CN109385464A - A kind of DNA methylation detection kit and method - Google Patents

Abstract

The present invention relates to a kind of DNA methylation detection kit and methods, more particularly to a kind of there is no the detection kit and method in the site CpG for flanking sequence, the kit and method have used and minor groove binding (minor groove binder, MGB) the Taqman probe combined, the island CpG (CpG Island can not only be measured, CGI methylation), can also measure this two sides sequences such as the CpG open sea in genosome does not have the isolated site CpG of CpG.The technology is not only very special and sensitive, but also easy to operate, does not need the DNA standard items of control reaction and exhaustive methylation as reference, so as to overcome their bring defects.In addition, it has repeatability more higher than existing MethyLight technology and accuracy.

Description

A kind of DNA methylation detection kit and method

Technical field

The invention belongs to analysis method technical field, it is related to a kind of DNA methylation detection kit and method, especially needle There is no the DNA methylation detection kit in the site CpG and method to flanking sequence.

Background technique

The methylation of cytimidine -5DNA present in cancerous tissue (m5C) is considered to have the DNA table of potential clinical value See modification [1].In vertebrate, m5C mainly appears on CpG dinucleotides.It is had confirmed in kinds of tumors, tumor suppressor gene The island CpG (CGI) abnormal methylation of promoter results in transcriptional inactivation [2].However, the CGI in promoter only represents methylation Sub-fraction, and the CpG open sea being predominantly located in genosome then represents DNA methyl most conservative in eucaryote Change target, but the function of region methylation is unclear.Nearest research disclose non-start up subregion (such as genosome and UTR) the synergistic effect to methylate to gene expression, genosome methylation may be potential therapy target [3] in cancer.This Outside, genosome methylation may be the potential mechanism [4] of RNA alternative splicing regulation, and can limit transcription initiation, to hinder Only aberrant transcription [5].We pass through EPIC methylation chip discovery, recurrence and non-recurrence colorectal cancer patients in previous research Differential methylation site (differential methylation positions, DMPs) in CGIs and promoter very It is few but many in open sea and genosome.We also have found in genosome one group relevant to gene overexpression high Methylated CpG site.

Since the methylation in analysis genome has very big value, the methods and techniques of many detection methylations are opened Hair.Methylation status of PTEN promoter (methylation-specific PCR, MSP) is a kind of end point analysis technology [6,7]；Later, The technology of second generation based on PCR --- MethyLight (quantitative MSP) is used for quantitative detection [8-10].Using being based on The MethyLight technology of AluC4 control reaction, has been widely used for the methylation [11,12] of CGI in detection tissue sample.So And all these existing DNA methylation detection techniques require that site to be measured is located at the area with cluster CpG dinucleotides Domain, such as the island CpG, to allow the primer and probe designed to cover enough sites CpG, detection is also not this position Point, but the situation to be methylated altogether by whole sites CpG that primer and probe covers, however, the prior art has the following problems:

(1) methylation of heterozygosis present in genome can not accurately be detected.If the site adjacent C G does not show as total methyl Change phenomenon, it can not primer and probe needed for successful design qMSP technology in this region.

(2) island CpG can not be detected with the methylation level of exterior domain.The island CpG includes open with exterior domain in genome The regions such as sea, CpG shore are predominantly located at gene body and intergenic region, this region site CG is less, seldom there is phase The adjacent site CG, primer and probe needed for qMSP technology can not be designed in this region.

(3) absolute quantitation of methylation level relies on methylation standard items, if to obtain certain site methylation level 100% methylation standard items need to be arranged in absolute value simultaneously, pass through the ratio calculation of sample and standard items methylation percentage parameter (percentage of methylation ratio,PMR).If methylation standard items production batch is different or presence can not be kept away The mass defect exempted from, such as 100% methylation, the spontaneous deamination of m5C are not converted into C, DNA degradation in the site CG, will cause methyl Change the quantitative gross error of horizontal absolute value.

Current DNA methylation assay is the methylation sites detection for the island CpG, unless using sequencing (bisulfites Pyrosequencing), it just can detecte such site, but it is excellent without cost-benefit in the queue verification of large sample Gesture.

And MSP and MethyLight cannot be analyzed and be mainly distributed in the regions such as the intracorporal open sea of gene, flank The isolated site CpG in the site sequence C pG.

Summary of the invention

The purpose of the present invention is to provide a kind of methylation detection kit and detection methods.

The purpose of the present invention is to provide a kind of DNA methylation inspections for any site CpG in whole gene sequence Test agent box and method.

It is another object of the present invention to for flanking sequence do not have the site CpG DNA methylation detection kit and Method.

It is another object of the present invention to provide a kind of DNA methylation detection kit without the standard items that methylate and Method.

High, cheap, convenient, rapid DNA methylation detection that it is another object of the present invention to provide a kind of accuracys Kit and method.

Above-mentioned purpose of the invention is realized by following technological means:

On the one hand, the present invention provides a kind of DNA methylation detection method, the method the following steps are included:

A) uracil is converted by the cytosine base of the non-methylation of DNA sample, and the cytosine base to methylate is protected It holds constant；

B) at least pair of primers and at least, a pair of oligonucleotide probe amplification step (1) for covering the site CpG to be measured turns DNA sample after change；Wherein, the oligonucleotide probe combination minor groove binding (minor groove binder, MGB)；

C) amplified production is analyzed, and from the methylation state of the Analysis of Existence of amplified production DNA to be measured.

The site CpG of the method detection is that the site CpG in heterozygosis methylation region or flank do not have CpG's The isolated site CpG, perhaps altogether methylate region the site CpG or be it is non-altogether methylate region the site CpG.As excellent The embodiment of choosing, the site CpG of method detection of the invention are that flank does not have the isolated site CpG of CpG.

Alternatively, the site CpG is the site CpG in the site CpG or the island CpG outside the island CpG.

Alternatively, the site CpG is located at genosome, intergenic region or promoter.

As preferred embodiment, the site CpG be positioned at CpG open sea of genosome or intergenic region, CpG shore、CpG shelf。

The method of existing a variety of detection DNA methylations, such as use MethyLight (methylation-specific quantitative PCR) It checks the methylation of the island CpG (CpG Island, CGI), is mainly based upon in the island promoter region Zhong CpG adjacent C pG Put generally existing the phenomenon that methylating altogether.The hypothesis of the DNA methylation assay technical basis of traditional based on PCR are as follows: complete in the island CpG The portion site CG occurs to methylate or do not methylate simultaneously simultaneously, and referred to as methylate region altogether.The primer and probe of design Cover multiple sites CG in the region, detection is these sites CG while the ratio that methylates, not CG single The high resolution detection technology of point.

Although full-length genome methylates, sequencing technologies are disclosed, and the total methylation phenomenon on the island CpG is generally existing, however, starting CGI in son methylates altogether only represents the sub-fraction of methylation, however it remains many is not the region of total methylation, wherein phase The methylation that the adjacent site CG has, some is without methylation, referred to as heterozygosis methylation region.Heterozygosis methylation region is primarily present in In CpG open sea, shore, shelf and the part island CpG.In addition, there are many more CGI is not located at, but it is located at base Because the flanking sequence of body, intergenic region does not have the isolated site CpG of CpG.Unfortunately, the technology of existing based on PCR can not Examining the site the CpG either flanking sequence of this heterozygosis methylation does not have the isolated site CpG of CpG.

Due to the high resolution detection technology that the present invention is the single site CG, significantly larger than traditional technology 3~10 CG The site the CG methylation water in the methylation of heterozygosis present in methylation group region can be effectively detected in the low resolution of point, the present invention Flat and flank does not have the isolated site CpG of CpG.

