circle amplification method amplification product that above step obtains and carry out spectral signal detection.
5. DNA methylation detection method according to claim 4, is characterized in that, described hydrosulphite is sodium bisulfite or Potassium hydrogen sulfite.
6. the detection method of DNA methylation according to claim 4, is characterized in that, is also included in the product obtaining after the backward fusion reaction of fusion reaction and adds DNA Digestive system, digests the not step of the DNA sequence dna of complete complementary pairing.
7. DNA methylation detection method according to claim 4, it is characterized in that, described to carry out that spectral signal detects be that the DNA product after using hyper-branched rolling circle amplification is mixed with SYBR Green I fluorescence dye, detects the transmitted wave intensity of mixture under fluorophotometer.
8. human non-small cell lung cancer's detection kit, is characterized in that, comprises detection probes, and the DNA sequence dna of described detection probes is SEQ ID NO:1.
9. human non-small cell lung cancer's detection kit according to claim 8, it is characterized in that, described test kit also comprises using hyper-branched rolling circle amplification primer, and the DNA sequence dna of the forward primer of described using hyper-branched rolling circle amplification and the DNA sequence dna of reverse primer are respectively SEQ ID NO:3 and SEQ ID NO:4.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210288700.6A CN103589777A (en) | 2012-08-14 | 2012-08-14 | DNA methylation detection probe, and detection method and detection kit thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210288700.6A CN103589777A (en) | 2012-08-14 | 2012-08-14 | DNA methylation detection probe, and detection method and detection kit thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN103589777A true CN103589777A (en) | 2014-02-19 |
Family
ID=50080090
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201210288700.6A Pending CN103589777A (en) | 2012-08-14 | 2012-08-14 | DNA methylation detection probe, and detection method and detection kit thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103589777A (en) |
Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104611453A (en) * | 2015-02-28 | 2015-05-13 | 基因科技（上海）有限公司 | Probe group for diagnostic gene methylation and application of probe group |
| CN106939335A (en) * | 2017-03-13 | 2017-07-11 | 福建医科大学 | The method that acute-on-chronic liver failure controls gene is resisted in a kind of detection human serum |
| CN106990084A (en) * | 2017-05-27 | 2017-07-28 | 山东师范大学 | A kind of being used for based on single quantum dot detects the nano-sensor of dnmt rna |
| CN107760762A (en) * | 2017-09-30 | 2018-03-06 | 山东师范大学 | A kind of fluorescence chemical sensor and its detection method of detection DNA adenine methyltransferases |
| CN108827935A (en) * | 2018-06-08 | 2018-11-16 | 南京师范大学 | It is a kind of based on the DNA methylation Surface Enhanced Raman Scattering Spectrum detection method of gold nano hole array and its application |
| CN109234388A (en) * | 2017-07-04 | 2019-01-18 | 深圳华大基因研究院 | Reagent, enrichment method and application for the enrichment of DNA hyper-methylation region |
| CN110195056A (en) * | 2019-05-31 | 2019-09-03 | 汪雪 | A kind of decoding for DTMF and its application |
| WO2019233451A1 (en) * | 2018-06-08 | 2019-12-12 | 深圳市圣必智科技开发有限公司 | Dna methylation detection probe |
| WO2019233450A1 (en) * | 2018-06-08 | 2019-12-12 | 深圳市圣必智科技开发有限公司 | Detection method for dna methylation |
| CN111676286A (en) * | 2020-05-29 | 2020-09-18 | 武汉爱基百客生物科技有限公司 | Multiplex PCR primer system for detecting free DNA methylation of lung cancer plasma, detection method and application |
| WO2024063036A1 (en) * | 2022-09-20 | 2024-03-28 | 国立大学法人東京農工大学 | Method for detecting methylation of genome dna |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1863927A (en) * | 2003-09-04 | 2006-11-15 | 人类遗传标记控股有限公司 | Nucleic acid detection assay |
| CN101985654A (en) * | 2010-10-21 | 2011-03-16 | 徐州医学院 | Multi-sample multi-site SNP detection method |
-
2012
- 2012-08-14 CN CN201210288700.6A patent/CN103589777A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1863927A (en) * | 2003-09-04 | 2006-11-15 | 人类遗传标记控股有限公司 | Nucleic acid detection assay |
| CN101985654A (en) * | 2010-10-21 | 2011-03-16 | 徐州医学院 | Multi-sample multi-site SNP detection method |
Non-Patent Citations (2)
| Title |
|---|
| CAO A ET AL.: "Sensitive and Label-Free DNA Methylation Detection by Ligation-Mediated Hyperbranched Rolling Circle Amplification", 《ANAL CHEM》 * |
| 郑小国等: "表观遗传学研究方法进展", 《生物技术通报》 * |
Cited By (14)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104611453A (en) * | 2015-02-28 | 2015-05-13 | 基因科技（上海）有限公司 | Probe group for diagnostic gene methylation and application of probe group |
| CN106939335A (en) * | 2017-03-13 | 2017-07-11 | 福建医科大学 | The method that acute-on-chronic liver failure controls gene is resisted in a kind of detection human serum |
