CN103031372A

CN103031372A - ZNF545 gene methylation quantitative detection method

Info

Publication number: CN103031372A
Application number: CN2012102847206A
Authority: CN
Inventors: 蔡林涛; 王朝晖; 龚萍; 苏献伟; 石碧华
Original assignee: Shenzhen Institute of Advanced Technology of CAS
Current assignee: Shenzhen Institute of Advanced Technology of CAS
Priority date: 2012-08-10
Filing date: 2012-08-10
Publication date: 2013-04-10

Abstract

The invention discloses a ZNF545 gene methylation quantitative detection method, which comprises the following steps of: introducing a first fluorescence indicator into an upstream primer or a downstream primer, introducing a second fluorescence indicator into a dCTP substrate, and labeling the two fluorescence indicators to a ZNF545 gene sequence in a sample to be detected in a PCR amplification process, wherein the methylation degree of ZNF545 genes in the sample to be detected can be quantitatively detected according to the signal strength ratio of the two fluorescence indicators after a fluorescence intensity standardization curve is established. The methylation quantitative detection method is not influenced by methylation sites, and the gene methylation is rapidly and accurately quantified; the sensitivity is high, and detection can be carried out by only a little amount of DNA samples; and moreover, a specific probe or a specific primer is not required to be design, the method is easy, convenient and feasible, and the adopted technology is easy to implement and low in cost.

Description

The ZNF545 gene methylation quantitative detection method

Technical field

The present invention relates to dna methylation and detect, relate generally to a kind of method of detection by quantitative dna methylation.

Background technology

The early diagnosis of cancer is most important to effective treatment of cancer patients.At present to the diagnosis Main Basis clinical symptom of cancer, direct palpation, picture signal detection and histopathological examination etc., but evening appears in most cancer clinical symptom, and living body sampling difficulty has been late period when diagnosing first, had a strong impact on patient's treatment and patient more after.Therefore seek the biomarker of malignant tumour, be applied to early diagnosis and examination, treatment and the prognosis of tumour patient had great importance.

The change of dna methylation degree and pattern is a tumorigenic important factor.These variations comprise hyper-methylation and the genomic dna hypomethylation state of part, CpG island.In normal cell, the CpG island that is positioned at the cancer suppressor gene promoter region is in low-level or methylation state not, and this moment, cancer suppressor gene was in normal open state, the generation of the continuous expression inhibiting tumour of cancer suppressor gene.And in tumour cell, this regional CpG island is by high methylation, and the chromatin conformation changes, the expression of cancer suppressor gene is closed, thereby causes cell to enter the cell cycle, the apoptosis forfeiture, the DNA rectification of defects, vasculogenesis and cell adhesion afunction etc. finally cause tumour to occur.Equally, for some genes and the tumor-necrosis factor glycoproteins that in normal cell, are in high methylation, if its decrease of methylation, these genes will be expressed and tumor-necrosis factor glycoproteins will activate, thereby cause Genomic Imprinting to be lost, and cell transition increases, inappropriate cell specific expression, genome fragility increases, and endoparasitism sequence (endoparasitic sequence) activation, finally also causes tumour to occur.

Because the local height on CpG island methylates will be early than the cell neoplasm, so its methylated detection can be used for the prediction of tumour, and the low-level methylation state of full genomic level then further reduces along with the increase of malignancy, makes its diagnosis that can be used for tumour and classification.In recent years, constantly there are the generation, the development that studies show that human tumor unusually relevant with dna methylation, and before clinical tumor is made a definite diagnosis, just can detect the aberrant methylation phenomenon of specific gene.The biomarker that can be used as the early diagnosiss such as tumour so methylate and prognosis evaluation index are to the examination of tumour and risk assessment, early diagnosis, somatotype, prognosis are judged and the treatment monitoring all has great importance by stages.