The flank does not have the isolated site CpG of CpG to be generally positioned in the site CpG outside the island CpG, is such as located at gene The CpG open sea of body or intergenic region, CpG shore, CpG shelf.

Wherein, in step a), the cytosine base of the non-methylation of DNA sample is converted to uracil, and is methylated Cytosine base remain unchanged, convert uracil for the cytosine base of non-methylation using transforming agent.Described Transforming agent is not particularly limited, and the reagent that the achievable cytimidine reported in the prior art to uracil converts can be with, as hydrazonium salt, Weight bisulfites and bisulfites (such as sodium metabisulfite, potassium bisulfite, bisulfite caesium, ammonium bisulfite etc.) One or more of.As a kind of exemplary embodiment, the transforming agent is selected from bisulfites.

Methylation is more methyl on cytimidine, by bisulfites or weight bisulfites or hydrazonium salt After equal transforming agents processing, the cytimidine of non-methylation will become urea pyrimidine, because of uracil and thymidine when being expanded Similar and can be identified as thymidine, being embodied on extension increasing sequence is exactly that there is no the cytimidines of methylation to become thymus gland The cytimidine (C) of pyrimidine (C becomes T), methylation will not then change.Therefore, after converting DNA sample using transforming agent, first The CpG of base is remained CpG (CG), and for deamination at TpG (TG), the CG/TG then used is special after the CpG of non-methylenation conversion The site specific probes combination CpG.

Wherein, in step b), the length of the primer amplification is 50~200bp.

The primer that amplification length is 50~200bp is designed, the cell not only cultivated in vitro or is mentioned in Fresh Frozen tissue In the high quality DNA taken, and fix in formalin-paraffin-embedded tissue (FFPE) in the fragmentation DNA that extracts, can Quantitative fluorescent PCR is effectively realized, thus the delicately methylation level of test sample.Amplification length is too long, not only PCR itself Amplification efficiency reduces rapidly, and probe 5 ' holds efficiency of fluorophor during PCR by archaeal dna polymerase cutting release also can It reduces, influences the accuracy of detection；In addition, the quality control standards such as integrality of primer pair DNA of long amplicon are more demanding, In the clinical common sample such as FFPE, DNA Chang Gaodu fragmentation.

As preferred embodiment, primer should avoid the site CG: this technology is especially suitable between genosome and gene Area, the site CG in the regions such as open sea, the site adjacent C G is less, and probe covers the site CG to be measured, and primer should avoid other CG Site, the influence to avoid heterozygosis methylation to testing result.

Wherein, in step b), described a pair covers the oligonucleotide probe in the site CpG to be measured, wherein a specificity In conjunction with CG sequence, another specific binding TG sequence, i.e., in a pair of of probe, one for combining the CpG of methylation Site, one for combining the site CpG of non-methylation.

As a preferred embodiment, the oligonucleotides that described a pair covers the site CpG to be measured is visited in the present invention Needle is a pair of Taqman probe.5 ' ends of every Taqman probe connect fluorophor, and 3 ' ends connect quencher and MGB base Group.And the fluorophor of two 5 ' ends of Taqman probe connection has different wavelength of transmitted light.It is finally complete with the site CpG site Probe structure is destroyed when full matching, to issue fluorescence and be detected.More specifically, wherein a probe is for non-methyl Change the TG-Taqman-MGB probe in the site CpG, another is for methylated CpG site CG-Taqman-MGB probe.

As preferred embodiment, the fluorophor is not particularly limited, and can be selected from probe in the prior art Fluorophor, such as each probe, can selected from as FAM, VIC, ROX, TAMRA, SYTO9, JOE/TET/HEX, One of Texas Red and NED/BODIPY/TMR-X etc., but need to keep in a pair of of probe, every probe institute The fluorophor of connection has different wavelength of transmitted light, so that they can distinguish in fluorescence detection.It needs to illustrate FAM and SYTO9 wavelength of transmitted light having the same, VIC and JOE/TET/HEX) wavelength of transmitted light having the same, ROX and Texas Red wavelength of transmitted light having the same, TAMRA and NED/BODIPY/TMR-X wavelength of transmitted light having the same, are answered It avoids selecting the identical fluorophor of above-mentioned two wavelength of transmitted light.

As preferred embodiment, the quencher is also not particularly limited, and fluorophor in the prior art is quenched It goes out one of agent, such as NFQ, BHQ1 and BHQ2.

The Taqman probe that the present invention uses can increase the annealing temperature of probe in the MGB group of 3 ' end connections.

MGB main application at present is that the Genotyping of DNA polymorphism detects.The present invention is in DNA methylation assay by MGB It is introduced into probe, dexterously by simply mode, makes full use of the single base mismatch close to MGB that annealing temperature is caused to may be up to The characteristic of 10 DEG C or more such as 17 DEG C of rapid drawdowns, by the methylation in site to be measured and the design of the non-site methylation CG in probe close to MGB Region, i.e., the island many years Fei CpG methylation sites test problems exist in the prior art beyond expectedly having captured.

It is a discovery of the invention that probe annealing temperature will reach 70 DEG C if being not connected to MGB, then probe will necessarily be grown very much, especially It is the region on the island Fei CpG, and CG content is low, may grow to 40bp.In so long probe, an only CG/TG Mispairing difference does not almost influence annealing temperature, so can not identify CG/TG.

As preferred embodiment, as far as possible reduction probe base quantity: probe annealing temperature should be higher than that primer annealing 10 degree of temperature, to ensure that the combination of probe and DNA profiling occurs before primer annealing extension.Probe is shorter, and probe identifies first The specificity in base and the non-methylation site CG is stronger.Probe length should be controlled in 10~20bp, embodiment party more preferably Formula, the probe length should be controlled in 12~18bp.

As preferred embodiment, the site CpG to be measured should be likely located among probe by the region at 3 ' ends.As more excellent The embodiment of choosing, the site CpG are located at by the one third region at the end probe 3'.

Compared with complete pairing, the single base mismatch in half region probe 3' (region MGB) has up to 17 DEG C of annealing Temperature difference, and it is located at the single base mismatch of 5 ' end regions, 2~10 DEG C of annealing temperature difference can only be generated.Therefore, CG to be measured The probe that site is located at 3 ' ends has higher specificity.

As preferred embodiment, the end of probe 5 ' should be held close to the 3 ' of forward primer, but can not be Chong Die with forward primer, 1 base or more can be spaced with 3 ' ends of forward primer.Probe can be Chong Die with reverse primer.

Since the exonuclease activity of archaeal dna polymerase is maximum at the initial stage of DNA synthesis chain extension, probe 5 ' is held as far as possible Ground can utmostly guarantee that the fluorophor of the end of probe 5 ' coupling can be cut by archaeal dna polymerase in extension close to forward primer It is free, discharge fluorescence, it is ensured that the accuracy of detection.In addition, also can archaeal dna polymerase polymerism and it is exo-acting maximum when, High efficiency cutting is incorporated in the probe on template strand, its caused DNA is avoided to extend the inaccuracy for terminating and detecting in advance.

The present invention cleverly design primer and probe, and using the probe for combining MGB, inverted dose of genomic DNA processing Afterwards, unmethylated Cytosines are uracil (UpG), and the cytosine residues to methylate are unaffected (mCpG), this makes Obtain the sequence difference that methylation dependence is generated in genomic DNA.Different transmitting light has been used during PCR amplification The TaqMan MGB probe of the fluorescent marker of wavelength handles the single base difference generated to distinguish transforming agent.In same reaction system In, using identical pair of primers, amplification methylation and unmethylated allele, sequence differentiation occur over just fluorescence probe The process of hybridization, this differentiation are to be matched completely based on sequence and the annealing temperature difference (Fig. 1) of mispairing.