| CN106990084A (en) * | 2017-05-27 | 2017-07-28 | 山东师范大学 | A kind of being used for based on single quantum dot detects the nano-sensor of dnmt rna |
| CN109234388A (en) * | 2017-07-04 | 2019-01-18 | 深圳华大基因研究院 | Reagent, enrichment method and application for the enrichment of DNA hyper-methylation region |
| CN109234388B (en) * | 2017-07-04 | 2021-09-14 | 深圳华大生命科学研究院 | Reagent for enriching DNA hypermethylated region, enrichment method and application |
| CN107760762A (en) * | 2017-09-30 | 2018-03-06 | 山东师范大学 | A kind of fluorescence chemical sensor and its detection method of detection DNA adenine methyltransferases |
| CN108827935B (en) * | 2018-06-08 | 2021-09-07 | 南京师范大学 | DNA methylation surface enhanced Raman scattering spectrum detection method based on gold nanopore array and application thereof |
| CN108827935A (en) * | 2018-06-08 | 2018-11-16 | 南京师范大学 | It is a kind of based on the DNA methylation Surface Enhanced Raman Scattering Spectrum detection method of gold nano hole array and its application |
| WO2019233451A1 (en) * | 2018-06-08 | 2019-12-12 | 深圳市圣必智科技开发有限公司 | Dna methylation detection probe |
| WO2019233450A1 (en) * | 2018-06-08 | 2019-12-12 | 深圳市圣必智科技开发有限公司 | Detection method for dna methylation |
| CN110195056A (en) * | 2019-05-31 | 2019-09-03 | 汪雪 | A kind of decoding for DTMF and its application |
| CN111676286A (en) * | 2020-05-29 | 2020-09-18 | 武汉爱基百客生物科技有限公司 | Multiplex PCR primer system for detecting free DNA methylation of lung cancer plasma, detection method and application |
| CN111676286B (en) * | 2020-05-29 | 2023-04-14 | 武汉爱基百客生物科技有限公司 | Multiplex PCR primer system for detecting free DNA methylation of lung cancer plasma, detection method and application |
| WO2024063036A1 (en) * | 2022-09-20 | 2024-03-28 | 国立大学法人東京農工大学 | Method for detecting methylation of genome dna |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103589777A (en) | DNA methylation detection probe, and detection method and detection kit thereof | |
| CN102719526B (en) | MicroRNA quantitative detection analytic method by utilizing isothermal amplification to synthesize fluorescent nano silver cluster probe | |
| Cheng et al. | A CRISPR-driven colorimetric code platform for highly accurate telomerase activity assay | |
| CN103305612B (en) | Lead ion detection kit based on constant-temperature cascade nucleic acid amplification and detection method of lead ion detection kit | |
| US9297047B2 (en) | Molecular beacon based assay for the detection of biomarkers for breast cancer metastasis | |
| CN105132524A (en) | Application of dual application reaction of Exo (exonuclease) III-assisted cycle and DNAzyme cycle to Hg<2+> detection | |
| WO2001012856A3 (en) | Analysis of sequence tags with hairpin primers | |
| CN105525010A (en) | Stem-loop structured combined probe and application thereof | |
| CN111778316B (en) | Fluorescent probe based on oxidative damage basic group, kit and method for directly detecting DNA methylation | |
| CN105112540A (en) | Method for detecting DAN methyltransferase activity based on strand displacement amplification and DNAzyme amplification | |
| Wang et al. | Target-mediated hyperbranched amplification for sensitive detection of human alkyladenine DNA glycosylase from HeLa cells | |
| CN104789667A (en) | Small RNA (ribonucleic acid) detection kit and quantitative method based on unbiased recognition and isothermal amplification | |
| Zhou et al. | A novel methyl-dependent DNA endonuclease GlaI coupling with double cascaded strand displacement amplification and CRISPR/Cas12a for ultra-sensitive detection of DNA methylation | |
| Liu et al. | A novel DNA methylation biosensor by combination of isothermal amplification and lateral flow device | |
| CN105154563A (en) | Triple-amplification-technology-based homogeneous-phase label-free method for detecting activity of telomerase | |
| CN115109859A (en) | Multi-mode composite functional helicobacter pylori nucleic acid detection test paper | |
| CN107083437B (en) | Method for simultaneously detecting multiple DNA glycosylases by using inherent fluorescent nucleotide ultrasensitiveness | |
| Cai et al. | Fabrication of on-bead functional nucleic acid nanowires based on cyclic ligation reaction-driven rolling circle amplification for label-free detection of long noncoding RNAs in breast and lung tissues | |
| CN116083575A (en) | Kit for detecting cervical cancer and application thereof | |
| EP3521450A1 (en) | Method for detecting small rnas or proteins associated with small rnas | |
| Wen et al. | An ATP-fueled nucleic acid signal amplification strategy for highly sensitive microRNA detection | |
| WO2019233451A1 (en) | Dna methylation detection probe | |
| Zhou et al. | Sensitive osteosarcoma diagnosis through five-base telomerase product-triggered CRISPR-Cas12a enhanced rolling circle amplification | |
| CN110511984B (en) | Rapid fluorescence detection method for EGFR gene exon 19 deletion mutation and application | |
| CN110577985A (en) | Method for detecting DNA methylation |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C12 | Rejection of a patent application after its publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20140219 |