The ZNF545 gene is the zinc finger gene that contains KRAB, the albumen of its coding can be combined with DNA, the ZNF545 gene is transcriptional regulator, studies have shown that ZNF545 is a kind of cancer suppressor gene (The KRAB-containing zinc finger protein ZNF545 is a functional tumor suppressor exerting proapoptotic and antiproliferation abilities with frequent epigenetic inactivation in multiple carcinomas, ChengYingduan, LiangPei and GengHua, 101st Annual Meeting of theAmericanAssociationforCancerResearch, 2010), in stomach cancer cell, suppress ribosome-RNA(rRNA) and transcribe (Zinc-finger protein 545 is a novel tumour suppressor that acts by inhibiting ribosomal RNA transcription in gastric cancer, Wang S, Cheng Y and Du W, Gut, 2012), the ZNF545 gene is as cancer suppressor gene, and the generation of its methylation and tumour is closely related.How CpG rich content in the ZNF545 gene order becomes a problem demanding prompt solution to its detection by quantitative that methylates.

Mainly contain following several method in the methylation analysis prior art for specific gene, the first prior art is susceptibility restriction enzyme-P C R/Southern (the methylation-sensitive restriction Endonuclease-PCR/Southern that methylates, MSRE-PCR/Southern), this method utilization methylate the susceptibility restriction enzyme to methylate the district the characteristic of not cutting, after the fragment of DNA digestion for different sizes, carry out Southern or pcr amplification separated product, clear and definite methylation state is analyzed again.The normal responsive restriction enzyme that methylates that uses has HpaII-Msp I (CCGG) and Sma-Xmal (CCCGGG) etc.

The second prior art is the bisulfite sequencing, the method at first makes with bisulfite and methylated cytosine(Cyt) deaminizating does not occur among the DNA is transformed into uridylic, and methylated cytosine(Cyt) remains unchanged, the performing PCR required fragment that increases, then uridylic all changes into thymus pyrimidine, at last, to the PCR product check order and with undressed sequence relatively, judge whether that the CpG site methylates.This method is that tolerance range is very high, the methylation state in each CpG site in the fragment that can have a definite purpose, but need a large amount of cloning and sequencings, process is comparatively loaded down with trivial details, expensive.

The third prior art is, the PCR (methylation-specificPCR, MS-PCR) of methylation-specific, and DNA processes with bisulfite first in the method, goes subsequently the PCR of primer specificity.It designs two pairs of primers, and the sequence complementary pairing after processing with bisulfite respectively is namely a pair of in conjunction with the methylate DNA chain after processing, the non-methylate DNA chain after another is processed combination.Detect the MS-PCR amplified production, if use the primer for methylate DNA chain after processing to amplify fragment, illustrate that then this detected site existence methylates; Vice versa.The method is fit to the situation that methylates in judgement characteristics site.

The 4th kind of prior art is the fluorescent method that methylates, and processes dna fragmentation to be measured in conjunction with bisulfite, designs the probe of an energy and site to be measured district's complementation, and 5 ' end of probe connects report fluorescence, and 3 ' end connects cancellation fluorescence, goes subsequently real-time quantitative PCR.If probe can be hybridized with DNA, then when PCR uses primer extension, TaqDNA polysaccharase 5 ' can be with the report fluorescence cutting-out of 5 ' end on the probe sequence to the 5 prime excision enzyme activity of 3 ' end, cancellation fluorescence no longer can suppress report fluorescence, the report fluorescence radiation is measured each circulation and is reported that the intensity of fluorescence can obtain the situation that methylates and the degree in this site like this.The method be fit to be analyzed the specific site situation that methylates.

The 5th kind of prior art is the tetra-sodium order-checking, the method is by the enzyme cascade chemiluminescence reaction in 4 kinds of same reaction systems of enzyme catalysis, take turns in the sequencing reaction at each, only add a kind of dNTP, if the pairing of this dNTP and template, polysaccharase just can join it in primer strand and the tetra-sodium (PPi) of mole number such as discharge.PPi can finally be converted into visible light signal, and is converted into a peak value by PyrogramTM, and its height is directly proportional with the Nucleotide number.When detecting when being used for methylating, can be regarded as the SNP change of C-T type through the sequence of bisulfite processing.

The 6th kind of prior art is the restriction enzyme enzyme process in conjunction with bisulfite, and this method is processed and pcr amplification the capable bisulfite of specimen dna, and former methylated cytosine(Cyt) is retained after processing, but not methylated cytosine(Cyt) becomes thymus pyrimidine.Subsequently with restriction enzyme to the characteristic that transforms the cutting of rear PCR product to identify the methylation status of former specimen dna.