The present invention uses a kind of MGB probe specificity combination methylated alleles sequence of the fluorescent marker of wavelength of transmitted light Column combine non-methylated alleles sequence using the MGB probe specificity of the fluorescent marker of another wavelength of transmitted light.With Pairing is compared completely, and the single base mismatch in half region probe 3' (region MGB) has up to 17 DEG C of annealing temperature difference.This It allows us to design probe and only covers single CpG dinucleotides, so as to measure the methylation level of isolated CpG.

In addition to detecting the individually isolated site CpG methylation, method of the invention can also detect multiple isolated simultaneously The methylation in the site CpG.At this time, it may be necessary to be directed to, corresponding pair of primers is designed in each site CpG and a pair of of covering is to be measured The oligonucleotide probe in the site CpG.In addition to this, the primer amplified region in each site CpG to be measured is not overlapped, to avoid Competition during multiplex PCR, and the fluorophor of 5 ' end of each probe connection has different wavelength of transmitted light.In addition, also The compatibility of multiple qMSP primer, probe combinations need to be analyzed: after the completion of primed probe design, should theoretically be excluded first multiple Secondary structure (http://www.cstl.nist.gov/ that may be present between whole primed probes in qMSP system Strbase/AutoDimerHomepage/AutoDimerProgramHomepage.htm), determine that the primer of multiple qMSP is visited Needle combination.

Wherein, in step c), by methylating, percentage parameter PMR analyzes the methylation state in DNA sample to be measured.It is existing Have in technology, the calculation method for the percentage parameter PMR that methylates are as follows: (methylation fluorescent value/internal reference fluorescent value)_{Sample to be tested}/ (first Base fluorescent value/internal reference fluorescent value)_{Exhaustive methylation standard items}

In the present invention, the methylation percentage parameter PMR are as follows:

Methylation/(methylation+non-methylation) × 100；

Specifically, are as follows:

Methylate fluorescent value/(methylation fluorescent value+non-methylation fluorescent value) × 100；

More particularly:

100/(1+1/2^-ΔCT), Δ CT=CT_{Methylate fluorescence}–CT_{Non- methylation fluorescence}

From calculation method as can be seen that the present invention compares the calculation method that traditional technology uses, more directly express The methylation ratio of sample to be tested, and without using internal reference reaction and exhaustive methylation standard items, while more easy, It also avoids reacting and exhaustive methylation standard items bring error using internal reference.

On the other hand, the present invention also provides a kind of kit of DNA methylation detection, the kit contains at least A pair of of oligonucleotide probe, the probe covers the site CpG, and the probe is the probe for being connected to MGB.

Kit of the invention does not contain exhaustive methylation standard items.

Wherein, in a pair of of oligonucleotide probe, a specific binding CG sequence, another specific binding TG sequence.

As preferred embodiment, a pair of of oligonucleotide probe in the kit is a pair of Taqman probe, every 5 ' ends of Taqman probe connect fluorophor, and 3 ' ends connect quencher and MGB group, and two 5 ' ends of Taqman probe The fluorophor of end connection has different wavelength of transmitted light.

Further, the fluorophor can be selected from FAM (or SYTO9), VIC (or JOE/TET/HEX), ROX In (or Texas Red), TAMRA (or NED/BODIPY/TMR-X) ... (other fluorophors that can choose please be supplement) Any two kinds.

Further, the quencher is selected from one of NFQ, BHQ1 and BHQ2 etc..

As preferred embodiment, the length of the probe is between 10~20bp；It is highly preferred that the length of probe Between 12~18bp；

As preferred embodiment, the site CpG to be measured is located among probe by the region at 3 ' ends；Preferably, The site CpG is located at the one third region that probe leans on the end 3'.

As preferred embodiment, the kit also contains at least pair of primers；The pair of primers is corresponding A pair of of probe.

Pair of primers includes forward primer and reverse primer, and a pair of of probe includes the probe in conjunction with CG and the spy for combining TG Needle, this must fully consider probe in this amplification subregion to primer, and in design, make probe as close to just To primer, but cannot overlap.This technology is applicable to multiplex PCR system, using two pairs or more to primer, and with its point Not corresponding two pairs or more to probe, detect the first in two or more site to be measured simultaneously in a reaction system Baseization is horizontal.

As preferred embodiment, the length of the primer amplification is 50~200bp.

As preferred embodiment, probe 5 ' is held close to 3 ' ends of forward primer.

Optionally, the kit also contains transforming agent, and the transforming agent is selected from hydrazonium salt, weight bisulfites and Asia One or more of disulfate；Preferably, the transforming agent is bisulfites.

Optionally, the kit stated also contains the reagent and also contains archaeal dna polymerase, dNTPs, Mg²⁺Ion and buffering One or more of liquid；Preferably, containing archaeal dna polymerase, dNTPs, Mg²⁺Ion and buffer.

In kit of the invention, when having multipair primer and multipair corresponding probe, each pair of primer amplification area Domain is not overlapped, and between primer and primer, between probe and probe, should be had compatibility between probe and primer, not generated two The secondary structures such as aggressiveness, hair clip spline structure, to guarantee the PCR amplification efficiency and specificity of multiple system.

As an alternative embodiment, kit of the invention include: be divided into receive in it one of reagent or Multiple containers.It such as include the first container for accommodating the probe in the specific binding site CpG；Accommodate the second container of amplimer； Receiving sensitively converts third container of transforming agent of unmethylated cytimidine etc..

The method and kit of DNA methylation detection of the invention, can be used for the diagnosis of non-disease, such as but unlimited In use species gene group methylation characteristic as marker cell line certification, tissue-derived identification, pre-natal diagnosis, microorganism Identification, new species Stock identification etc..

Beneficial effects of the present invention:

1. can be detected for the methylation level of single CpG:

Present invention firstly provides a kind of novel QASM (quantitative analysis of single-CpG Methylation) technology has used the probe combined with minor groove binding (minor groove binder, MGB), has utilized base Accurate quantitative analysis is carried out to the methylation level of single CpG in the real-time quantitative PCR of fluorescence probe, solving traditional qMSP can not be right The site CG outside the island CpG carries out the quantitative problem that methylates.

2. better repeatability and accuracy:

Firstly, the detection design of this technology is more easy, without independent input referring to reaction, therefore from input reference The inevitable deviation and error of reaction will not accumulate, and will not and influence final result.As shown in Figure 2 E, independent twice The standard deviation of measurement, which is significantly less than, uses Alu-C4 reaction as the standard deviation of input reference.

Secondly because the PMR of this technology is determined by methylation/(methylation+non-methylation) ratio, complete first is not needed The DNA of base is used as referring to calculating.If determined referring to the non-exhaustive methylation of DNA by sample to be tested and with reference to the ratio of DNA PMR is clearly incorrect, this also explains why this technology and pyrosequencing result it is almost consistent, and it is traditional Even if MethyLight is linearly related with the result of pyrosequencing, but there are certain deviations by PMR.

Finally, single CpG resolution ratio of this technology influences result by the methylation variation of CpGs in flanking sequence. Therefore, the methylation percentage that the PMR measured by this technology is almost determined with pyrosequencing is identical.