More than every prior art still lack good solution aspect quantitatively accurately and efficiently in that the specific gene regional DNA is methylated.Because ZNF545 gene GpC content is higher, and the CpG site methylates and has randomness, especially occur in the very low situation of early stage methylation in tumour, method for specifically methylate site design Auele Specific Primer or probe has very large limitation, can not react exactly the situation that methylates of CpG site accumulation area in the whole ZNF gene.In addition, the tumor development stage mainly is relevant with the methylation of the district occurred frequently that methylates, and irrelevant with concrete which CpG site.Therefore need to solve existing quantitative methylation analysis method and be subjected to the problem that methylates at random and affect, proposes a kind of new easy, obtain the quantitative methylation analytical procedure direct relevant with tumor development reliably, fast.

Summary of the invention

The method that the purpose of this invention is to provide a kind of detection by quantitative dna methylation can be obtained the methylation analysis direct relevant with tumor development easy, reliably, fast.

The method of a kind of ZNF gene methylation detection by quantitative provided by the invention may further comprise the steps:

Step 1, set up the standardized curve of ZNF545 gene by fluorescence intensity:

It is that 100% positive quality control product and methylation are 0% negative quality control product that the ZNF545 gene is set up respectively methylation, be to mix in 0: 1,1: 3,1: 1,3: 1,1: 0 the positive and negative quality control product according to mass ratio, obtain one group of standard substance, the methylation of standard substance is respectively 0%, 25%, 50%, 75%, 100%;

With upstream primer or the downstream primer of the first fluorescent mark substance markers ZNF545 gene, with the second fluorescent mark substance markers dCTP;

With dTTP, dGTP, dATP and the dCTP that is marked with the second fluorescent marker as substrate, with the primer that is marked with the first fluorescent marker standard substance are carried out pcr amplification, detect the first fluorescent marker in the product of described standard substance pcr amplification and the fluorescence intensity of the second fluorescent marker;

Wherein, the fluorescence intensity of the first fluorescent marker is M, and the fluorescence intensity of the second fluorescent marker is N,

Take N/M as ordinate zou, the methylation of standard substance is X-coordinate, obtains the typical curve equation of ZNF545 gene methylation;

Step 2: testing sample ZNF545 gene methylation detection by quantitative

The poba gene group DNA of extracting sample to be tested carries out the hydrosulphite conversion processing to genomic dna;

Upstream primer or downstream primer with the first fluorescent mark substance markers ZNF545 gene, with the second fluorescent mark substance markers dCTP, with dTTP, dGTP, dATP and the dCTP that is marked with the second fluorescent marker as substrate, carry out pcr amplification with the primer that is marked with the first fluorescent marker sample to be tested genomic dna after to bisulf iotate-treated, the first fluorescent marker in the product behind the detection pcr amplification and the strength of signal of the second fluorescent marker, the strength of signal of the first fluorescent marker is M1, and the strength of signal of the second fluorescent marker is N1;

Step 3, with the typical curve equation of N1/M1 substitution step 1, calculate the value of corresponding X-coordinate, be ZNF545 gene methylation degree in the sample to be tested.

The present invention also provides the method for another kind of ZNF545 gene methylation detection by quantitative, it is characterized in that, may further comprise the steps:

Step 1, set up the standardized curve of ZNF545 gene by fluorescence intensity:

With the upstream primer of the first fluorescent mark substance markers ZNF545 gene, with the 3rd marker mark downstream primer or with the downstream primer of the first fluorescent mark substance markers ZNF545 gene, with the 3rd marker mark upstream primer; With the 3rd marker mark with the second fluorescent mark substance markers dCTP;

With dTTP, dGTP, dATP and the dCTP that is marked with the second fluorescent marker as substrate, with the upstream primer primer that is marked with the first fluorescent marker and the downstream primer that is marked with the 3rd marker or with the downstream primer primer that is marked with the first fluorescent marker and the upstream primer that is marked with the 3rd marker standard substance are carried out pcr amplification, described the 3rd marker is affinity tag; Pcr amplification product is joined in the enzyme plate that is fixed with avidin, pcr amplification product is fixed on the enzyme plate by the effect of the 3rd marker and avidin, measure the strength of signal of the first fluorescent marker and the second fluorescent marker with microplate reader;

Take N/M as ordinate zou, the methylation in the standard substance is X-coordinate, obtains the typical curve equation of ZNF545 gene methylation;