3. cheaper, convenient, rapid:

It is worth noting that, by with the bisulfites coke phosphorus that is widely regarded as most reliable methylation assay method Acid sequencing compare, accuracy of the invention it is consistent with pyrosequencing with it, it can be achieved that it is point-device methylation quantify.This The high-resolution methylation information of kind can only use bisulfites gene order-checking or bisulfites pyrosequencing in the past It could obtain, but this does not have in the DMPs large sample queue verification in the fixed paraffin-embedded tissue of formalin Cost-effectiveness advantage or feasibility.And method of the invention accuracy with its under pyrosequencing unanimous circumstances, than Pyrosequencing is cheaper, convenient, rapid.Pyrosequencing is the goldstandard technology for detecting DNA methylation level, it can also be used to The detection for the isolated site the CG methylation level that this technology is directed to.But pyrosequencing needs special pyrosequencing instrument, it is more difficult to It obtains (not finding that there is this equipment in Guangzhou temporarily), is sequenced somewhat expensive (about 300/ sample), the sequencing period is long (about January)；And this skill Art only needs a conventional fluorescent quantitative PCR apparatus, and testing cost is cheap, can obtain data immediately.

4. it is applied widely, DNA methylation assay is able to achieve to the single site CpG of CGI and non-CGI:

Traditional MethyLight technology be whole CpG based on CGI exist simultaneously methylation or non-methylation it is assumed that It is the percentage to methylate altogether that is measured, which is whole sites CpG of primed probe covering,.However, if site to be measured and flank sequence The CpGs that primer and probe covers in column does not appear as total methylation, and original technology will be unable to the methyl for accurately checking region to be measured Change state.Since this technology shows the high susceptibility to single CpG measurement, specificity and accuracy.Method of the invention For measuring the methylation level of single CpG dinucleotides, but it is not limited to the open sea that flanking sequence lacks CpG, such as in CGI In can design primed probe probe is made only to cover CpG to be measured, method of the invention can more accurately detect the region to be measured CGI Methylation level.

Detailed description of the invention

Fig. 1 is testing principle of the invention.

Fig. 2 is specificity of the invention, sensitivity, repeatability and dosing accuracy；

A. using tri- positions FAT3, FHIT and KIAA1026 in this technology detection exhaustive methylation and unmethylated DNA The methylation level of point；

B. after handling HCT116 cell using DNA demethylation reagent 5- azepine -2'- deoxycytidine (5-aza)；Use this The methylation level of FAT3, FHIT and the KIAA1026 of technology detection reduce as expected；

C. repeatability of this technology in tumour heterogeneous samples；

Repeatability of the C1.FAT3 in tumour heterogeneous samples；

Repeatability of the C2.FHIT in tumour heterogeneous samples；

Repeatability of the C3.KIAA1026 in tumour heterogeneous samples；

D. the detection range and dosing accuracy of this technology are examined by standard curve；

E. repeatable with the Technical comparing for using Alu-C4 to react correction.

Fig. 3 is the methyl with tri- sites bisulfites pyrosequencing comparative assessment FAT3, FHIT and KIAA1026 Change dosing accuracy；

It A. is the methylation dosing accuracy with the site bisulfites pyrosequencing comparative assessment FAT3；

It B. is the methylation dosing accuracy with the site bisulfites pyrosequencing comparative assessment FHIT；

It C. is the methylation dosing accuracy with the site bisulfites pyrosequencing comparative assessment KIAA1026.

Fig. 4 is the selection result that the EPIC microarray in 45 tumor samples is verified using this technology.

Fig. 5 PMR calculation method (A) of the present invention is compared with tradition PMR calculation method (B) accuracy.

Specific embodiment

Technical solution of the present invention is further illustrated below by way of specific embodiment, and specific embodiment does not represent to this hair The limitation of bright protection scope.Other people according to the present invention theory made it is some it is nonessential modification and adjustment still fall within this hair Bright protection scope.

In the present invention, " site CpG in heterozygosis methylation region " and " site CpG in the non-region that methylates altogether " is a meaning Think.For example having 10 CpG in the region of DNA domain of a 100bp, methyl region of chemistry would generally assume that this 10 CpG are to send out simultaneously Raw methylation or non-methylation simultaneously, referred to as " methylate region altogether ", many regions of genome are such " methylations altogether Region ", traditional technology are namely based on such maximum probability " methylation altogether ", and design primer and probe cover in the region 100bp Multiple sites CpG, detection is multiple sites the CpG while cell to methylate accounts for all cells in this region in fact Ratio.

Genosome: gene is complete nucleotide sequence needed for generating a polypeptide chain or function RNA, genosome, that is, gene Main part, typically refer to a gene and remove its promoter region (to typically refer to transcription initiation site upstream and downstream The region 2000bp) complete nucleotide sequence.

Intergenic region: intergenic region refers to the intervening sequence between gene and gene, is not have hereditary effect in genome Segment, be not belonging to gene structure.

The island CpG: distribution of the CpG dinucleotide in human genome is very inhomogenous, in certain sections of genome, CpG Keep or be higher than normal frequency.The island CpG is predominantly located at the promoter and exon region of gene, is rich in CpG dinucleotides Some regions, length are 300-3000bp.Generally defined as G/C content is more than 55%, and actually with expected CpG double-core glycosides The quantity ratio of acid is greater than 65%, it is contemplated that the calculation method of CpG dinucleotide quantity is (C quantity * G quantity)/sequence length.

CpG shore: in the two side areas on the island CpG, length about 100~3000bp, the appearance of CpG dinucleotide The requirement that the island CpG defines is not achieved in frequency, but is higher than other regions of genome, relative to the island CpG, these adjacent flanks Region is known as " beach " (shore) the CpG open sea on the island CpG: in the genome area far from the island CpG, CpG dinucleotide The frequency of occurrences be far below the island CpG, relative to the island CpG, the widest region of these genomes is referred to as " high sea " (open sea)

CpG shelf: in the two side areas of CpG shore, the frequency of occurrences of CpG dinucleotide is lower than shore, but Higher than open sea, relative to the island CpG, " beach " and extensive " high sea ", these are from the island CpG and " beach " closer side Wing area is known as CpG " submerged reef " (shelf)

Flanking sequence does not have the isolated site CpG of CpG: in the region CpG open sea and shore, CpG dinucleotide The frequency of occurrences it is low, the site CG usually isolates, and in the PCR amplification sequence of 100~200bp of its two sides, usually lacks other CpG Site.

The DNA methylation level of tissue has potential clinical value.There are many detection DNA methylations at present Method, such as the island CpG (CpG Island, CGI) is checked using MethyLight (methylation-specific quantitative PCR) Methylation.However, the CGI in promoter only represents the sub-fraction of methylation group, gradually discovery is predominantly located at genosome now In CpG open sea play a significant role in disease development and abnormal intracellular molecules event.Unfortunately, The technology of existing based on PCR can not examine this two sides sequence not have the isolated site CpG of CpG.Therefore, the present invention provides A kind of new QASM analysis method, the method use what is combined with minor groove binding (minor groove binder, MGB) Taqman probe.

Specifically, after inverted dose of genomic DNA processing, unmethylated Cytosines are uracil (UpG), and first The cytosine residues of base are unaffected (mCpG), this makes the sequence difference that methylation dependence is generated in genomic DNA. We have used the MGB of two different fluorescent markers during PCR amplificationProbe distinguishes bisulfites Handle the single base difference generated.In same reaction system, using identical pair of primers, amplification methylates and does not methylate Allele, sequence distinguishes the process of fluorogenic probe hybridzation of occurring over just, this differentiation is match completely based on sequence and mistake The annealing temperature difference (Fig. 1) matched.

The present invention cuts double labeling probe using the 5' exonuclease activity of archaeal dna polymerase, processed with transforming agent DNA CG/TG sequence hybridization.The cutting of 5' exonuclease separates 5'- fluorogen and 3'- quencher, releases fluorogen It puts and generates detectable fluorescence signal.Minor groove binding group (minor groove binding, MGB) and 3'- quencher knot Conjunction allows to the probe shorter using sequence, and has hypersensitivity and specificity to single base mismatch.Methylation CpGs remains CpG in transforming agent processing, and the CpG of non-methylenation deamination after transforming agent conversion therefore is used at TpG CG/TG the specific probe for carrying two different fluorogens, can carry out single step DNA methylation assay, compared with complete pairing, Single base mismatch in half region probe 3' (region MGB) has up to 17 DEG C of annealing temperature difference.This is allowed us to Design probe only covers single CpG dinucleotides, so as to measure the methylation level of isolated CpG.PMR is by amplification cycles The CT threshold rate of interior two kinds of fluorescence probes determines that the ratio between methylation probe and the signal for the probe that do not methylate can It realizes the accurate quantification of site methylation level to be measured, and does not need amplification crt gene, such as Alu-C4 and ACTB, with reflection With the amount and integrality of correction starting DNA profiling；In addition, also no longer needing the DNA sample using CpGenome exhaustive methylation As referring to the PMR sample for calculating each sample.

QASM detection method of the present invention and traditional MethyLight the difference is that, it detects the methyl in genome Change situation has very high resolution ratio, can detecte the methylation level of single CpG.It is this to be based on methylation probe and non-methylation The detection of probe ratio can improve the sample standard method in the detection technique of based on PCR, since it is without using the positive Control calculates PMR (DNA of exhaustive methylation), is also not required to input control correction DNA profiling total amount (Alu-C4PCR reaction), because This, not vulnerable to the influence by the relevant copy number variation of cancer, this advantage is more bright in the complex clinical tissue of large sample It is aobvious.

We design and apply this QASM technology to detect positioned at FAT3, FHIT and KIAA1026 genosome CpG open The methylation level in the isolated site CpG of three of sea.We with " goldstandard " bisulfites pyrosequencing by comparing Compared with having evaluated testing result of this QASM technology in Tumour DNA sample.In addition, we utilize methylation/non-methylation ratio Rate calculates PMR, corrects the total amount of different sample DNA templates so as to avoid another independent PCR reaction is increased.We It also developed a kind of calculation method, so that researcher is used as control without using the DNA sample of exhaustive methylation, so that it may measure The percentage of methylated alleles in PCR reaction system.We further describe in this technology PCR condition and how Design primer and pairs of probe, faster, more accurately to measure the methylation level of isolated CpG.

The technology is not only very special and sensitive, but also easy to operate, does not need the DNA of control reaction and exhaustive methylation Standard items are as reference, so as to overcome their bring defects.In addition, it has more than existing MethyLight technology High repeatability and accuracy.We are determined using this technology positioned at CpG open in 45 colorectal cancer samples The isolated site CpG of three of sea, discovery the method can be used for the methylation quantitative detection of clinical complex samples.

QASM detection method technically securely enough to detects the methylation level in the isolated site CpG.It is suitable such as to design Primer and probe, can be also used for the methylation level for more accurately detecting CGI.

Material

Fresh Frozen tumor tissues sample is obtained from 45 primary colorectal adenocarcinoma patients.Patient includes 32 male Property and 13 women.In these patients, 17 are I phase tumour, and 28 are II phase tumour；18 neurological outcomes are to recur, 27 Neurological outcomes do not recur.See Table 1 for details.

1 tumor sample of table

The DNA methylation quantitative approach in 1 site CpG of embodiment

DNA separation and bisulfite conversion

Use QIAamp DNA Mini Kit (Qiagen, 51306) and EZ DNA methylation kit (Zymo Research, D5002), extract to specifications the genomic DNA in above-mentioned sample and carry out it is bisulphite modified [7, 14]。

QASM detection

After bisulfite conversion, real-time fluorescence quantitative PCR amplifying genom DNA is carried out.In brief, using be located at draw Genomic DNA after object and a pair of oligonucleotide probe amplification bisulfite conversion for covering the site CpG to be measured, each widow's core The thuja acid end probe 5' connects fluorescent reporter dye 6FAM or VIC (specifically binding CG sequence and TG sequence respectively), the coupling of the end 3' - MGB group (MGB-NFQ) (Fig. 1) is quenched.When DNA extends, 5' to 3' exonuclease activity will be cut Taq archaeal dna polymerase It cuts probe and discharges reporter gene, fluorescence can pass through the real-time PCR of Applied Biosystems QuantStudio 7Flex System detection.Initial DNA template concentration can obtain [15] by CT (cycle threshold) value of fluorescence signal.By exhaustive methylation With complete non-methylation standard sample, QASM detection is carried out after mixing according to a certain percentage, to describe standard curve.We use The reaction system of 20uL, system include 500nM primer, 150nM probe, each 200nM of dATP, dCTP, dGTP and dTTP, 2.25mM MgCl₂, 0.75U HotStar Taq enzyme, 1X PCR buffer.Reaction condition are as follows: after bisulfite conversion DNA, 95 DEG C 15 minutes, followed by 94 DEG C 30 seconds, 56~60 DEG C of 1 minute and 72 DEG C of 50 of 1 minute circulations.

Primer and probe sequence

For FAT3 (gene I/D: 120114), FHIT (gene I/D: 2272) and KIAA1026 (gene I/D: 23254) gene In three sites to be measured, we devise three groups dedicated for primer and probe (table 2) of the invention.Three to be measured isolated The 200bp flanking sequence in the site CpG is shown in Table 3.

Table 2 is used for primer and probe sequence of the invention

The flanking sequence of 3 three isolated CpGs of table

The percentage that methylates calculates

The QASM detection data in the site CpG to be measured is indicated with PMR, but calculation method is different from the method for report before [8].In the method for invention, the PMR of each sample is equal to methylation/(methylation+non-methylation) × 100, when specifically calculating, We use following formula: PMR=100/ (1+1/2^-ΔCT), Δ CT=CT_FAM-CT_VIC

DNA methylation assay result

Such as following table 4- table 6, tri- sites FAT3, FHIT and KIAA1026 in respectively 10 clinical Colorectal Carcinomas Testing result.

The DNA methylation assay result of 4 FAT3 of table

The DNA methylation assay result of 5 FHIT of table

The DNA methylation assay result of 6 KIAA1026 of table

The technical identification of QASM

Inventor has filtered out the difference first between recurrence and non-recurrence colorectal cancer sample by EPIC microarray Base promoter (Differentially Methylated Positions, DMPs).DMPs in CGIs and promoter compared with It is few but many in open sea and genosome.However, traditional MethyLight analysis can not be examined in CpG open sea Survey the CpG methylation level of no flank CpG cluster.We select to be located at FAT3, the open of FHIT and KIAA1026 genosome 3 isolated sites CpG in sea, in people's colorectal cell system HCT116 and CpGenome methylation and non-methylate DNA Every characteristic of this technology is examined in (Millipore S7821& S7822).

The specificity (table 2) of detection primer and methylation and non-methylation probe first.The PMR of methylate DNA standard items Close to 100%, with its exhaustive methylation state consistency.On the contrary, the PMR of non-methylate DNA standard items is close to zero (Fig. 2A).This Outside, it after handling HCT116 cell with DNA demethylation reagent 5- azepine -2'- deoxycytidine (5-aza), is detected with this technology FAT3, FHIT and KIAA1026 methylation level are declined (P < 0.05, Fig. 2 B) as expected.This shows that method of the invention can Specifically to distinguish methylation and unmethylated gene loci.