Step 2: testing sample ZNF545 gene methylation detection by quantitative

With dTTP, dGTP, dATP and the dCTP that is marked with the second fluorescent marker as substrate, with the upstream primer primer that is marked with the first fluorescent marker and be marked with the downstream primer of the 3rd marker or with the downstream primer primer that is marked with the first fluorescent marker and the upstream primer that is marked with the 3rd marker to the sample to be tested genomic dna after the bisulf iotate-treated is carried out pcr amplification, described the 3rd marker is affinity tag; Pcr amplification product is joined in the enzyme plate that is fixed with avidin, pcr amplification product is fixed on the enzyme plate by the effect of the 3rd marker and avidin, measure the strength of signal of the first fluorescent marker and the second fluorescent marker with microplate reader, the strength of signal of the first fluorescent marker is M1, and the strength of signal of the second fluorescent marker is N1;

Step 3, with the typical curve equation of N1/M1 substitution step (1), calculate the value of corresponding X-coordinate, be ZNF545 gene methylation degree in the sample to be tested.Preferably, the process of the described ZNF of foundation gene masculine quality control product is:

Cultivate breast cancer cell line MB231 cell, extract the MB231 cell genomic dna, the genomic dna that extracts is carried out the hydrosulphite conversion processing, carry out pcr amplification with the ZNF545 gene as the DNA of purpose fragment after to bisulf iotate-treated, the PCR product is connected with carrier after reclaiming purifying, be transformed in the E.coli DH5 α competent cell, recombinant clone is checked order, selecting the CG site does not have the sequence of change as positive quality control product.

Preferably, the process of the negative quality control product of the described ZNF of foundation gene is:

Cultivate normal immortal continuous cell line HEK293 cell, extract the HEK293 cell genomic dna, the genomic dna that extracts is carried out the hydrosulphite conversion processing, carry out pcr amplification with the ZNF545 gene as the DNA of purpose fragment after to bisulf iotate-treated, the PCR product is connected with carrier after reclaiming purifying, be transformed in the E.coli DH5 α competent cell, recombinant clone is checked order, select the CG site and all become the sequence of TG as positive quality control product.

Preferably, the pcr amplification primer sequence of described ZNF gene is:

Upstream primer: 5-TTATAGGGTTTTTTAGTTGGTAT-3 ' (SEQ ID No:4);

Downstream primer: 5 '-CCTCTCTCTTTACCCCCTAA-3 ' (SEQ ID No:5).

Preferably, described the first fluorescent marker is FITC.

Preferably, described the second fluorescent marker is Cy5.

Preferably, the reaction conditions of described pcr amplification is: initial 95 ℃ of 5min; Then 95 ℃ of 30s, 60 ℃ of 30s, 72 ℃ of 30s carry out 40 circulations; Last 72 ℃ of 10min.

Preferably, described the 3rd marker is vitamin H.

Beneficial effect of the present invention is, the first, and the do not methylated impact in site of the method for the detection by quantitative that methylates of the present invention fast, is carried out quantitatively gene methylation accurately; The second, sensitivity of the present invention is higher, only needs the DNA sample of trace just can detect; The 3rd, the present invention need not to design specific probe or Auele Specific Primer, and method is simple, and the technology that adopts simply is easy to realize that cost is low.

Description of drawings

Fig. 1 is the standardized curve of ZNF545 gene by fluorescence intensity among the embodiment 1.

Embodiment

The sequence of ZNF545 gene is shown in SEQ ID No:1, and ZNF545 gene CpG site is more, and is larger for the difficulty of each CpG designing probe.

ZNF gene methylation quantitative detection method of the present invention, step is as follows:

Step 1, set up the standardized curve of ZNF545 gene by fluorescence intensity:

Step 2: testing sample ZNF545 gene methylation detection by quantitative

(1) the poba gene group DNA of extracting sample to be tested;

(2) genomic dna is carried out the hydrosulphite conversion processing;

Step 3, with the typical curve equation of N1/M1 substitution step (1), calculate the value of corresponding X-coordinate, be ZNF545 gene methylation degree in the sample to be tested.