It further analyzes this technology and detects the repeatable of methylation level in 10 colorectal cancer DNA samples (table 1) Property.Repeatability is detected by carrying out two independent qPCR reactions to each site to be measured.As shown in Figure 2 C, three it is to be measured The result in site is CT obtained in measurement twice_FAM-CT_VICValue, the standard deviation independently measured twice is lower, and first Secondary and second of measurement delta CT value is linearly related, R²>0.999.In short, these the result shows that this technology complicated Repeatedly measurement can generate reproducible result in clinical heterogeneous samples.

Finally, we examine the detection range and dosing accuracy of this technology by standard curve.The general first of CpGenome Base DNA is mixed with general non-methylate DNA according to different proportion, tracks FAT3, FHIT and KIAA1026 tri- using this technology Change of the methylation level in a site to be measured in the standard items of different mixing proportion.The results show that even if 10,000 times After non-methylate DNA dilution, the methylation level in three sites can be still reliably detected.We calculate dilution every time and obtain PMR, to determine the quantitative precision of this technology, the results showed that methylation of the technology on four orders of magnitude is quantitatively rendered as Linearly (Fig. 2 D).These are the result shows that this technology has very high sensitivity and dosing accuracy.

2 bisulfites pyrosequencing comparative assessment dosing accuracy of embodiment

Use pyrosequencing as reference, further examines the accuracy of present invention methylation quantitative approach.We In 10 Colorectal Carcinomas (table 1), while detecting using pyrosequencing and this technology the methyl in above-mentioned 3 sites CpG Change horizontal.The primer of pyrosequencing is shown in Table 2.The PMR measured through the invention and the methylation obtained by pyrosequencing Percentage linear correlation (Fig. 3；FAT3 is 0.9690, FHIT 0.9954, KIAA1026 0.8755, equal P < 0.001).No Only in this way, the methylation percentage of PMR and pyrosequencing has surprising consistency.This is than the tradition reported in the past MethyLight is more accurate [11,12].Therefore, this technology has identical with bisulfites pyrosequencing quantitative accurate Property, and operation is easier, is easier acquisition, is cheaper.

Embodiment 3 reacts the Technical comparing repeatability of correction with using Alu-C4

Past, all detection techniques based on qPCR are required to internal reference gene and are quantified.Traditional MethyLight It is widely used that AluC4 as control reaction, the total amount [8,11,12] of assessment and correction starting DNA profiling.It is singly copied with other Shellfish crt gene is compared, and this high copy control amplicon is less susceptible to the influence of cancer related gene change.Unfortunately, entirely The influence of genome copies number variation and mutation to AluC4 stability still can not avoid completely.It is an advantage of the invention that being not required to This internal reference is wanted to react, each independent sample can obtain the accurate PMR unrelated with DNA profiling total amount is originated.In addition, we Compare repeatability (the specific steps reference of traditional MethyLight of both detection methods in 10 clinical tumor samples Eads CA,Danenberg KD,Kawakami K,et al.MethyLight:a high-throughput assay to measure DNA methylation.Nucleic Acids Res.2000.28(8):E32.).We have found that using Alu- In traditional MethyLight of the C4 reaction as input control, the PMR standard deviation independently measured twice is than using first in this technology Base/non-methyl signal is higher (Fig. 2 E) than with the PMR standard deviation for correcting input difference.It is observed in traditional MethyLight To higher standard difference may be from the tired of random PCR [16] under low template concentrations and two independent reaction large deviations Product.However, this technology is lower a possibility that influence by these factors, because the signal from methylation and non-methylation probe is It is generated in identical reaction system using identical primer amplification.

The quantitative reliability of 4 EPIC microarray comparative assessment of embodiment

It is verified using this technology in Illumina MethylationEPIC (EPIC) BeadChip microarray In the methylation state of DMPs that filters out.EPIC array is the second generation product of Illumina HM450 microarray, Coverage rate significant increase of the detection probe in CpG open sea, to filter out the separation with biology and clinical meaning CpGs provides a kind of valuable tool [17].45 people's colorectal adenocarcinoma tissues are used for this experiment (table 1).We make With this technology detection compared in EPIC microarray cg00561674 (FAT3), cg05704547 (FHIT) and The methylation percentage of cg06887407 (KIAA1026), to determine the reliability of EPIC microarray.Pass through EPIC (technology of EPIC microarray is referring to Pidsley R, Zotenko E, Peters TJ, et by microarray al.Critical evaluation of the Illumina MethylationEPIC BeadChip microarray For whole-genome DNA methylation profiling.Genome Biol.2016.17 (1): it 208.) detects To opposite methylation level (β value) with by the PMR that this technology obtains have good linear dependence (cg21101720R= 0.9208；Cg21101720R=0.29790；All P of cg14215472 0.8920. are < 0.001) (see Fig. 4).

The PMR calculation method of the present invention of embodiment 5 is compared with tradition PMR calculation method accuracy

We compare this technology computing system and traditional technology calculation method obtain in 10 clinical tumor samples The PMR value in the site KIAA1026, and be compared with pyrosequencing result.The experiment of this technology group and calculation method are as above It is described；In traditional technology group, an exhaustive methylation standard items must be increased as External reference, two independences need to be arranged in each sample QPCR reaction system, detect the total amount of methylation sites and AluC4 internal reference gene to be measured respectively, count with the following method Calculate PMR:(methylation fluorescent value/internal reference fluorescent value)_{Sample to be tested}/ (methylation fluorescent value/internal reference fluorescent value)_{Exhaustive methylation standard items}。

Although the PMR for showing that the computing system of this technology and traditional technology obtains compared with the result of pyrosequencing is equal The methylation ratio correlation obtained with pyrosequencing is good, but the linearly related degree of this technology is better than traditional technology (Fig. 5 A Vs.B, R=0.8412VS.0.7698).In addition, using AluC4 and exhaustive methylation standard items respectively as the biography of interior External reference In MethyLight technology of uniting, the methylation ratio goodness of fit that the PMR value and pyrosequencing being calculated obtain can not show a candle to this The PMR value (Fig. 5 A vs.B) that technology is calculated.

Bibliography

[1]Feinberg AP.The Key Role of Epigenetics in Human Disease Prevention and Mitigation.N Engl J Med.2018.378(14):1323-1334.

[2]Luo Y,Wong CJ,Kaz AM,et al.Differences in DNA methylation signatures reveal multiple pathways of progression from adenoma to colorectal cancer. Gastroenterology.2014.147(2):418-29.e8.

[3]Yang X,Han H,De Carvalho DD,Lay FD,Jones PA,Liang G.Gene body methylation can alter gene expression and is a therapeutic target in cancer. Cancer Cell.2014.26(4):577-90.

[4]Flores,K,et al.Genome-wide association between DNA methylation and alternative splicing in an invertebrate.BMC Genomics 2012；13:480.

[5]Neri,F,et al.Intragenic DNA methylation prevents spurious transcription initiation.Nature 2017；543:72-77.

[6]Herman JG,Graff JR, S,Nelkin BD,Baylin SB. Methylation- specific PCR:a novel PCR assay for methylation status of CpG islands.Proc Natl Acad Sci U S A.1996.93(18):9821-6.

[7]Bosch LJ,Luo Y,V LV,et al.WRN Promoter CpG Island Hypermethylation Does Not Predict More Favorable Outcomes for Patients with Metastatic Colorectal Cancer Treated with Irinotecan-Based Therapy.Clin Cancer Res. 2016.