The C of sample to be tested genomic dna on methylated CpG site after the bisulf iotate-treated keeps, the C in other site is converted into U, in the process of dna replication dna, become T, that is to say, after the bisulf iotate-treated in the ZNF gene quantity of Nucleotide C identical with the methylated quantity of CpG, the dCTP of the second fluorescent mark substance markers can enter methylated CpG site in the PCR process, also can with dna sequence dna in G pairing, because the G content in the whole dna sequence dna is determined, therefore the quantity variance that is mainly the methylated CpG site that causes the second fluorescent marker strength difference, therefore, directly characterize the content of methylated CpG with the strength of signal (N) of the second fluorescent marker, the strength of signal of the first fluorescent marker (M) characterizes the content of DNA, and N/M characterizes methylation.

Further, mark can be used for mark such as the biotin of affine absorption on fluorescently-labeled another primer, avidin in the pcr amplification product is combined with the enzyme plate that contains the avidin acceptor, be fixed on the enzyme plate, not only can carry out purifying behind the pcr amplification, also available fluorescence microplate reader is directly measured the fluorescence intensity of the first fluorescent marker and the second fluorescent marker.

Below in conjunction with drawings and Examples the present invention is further elaborated.

Embodiment 1

(1), the preparation of ZNF545 gene masculine and negative quality control product

1, methylation is the preparation of 100% ZNF545 gene masculine quality control product

I, cultivation breast cancer cell line MB231 cell extract MB231 cell (referred to as MB231) genomic dna according to the described method of following experimental technique (1);

Ii, according to the described method of following experimental technique (2) the MB231 genomic dna is carried out bisulf iotate-treated;

Iii, gained DNA among the step I i is carried out pcr amplification, primer is that specifying information is as follows for the Auele Specific Primer of ZNF545 gene design:

Upstream primer: 5-TTATAGGGTTTTTTAGTTGGTAT-3 ' (SEQ ID No:4);

Downstream primer: 5 '-CCTCTCTCTTTACCCCCTAA-3 ' (SEQ ID No:5)

Carry out pcr amplification according to the ZNF545 gene amplification PCR reaction system of table 1 and the reaction conditions of table 2, obtain the ZNF545 gene, pCR-TOPO4 with Invitrogen company connects test kit with the rear Transformed E .coliDH5 α competent cell of PCR product connection, be inoculated into and contain on the antibiotic LB solid medium, in 37 ℃ of incubators, cultivate 12h;

Table 1ZNF545 gene amplification PCR reaction system

Table 2ZNF545 gene expands once PCR reaction conditions

Iv, to independent in the E.coli bacterium colony of gained among the step I ii, full white colony as recombinant clone, add and contain in the antibiotic LB liquid nutrient medium 37 ℃, 200rpm, incubated overnight;

V, to checking order behind the gained bacterium liquid extracting plasmid among the step I v;

Vi, the former sequence of ZNF545 gene in sequencing result and the genome (SEQ ID No:1) is compared, select the CG site do not become fully TG as positive quality control product (SEQ ID No:2).

2, methylation is the preparation of the negative quality control product of 0% ZNF545 gene

I, the normal immortalized cell line HEK293 cell of cultivation extract HEK293 cell (referred to as HEK293) genomic dna according to the described method of following experimental technique (1);

Ii, according to the described method of following experimental technique (2) the HEK293 genomic dna is carried out bisulf iotate-treated;

Upstream primer: 5-TTATAGGGTTTTTTAGTTGGTAT-3 ' (SEQ ID No:4);

Downstream primer: 5 '-CCTCTCTCTTTACCCCCTAA-3 ' (SEQ ID No:5)

Vi, the former sequence of ZNF545 gene (SEQ IDNo:1) in sequencing result and the genome is compared, select the negative quality control product (SEQ ID No:3) of conduct that the CG site becomes TG fully.

Experimental technique:

(1), the extracting method of genomic dna

The concrete steps of DNA extraction are as follows:

I, TRIzol is joined in the cell solution, ratio is 1 * 10 ⁷Individual cell adds 3mL TRIzol, and cell is fully suspended, and with the concussion of vortex vibrator cell is evenly suspended, and places 5min under the room temperature;

Ii, add chloroform, the volume of chloroform is 0.2 times of TRIzol reagent in the step I, with the abundant mixing of vortex vibrator, places 3min under the room temperature, then at 4 ℃, and centrifugal 15min under the 12000rpm condition;