[8]Weisenberger DJ,Campan M,Long TI,et al.Analysis of repetitive element DNA methylation by MethyLight.Nucleic Acids Res.2005.33(21):6823-36.

[9]Eads CA,Danenberg KD,Kawakami K,et al.MethyLight:a high-throughput assay to measure DNA methylation.Nucleic Acids Res.2000.28(8):E32.

[10]Weisenberger DJ,Trinh BN,Campan M,et al.DNA methylation analysis by digital bisulfite genomic sequencing and digital MethyLight.Nucleic Acids Res.2008.36(14):4689-98.

[11]Luo Y,Kaz AM,Kanngurn S,et al.NTRK3 is a potential tumor suppressor gene commonly inactivated by epigenetic mechanisms in colorectal cancer. PLoS Genet.2013.9(7):e1003552.

[12]Luo Y,Tsuchiya KD,Il PD,et al.RET is a potential tumor suppressor gene in colorectal cancer.Oncogene.2013.32(16):2037-47.

[13]Kutyavin IV,Afonina IA,Mills A,et al.3'-minor groove binder-DNA probes increase sequence specificity at PCR extension temperatures.Nucleic Acids Res.2000.28(2):655-61.

[14]Frommer M,McDonald LE,Millar DS,et al.A genomic sequencing protocol that yields a positive display of 5-methylcytosine residues in individual DNA strands.Proc Natl Acad Sci U S A.1992.89(5):1827-31.

[15]Schmittgen TD,Livak KJ.Analyzing real-time PCR data by the comparative C(T)method.Nat Protoc.2008.3(6):1101-8.

[16]Warnecke PM,Stirzaker C,Melki JR,Millar DS,Paul CL,Clark SJ.Detection and measurement of PCR bias in quantitative methylation analysis of bisulphite-treated DNA.Nucleic Acids Res.1997.25(21):4422-6.

[17]Pidsley R,Zotenko E,Peters TJ,et al.Critical evaluation of the Illumina MethylationEPIC BeadChip microarray for whole-genome DNA methylation profiling.Genome Biol.2016.17(1):208。

Sequence table

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<120>a kind of DNA methylation detection kit and method

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ttgggattgt agaattgtag ataaaga 27

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<213>artificial sequence (Artificial Sequence)

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cacctacaac ccccaacttc 20

<210> 7

<211> 23

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 7

tgaatttata tatcggttat agg 23

<210> 4

<211> 24

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 4

tgaatttata tattggttat aggt 24

<210> 5

<211> 34

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 5

attcgatgag tttattgtat tgtttattta tttg 34

<210> 6

<211> 27

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 6

tcatacccca aacataaaca tcataca 27

<210> 7

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<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 7