Iii, remove supernatant liquid (for water), keep lower floor's liquid (organic phase), add 100% ethanol, the volume of 100% ethanol is 0.3 times of TRIzol reagent in the step I, and mixing is placed 3min under the room temperature, then at 4 ℃, centrifugal 5min under the 2000rpm condition;

Iv, remove supernatant liquid, with 0.1M Trisodium Citrate-10% ethanolic soln flushing lower sediment, the volume of used 0.1M Trisodium Citrate-10% ethanolic soln is identical with the volume of TRIzol reagent in the step I, at room temperature place 30min, then at 4 ℃, centrifugal 5min under the 2000rpm condition repeats above step 1 time again;

V, with the precipitation Eddy diffusion that obtains among the step I v in 75% ethanol, the volume of 75% ethanol be TRIzol reagent in the step I 1.5-2 doubly, place 20min under the room temperature, then at 4 ℃, centrifugal 5min under the 2000rpm condition;

Vi, be deposited under the room temperature centrifugal rear gained among the step v air-dry, add 8mM NaOH, the volume of 8mM NaOH is the sixth of TRIzol reagent volume in the step I, 4 ℃ of lower 12h that place, make fully dissolving of precipitation, with hydroxyethyl piperazine second thiosulfonic acid damping fluid (HEPES buffer, Gibco BRL company), gained DNA sample is preserved under 4 ℃ of conditions.

(2), the method for bisulf iotate-treated genomic dna, make methylated cytosine(Cyt) deaminizating does not occur in the described genomic dna to change uridylic into.

Concrete steps are as follows:

The pruning of i, genomic dna

5 μ g DNA are dissolved in the 30 μ L TE damping fluids, add 3M NaOH3.3 μ L, the concentration that makes NaOH in the solution is 0.3M, then 37 ℃ of lower 15min that place;

Ii, bisulfite reaction

Bisulfite solution preparation: take by weighing the 1.82g Sodium Pyrosulfite, add 2.8mLH2O and 430 μ L3M NaOH solution, solid is fully dissolved, add 210 μ L10mM Resorcinol solution, regulating the pH value is 5.0-5.2, the pyrosulphite na concn is 2.4M in the gained solution, is equivalent to sodium sulfite solution 4.8M.

Add in the dna solution of 333 μ L bisulfite solutions gained in the step I, under 55 ℃ of conditions, dark reaction 4h, in reacted liquid, add 1mL QX1 (dissolving and binding buffer liquid, Qiagen company), add 40 μ LQiaex II solution (Qiaex II glue reclaims the damping fluid Qiaex II in the test kit), rotate 1h under the room temperature, reclaim test kit with Qiaex II glue again and carry out the DNA purifying to reacting rear solution, with 50 μ LTE dissolving purifying gained DNA, place 5min under the room temperature, will note keeping static when placing under the room temperature.

Iii, bisulfite reaction aftertreatment

In step I i gained solution, add 5.56 μ L3M NaOH solution, place 15min for 37 ℃, rap mixing with finger, add again 27.78 μ L pH values and be 7.0 Spirit of Mindererus, finger raps mixing, add again 27.78 μ LpH values and be 5.2 sodium acetate soln, at this moment, the pH value of step I i gained solution adds 1mL QX1 solution again less than 7.0, rotate 20min under the room temperature, reclaim test kit with Qiaex II glue above gained solution is carried out the DNA purifying, add first 60 μ LTE damping fluid room temperatures behind the purifying and transfer and put 5min, add again 37 ℃ of 40 μ LTE damping fluids and place 10min, obtain the DNA of bisulf iotate-treated, under-20 ℃ of conditions, preserve.

(2), set up ZNF545 gene methylation typical curve

1, the preparation of standard model

Positive and the negative quality control product of will methylating carries out pcr amplification, and (primer is the primer of use in () 1, reaction system sees Table 1, reaction conditions sees Table 2), obtain positive and negative amplified production according to positive amplified production: negative amplified production mass ratio mixed in 0: 1,1: 3,1: 1,3: 1,1: 0, be respectively methylation 0%, 25%, 50%, 75% and 100% standard model.

2, the pcr amplification of standard model

(1) standard model primer mark

5 ' end of upstream sequence primer (SEQ ID No:4) is introduced FITC, upstream primer is carried out fluorescent mark, downstream primer (SEQ ID No:5).