tgatttcggg gtgtata 17

<210> 8

<211> 18

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 8

tttgattttg gggtgtat 18

<210> 9

<211> 30

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 9

ttggaagagg agttataatg taggatgttg 30

<210> 10

<211> 30

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 10

cctcttaaat cccttcaaac cttttcttat 30

<210> 11

<211> 19

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 11

ttaaggtagg taagcgtgg 19

<210> 12

<211> 20

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 12

aaggtaggta agtgtggtgt 20

<210> 13

<211> 28

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 13

ttgggattgt agaattgtag ataaagag 28

<210> 14

<211> 30

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 14

ccaaaaactt tcctctaaaa aaaacctata 30

<210> 15

<211> 20

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 15

tgtagataaa gagttgtgaa 20

<210> 16

<211> 30

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 16

gataaatttg ttaaaagaaa gaggaagtat 30

<210> 17

<211> 28

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 17

cccaaacata aacatcatac aatatacc 28

<210> 18

<211> 15

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 18

cccacatata caccc 15

<210> 19

<211> 28

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 19

gggattttaa ttgttattgg agaatagg 28

<210> 20

<211> 30

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 20

aaaacattaa ttcccaaata cttaaattct 30

<210> 21

<211> 17

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 21

caaaaacaca ccaaaac 17

<210> 22

<211> 1280

<212> DNA

<213> Homo sapiens

<400> 22

atccttgatc gttttagaag tatatgtatg taatctcaag caagtgatat tctaattaca 60

gatcagtctt acttattcat tagagcaaat ctcccgagga ctttaccagg aaatacttaa 120

ctgattttga atcgttggat gtaatttaaa gtaataaagc ggcatttatt tttaaatact 180

ctgtgattct gactaatttc cggctgtcct cacctctatc tgttcaaatt aggtttcgtt 240

tggctacatt ttaaaaggta gattaccaaa aatataggtg attggacctt ttaaatatca 300

gaaaaattgc tgaaatagag aaaagtgaaa aagaaggtct atgttctcaa ataatactaa 360

gctaagaaag aataagactg aaacacttgc cttataatta tactcaaagt gagtcatgga 420

ttgctttaaa gagaataaaa ttgatttaat tatttttcat tagaatgctt tagtcattct 480

tcctgaaaat ctacatgagt attagccttt aacataagca cagaatgttg aagacatgta 540

gcccttcacc cacccacctg cagcccccag cttcccagag ggaccaaaaa ttactgtagc 600

cattagagga aaatgtcaga accaaagact ttcctctggg gaaaacctat aaccgatata 660

tagattcaca gctctttgtc tgcaattcta caatcccaaa agctctgaga acttaaaggg 720

tttggagtat gttagtggca aactaatttg gtggcaaaac ctgttcaaaa ctgttttgag 780

gctctttcta ctctttattt ctctcattta gtgtgaatat ccatatgttt cactgcagaa 840

acactggtat ttttgattat gggatactac ggccagcacc aatttctgtg agggggagga 900

gtattttaca taatgtgaaa gatatgtgct gcatgtcctt gctaaaactg cacaatttta 960

aattctgaga tgcatctgct cccgcggatt tggagtccag taccttttca atagcatctg 1020

ttattctttc tctttctcct cctctcctct cccacctcag tctcttcctt tcccccaacc 1080

caggtaggaa aggaagacct gggagagatt gggttgggag gaacacctta aggcaaggtg 1140

atgacttggg gaccacagtg gccacagaaa tgcctaaatt aactccccct cccccccccc 1200

accaaccatc tttgtcctgc ttcatctgta ccctttaggt gcatcgtttt cttttaaaga 1260

tgtctgtgtt ttctcgagtt 1280

<210> 23

<211> 601

<212> DNA

<213> Homo sapiens

<400> 23

aaggaaaacc cctagtttat tatagatctg tggggtgtgt gaatatgtgt gtgtgttttg 60

agagatatat acaggaccct aatcagagag agtgccagga ataacagcca tgcatgccag 120

gcatcttttc ctgtttcttt ttcagttact tgaatccacc tgtgataaac ttgccaaaag 180

aaagaggaag tacaataacc acaaacgtta agtgattttt ttttttgttc tacaaactcg 240

atgagttcac tgcattgtct acttatctgt ttttgtaatt tcaactttta tttttgattt 300

cggggtgcac atgtgggttt gttccatagg tatattgcat gatgctcatg tttggggtat 360

gattaatacc atcacccagg tagtgagcat agtacccaat agctcttttt caatccttga 420

ccccctcctt ccctcccact agtccccagt gtttatggct gccattttca tgtccatgtg 480

taccatgttt gtctgtttaa ctctcactta taagagagaa catgtagtat ttggttttct 540

gttcctgtgt taatttgctt aggataatga cctgcagctg cattcatggt gctgcaacag 600

a 601

<210> 24

<211> 601

<212> DNA

<213> Homo sapiens

<400> 24

cgagcattgg ttggtttcag gcaaagttgt gcctggggga acctcagcct gaaccaactt 60

ggagtgtaag ttacacccac ctggaatgta agttacacct cggggtgtgt cctgaggtga 120

ggcaagggag cgctgctttc aaactcacag gtgggtcagt cacgggagga ggggctgccc 180

tgaggggaca ttaattccca ggtacttaag ttctcggagg ccgggaggca aagggagagt 240

cctggaagag gagccacaat gcaggatgct gaaatcagaa gcacaccaag gcaggcaagc 300

gtggtgcaca agaaaaggct tgaagggatc taagaggatc tggacggtat tgtctgctac 360

acatactcat tgttcagata aggaaactga ggctcatgga caccgacctg ttctccaatg 420

gcagttggga tcccaatccc cgtctgactc cagatcgcat cctctgctct ttcctagcaa 480

acccctcctt cctcctgcca gtggtcatga aatattaaag ccgtctgatg cttcctatgg 540

catgaaataa tcctgcaggt acgctggcca cactctcttg cagactgtct tttagtgcct 600

a 601

Claims (17)

1. a kind of DNA methylation detection method, which comprises the following steps:

A) uracil is converted by the cytosine base of the non-methylation of DNA sample, and the cytosine base to methylate is kept not Become；

B) at least pair of primers and at least after a pair of oligonucleotide probe amplification step (1) conversion for covering the site CpG to be measured DNA sample；Wherein, the oligonucleotide probe combination MGB；

C) amplified production is analyzed, and from the methylation state of the Analysis of Existence of amplified production DNA to be measured.

2. the method according to claim 1, wherein the site CpG is the position CpG in heterozygosis methylation region Point or flank do not have the isolated site CpG of CpG, or the site CpG in the region that methylates altogether；It or is non-methyl altogether Change the site CpG in region；

Or preferably, the site CpG is the site CpG in the site CpG or the island CpG outside the island CpG；

Or preferably, the site CpG is located at genosome, intergenic region or promoter；

It is highly preferred that the site CpG is located at CpG open sea, CpG shore, the CpG of genosome or intergenic region shelf。

3. described turns the method according to claim 1, wherein being converted in step a) using transforming agent Agent is selected from hydrazonium salt, weight one or more of bisulfites and bisulfites；

Preferably, the transforming agent is bisulfites.

4. the method according to claim 1, wherein in step b), the length of the primer amplification is 50~ 200bp。

5. the method according to claim 1, wherein described a pair covers the site CpG to be measured in step b) Oligonucleotide probe, wherein a specific binding CG sequence, another specific binding TG sequence.

6. according to the method described in claim 5, it is characterized in that, described a pair covers the oligonucleotides in the site CpG to be measured Probe is a pair of Taqman probe, and 5 ' ends of every Taqman probe connect fluorophor, and 3 ' ends connect quencher and MGB Group, and the fluorophor of two 5 ' ends of Taqman probe connection has different wavelength of transmitted light.

7. according to the method described in claim 6, it is characterized in that, for each Taqman probe, the fluorophor It is any one in FAM, VIC, ROX, TAMRA, SYTO9, JOE/TET/HEX, Texas Red and NED/BODIPY/TMR-X Kind；

Preferably, the quencher is selected from one of NFQ, BHQ1 and BHQ2.

8. according to the method described in claim 6, it is characterized in that, the length of probe is between 10~20bp；Preferably, probe Length between 12~18bp.

9. according to the method described in claim 6, it is characterized in that, the site CpG to be measured is located among probe by 3 ' ends Region；Preferably, the site CpG is located at by the one third region at the end probe 3'；

Preferably, probe 5 ' is held close to 3 ' ends of forward primer.

10. according to the method described in claim 1, each site CpG is contained when detecting multiple isolated sites CpG simultaneously Corresponding pair of primers and a pair of oligonucleotide probe for covering the site CpG to be measured, and the primer in each site CpG to be measured Amplification region is not overlapped.

11. the method according to claim 1, wherein passing through percentage parameter PMR points of methylation in step c) Analyse the methylation state of DNA to be measured；

Preferably, the methylation percentage parameter PMR is methylation/(methylation+non-methylation) × 100；

It is highly preferred that the methylation percentage parameter PMR=methylation fluorescent value/(methylation fluorescent value+non-methylation is glimmering Light value) × 100；

It is more preferred still that the methylation percentage parameter PMR=100/ (1+1/2^-ΔCT), Δ CT=CT_{Methylate fluorescence}– CT_{Non- methylation fluorescence}。

12. a kind of kit of DNA methylation detection, which is characterized in that the kit contains at least a pair of of oligonucleotides Probe, the probe are the probe for being connected to MGB；

Preferably, the kit does not contain exhaustive methylation standard items；

Preferably, a pair of of oligonucleotide probe, wherein a specific binding CG sequence, another specific binding TG Sequence；

Preferably, a pair of of oligonucleotide probe is a pair of Taqman probe, 5 ' the ends connection of every Taqman probe Fluorophor, 3 ' ends connect quencher and-MGB group, and the fluorophor of two 5 ' ends of Taqman probe connection has Different wavelength of transmitted light；

Preferably for each Taqman probe, the fluorophor be selected from FAM, VIC, ROX, TAMRA, SYTO9, Any one in JOE/TET/HEX, Texas Red and NED/BODIPY/TMR-X；；

Preferably, the quencher is selected from one of NFQ, BHQ1 and BHQ2；

Preferably, the length of the probe is between 10~20bp；It is highly preferred that the length of probe is between 12~18bp；

Preferably, the site CpG to be measured is located among probe by the region at 3 ' ends；Preferably, the site CpG is leaned on positioned at probe The one third region at the end 3'.

13. kit according to claim 12, which is characterized in that the kit also contains at least pair of primers； Corresponding a pair of of the probe of the pair of primers；

Preferably, the length of the primer amplification is 50~200bp；

Preferably, probe 5 ' is held close to 3 ' ends of forward primer.

14. kit according to claim 12, which is characterized in that the kit also contains transforming agent, described Transforming agent is selected from hydrazonium salt, weight one or more of bisulfites and bisulfites；Preferably, the transforming agent is Asia Disulfate.

15. kit according to claim 12, which is characterized in that the kit also contain archaeal dna polymerase, dNTPs、Mg²⁺One or more of ion and buffer；

Preferably, containing archaeal dna polymerase, dNTPs, Mg²⁺Ion and buffer.

16. kit according to claim 13, which is characterized in that when having multipair primer and multipair corresponding probe When, each pair of primer amplified region is not overlapped.

17. a kind of system for detecting DNA methylation, which is characterized in that the system contains:

A) DNA methylation detection means, and,

B) result judges component；

The DNA methylation detection means contain any kit of claim 12-16；

Preferably, the result judges DNA methylation of the component for being detected according to detection means as a result, output methylation hundred Divide than parameter PMR risk probability of illness or the type of disease；

Preferably, the methylation percentage parameter PMR be methylation/(methylation+non-methylation) × 100 it is highly preferred that The methylation percentage parameter PMR=methylation fluorescent value/(methylation fluorescent value+non-methylation fluorescent value) × 100；

It is more preferred still that the methylation percentage parameter PMR=100/ (1+1/2^-ΔCT), Δ CT=CT_{Methylate fluorescence}– CT_{Non- methylation fluorescence}；

Preferably, the result judge component be the methylation by comparing sample to be tested and normal sample as a result, according to The difference or difference value of test sample sheet and normal sample export risk or probability of illness.