(2) standard model pcr amplification

Substrate is dATP, dTTP, and the mixture of dGTP and Cy5-dCTP (with the triphosphoric acid cytosine(Cyt) deoxynucleoside of Cy5 fluorescence molecule mark), the PCR reaction system of standard model sees Table 3, and the PCR reaction conditions of standard model sees Table 4.

Table 3 standard model PCR reaction system

Table 4 standard model PCR reaction conditions

(3) standard model PCR product purification

Reclaim test kit with Qiaex II glue the pcr amplification product of standard model is carried out the DNA purifying.

(4) the standard model pcr amplification product behind the purifying is carried out fluorescence spectrum scanning, when being 494nm, excitation wavelength obtains FITC fluorescence intensity (value is M), obtain Cy5 (value is N) fluorescence intensity when excitation wavelength is 648nm, the fluorescent value of FITC and Cy5 and the value of Cy5/FITC are shown in Table 5.

FITC fluorescent value and the Cy5 fluorescent value of the different methylation standard model of table 5 pcr amplification product

Take N/M as ordinate zou, methylation is X-coordinate, carries out simple linear regression analysis, obtains linear ZNF545 Standardization curve for fluorescence intensity Fig. 1 and equation of linear regression: y=0.3474x+0.4679 (R ²=0.9963).

(3) the testing sample detection by quantitative that methylates

1, the preparation of testing sample

Extracting testing sample poba gene group DNA carries out bisulf iotate-treated with the described method of above-mentioned experimental technique (2) with the testing sample genomic dna.

2, the pcr amplification of testing sample

(1) primer mark

(2) testing sample pcr amplification

Substrate is dATP, dTTP, and the mixture of dGTP and Cy5-dCTP (with the triphosphoric acid cytosine(Cyt) deoxynucleoside of Cy5 fluorescence molecule mark), the PCR reaction system of testing sample sees Table 3, and the PCR reaction conditions of standard model sees Table 4.

(3) testing sample PCR product purification

Reclaim test kit with Qiaex II glue the pcr amplification product of testing sample is carried out the DNA purifying.

(4) the testing sample pcr amplification product behind the purifying is carried out fluorescence spectrum scanning, when being 494nm, excitation wavelength obtains FITC fluorescence intensity (value is M1), when being 648nm, excitation wavelength obtains Cy5 (value is N1) fluorescence intensity, calculate the value of N1/M1, N1/M1 is updated to as the y value calculates the x value among the typical curve equation y=0.3474x+0.4679 and be the testing sample methylation.

Embodiment 2

Two primer marks

The concrete operation step of all processes is seen (one) part among the embodiment 1.

(2), set up ZNF545 gene methylation typical curve

1, the preparation of standard model

The concrete operation step of all processes is seen (two) 1 parts among the embodiment 1

2, the pcr amplification of standard model

(1) design of primers

5 ' end of upstream sequence primer (SEQ ID No:4) is introduced FITC, upstream primer is carried out fluorescent mark, with 5 ' end primer vitamin H (biotin) of downstream primer (SEQ ID No:5).

(2) standard model pcr amplification

Substrate is dATP, dTTP, and the mixture of dGTP and Cy5-dCTP (with the triphosphoric acid cytosine(Cyt) deoxynucleoside of Cy5 fluorescence molecule mark), the PCR reaction system of standard model sees Table 6, and the PCR reaction conditions of standard model sees Table 4.

Table 6 standard model PCR reaction system

(3) standard model PCR product is fixed

Pcr amplification product joined in the enzyme plate that is fixed with avidin and placed 4 hours under 37 ℃ of conditions, pcr amplification product is fixed on the enzyme plate, again with phosphoric acid buffer flushing enzyme plate three times.

(4) enzyme plate that contains pcr amplification product that obtains in the step (3) is put into microplate reader and carry out fluorometric assay, when excitation wavelength is 494nm, obtain FITC fluorescence intensity (value is M), when excitation wavelength is 648nm, obtain Cy5 (value is N) fluorescence intensity.

Take N/M as ordinate zou, methylation is X-coordinate, carries out simple linear regression analysis, obtains linear ZNF545 Standardization curve for fluorescence intensity and equation of linear regression.

(3) the testing sample detection by quantitative that methylates

(1) design of primers

(2) standard model pcr amplification

Substrate is dATP, dTTP, and the mixture of dGTP and Cy5-dCTP (with the triphosphoric acid cytosine(Cyt) deoxynucleoside of Cy5 fluorescence molecule mark), the PCR reaction system of testing sample sees Table 6, and the PCR reaction conditions of testing sample sees Table 4.

(3) testing sample PCR product is fixed

(4) enzyme plate that contains pcr amplification product that obtains in the step (3) is put into microplate reader and carry out fluorometric assay, when being 494nm, excitation wavelength obtains FITC fluorescence intensity (value is M2), when being 648nm, excitation wavelength obtains Cy5 (value is N2) fluorescence intensity, calculate N2/M2 ratio, be updated to typical curve Equation for Calculating testing sample methylation.

Above-described embodiment of the present invention does not consist of the restriction to protection domain of the present invention.Any modification of doing within the spirit and principles in the present invention, be equal to and replace and improvement etc., all should be included in claim protection domain of the present invention it.

Claims

1. a ZNF545 gene methylation quantitative detection method is characterized in that, may further comprise the steps:

Step 1, set up the standardized curve of ZNF545 gene methylation:

Wherein, the fluorescence intensity of the first fluorescent marker is M, and the fluorescence intensity of the second fluorescent marker is N;

Step 2: testing sample ZNF545 gene methylation detection by quantitative:

2. a ZNF545 gene methylation quantitative detection method is characterized in that, may further comprise the steps:

Step 1, set up the standardized curve of ZNF545 gene methylation:

With dTTP, dGTP, dATP and the dCTP that is marked with the second fluorescent marker as substrate, with the upstream primer primer that is marked with the first fluorescent marker and the downstream primer that is marked with the 3rd marker or with the downstream primer primer that is marked with the first fluorescent marker and the upstream primer that is marked with the 3rd marker standard substance are carried out pcr amplification, described the 3rd marker is affinity tag; Pcr amplification product is joined in the enzyme plate that is fixed with the avidin acceptor, pcr amplification product is fixed on the enzyme plate by the effect of the 3rd marker and avidin acceptor, measures the strength of signal of the first fluorescent marker and the second fluorescent marker with microplate reader;

Step 2: testing sample ZNF545 gene methylation detection by quantitative:

With dTTP, dGTP, dATP and the dCTP that is marked with the second fluorescent marker as substrate, with the upstream primer primer that is marked with the first fluorescent marker and be marked with the downstream primer of the 3rd marker or with the downstream primer primer that is marked with the first fluorescent marker and the upstream primer that is marked with the 3rd marker to the sample to be tested genomic dna after the bisulf iotate-treated is carried out pcr amplification, described the 3rd marker is affinity tag; Pcr amplification product is joined in the enzyme plate that is fixed with the avidin acceptor, pcr amplification product is fixed on the enzyme plate by the effect of the 3rd marker and avidin acceptor, measure the strength of signal of the first fluorescent marker and the second fluorescent marker with microplate reader, the strength of signal of the first fluorescent marker is M1, and the strength of signal of the second fluorescent marker is N1;

3. ZNF gene methylation quantitative detection method according to claim 1 and 2 is characterized in that, the process of the described ZNF of foundation gene masculine quality control product is:

4. ZNF gene methylation quantitative detection method according to claim 1 and 2 is characterized in that, the process of the negative quality control product of the described ZNF of foundation gene is:

5. ZNF gene methylation quantitative detection method according to claim 1 and 2 is characterized in that, the pcr amplification primer sequence of described ZNF gene is:

Upstream primer: 5-TTATAGGGTTTTTTAGTTGGTAT-3 ';

Downstream primer: 5 '-CCTCTCTCTTTACCCCCTAA-3 '.

6. ZNF gene methylation quantitative detection method according to claim 1 and 2 is characterized in that, described the first fluorescent marker is FITC.

7. ZNF gene methylation quantitative detection method according to claim 1 and 2 is characterized in that, described the second fluorescent marker is Cy5.

8. ZNF gene methylation quantitative detection method according to claim 1 and 2 is characterized in that, the reaction conditions of described pcr amplification is: initial 95 ℃ of 5min; Then 95 ℃ of 30s, 60 ℃ of 30s, 72 ℃ of 30s carry out 40 circulations; Last 72 ℃ of 10min.

9. ZNF gene methylation quantitative detection method according to claim 2 is characterized in that, described the 3rd marker is vitamin H.