CN106916893A - A kind of gene methylation degree quantitative approach based on digital pcr chip - Google Patents
A kind of gene methylation degree quantitative approach based on digital pcr chip Download PDFInfo
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- CN106916893A CN106916893A CN201710183073.2A CN201710183073A CN106916893A CN 106916893 A CN106916893 A CN 106916893A CN 201710183073 A CN201710183073 A CN 201710183073A CN 106916893 A CN106916893 A CN 106916893A
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- digital pcr
- methylation
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6851—Quantitative amplification
Abstract
The present invention relates to a kind of gene methylation degree quantitative approach based on digital pcr chip, including：(1) for genes of interest to be detected, it is determined that the region rich in methylation sensitive restriction enzyme cleavage sites, and to region design primer and taqman probes；(2) sample to be detected is divided into two parts, through methylation sensitive restriction enzyme ferment treatment, another is not processed a copy of it；(3) quantitative determination is carried out to two parts of samples respectively using digital pcr chip, finally calculates the methylation of genes of interest in sample.The present invention realizes the method to gene methylation degree absolute quantitation, and the method can accurately quantify the percentage methylated shared by copy number of cls gene to be checked；Detection sensitivity is high；Detection process is simple；And detection time is short.
Description
Technical field
The invention belongs to gene methylation detection field, more particularly to a kind of gene methylation based on digital pcr chip
Degree quantitative approach.
Background technology
DNA methylation is primarily referred to as methylating for 5 carbon of cytimidine (C), is formedm5C.In mammalian DNA, in methylating
The base-pair of statem5CG can play restriction effect to the expression of gene.
Research in recent years finds that DNA methylation occurs closely related with development with malignant tumour.DNA methylation level
Change in tumor development is mainly shown as the hypomethylation of local hyper-methylation and genome.First it is tumor suppressor gene
Promoter region, the region is in hypomethylation or non-methylation state in normal cell, gene can with normal expression, from
And constantly suppress tumour generation.And in cancer cell, the promoter region of gene can be presented high methylation state, it is suppressed that
The expression of gene, and ultimately result in the generation of cancer.Next to that for some bases in high methylation in normal cell
Cause and repetitive sequence, its methylation level reduction will cause the activation of these genes, cell hyperproliferation, and ultimately result in cancer
Disease.
In traditional methylation detecting method, although the method testing result based on sequencing is often considered as goldstandard, its
The operating process of the instrument and complexity of costliness is too dependent on, and cannot detect that trace methylation is copied.And other methods,
Such as methylation-specific high-resolution solubility curve method, fluorescence quantitative PCR method etc., although simple and efficient, but testing result is not
Enough accurate, insufficient sensitivity is high.The above shortcoming all limits application valency of the above-mentioned detection means in actual clinical detection
Value.
Digital pcr chip technology, as a kind of technology that can be accurately detected single DNA molecules, with lot of advantages.
For example：
First, absolute quantitation can be carried out to initial sample, without relying on standard items, it is not necessary to which pre-rendered standard is bent
Line.
2nd, sensitivity is high, and test limit can reach 101The order of magnitude is copied.
3rd, can be changed with the small copy number of accurate quantification, accurately identify the such small concentrations difference of DNA molecular.
These advantages all cause that digital pcr chip has the performance more superior than traditional quantitative methods, so as to more be applicable
In biochemistry detection.
Methylation sensitive restriction enzyme, is limit that a class is capable of specific recognition its cleavage site methylation state
Property restriction endonuclease processed, when its cleavage site state is to methylate, cutting does not occur.When its cleavage site state methylates for non-
When, cutting occurs.
The content of the invention
The technical problems to be solved by the invention are to provide a kind of gene methylation degree based on digital pcr chip and quantify
Method, the method achieve the method to gene methylation degree absolute quantitation, and the method can accurately quantify to be detected
The percentage methylated shared by copy number of gene；Detection sensitivity is high；Detection process is simple；And detection time is short.
A kind of gene methylation degree quantitative approach based on digital pcr chip of the invention, including：
(1) for genes of interest to be detected, it is determined that the area rich in methylation sensitive restriction enzyme cleavage sites
Domain, and to region design primer and taqman probes；
(2) sample to be detected is divided into two parts, a copy of it through methylation sensitive restriction enzyme ferment treatment, another
Do not process；
(3) quantitative determination is carried out to two parts of samples respectively using digital pcr chip, finally calculates genes of interest in sample
Methylation.
FAM fluorescence molecules and BHQ fluorescent quenching agent molecules are modified in taqman probes two ends in the step (1) respectively.
Digital pcr chip in the step (3) is the array digital pcr micro-fluid chip made using PDMS.
The array digital pcr micro-fluid chip made using PDMS is independent of special digital pcr equipment, point liquid and reading
Take and completed on chip, greatly reduce the digital pcr testing cost for methylating.
Pcr chip reaction system in the step (3) is：10 μ L Roche 480probs master mix, 0.5 μ L are dense
It is 10 μM of preceding primers and rear primer to spend, and 0.5uL concentration is 10 μM of taqman probes, and adds water to cumulative volume for 20 μ L.
Computational methods in the step (3) are：Copy number in two parts of samples of statistics, from determining for undressed sample
Amount result obtains methylating and non-methylated alleles sum, from the quantitative result of the sample through processing obtain sample in first
Base copy number, both are divided by, and finally give the percentage of methylated alleles in sample, the i.e. methylation of sample.
Technical scheme is specially：
The first step：The genes of interest to be detected searches out the area rich in methylation sensitive restriction enzyme cleavage sites
Design specific primer and taqman probes in domain.First, it is determined that the corresponding region of the genes of interest to be studied, looks in this region
To the sequence rich in methylation sensitive restriction enzyme cleavage sites, design of primers is carried out by template of the sequence, and together
When design corresponding taqman probes.
Second step：Restriction enzyme ferment treatment is carried out to sample.Sample to be detected is, from cell, to organize, and is carried in blood etc.
The DNA for taking, equal two parts are divided into by sample to be detected：Part A and part B.According to corresponding methyl-sensitive in part B
The operation instruction of property restriction enzyme, carries out restriction enzyme ferment treatment, the non-methyl in part B sample by part B sample
Changing copy will be digested by restriction enzyme cleavage, only the remaining copy that methylates.Part A does not use methylation sensitive restricted
Inscribe ferment treatment.Two parts sample preserves stand-by after above-mentioned steps have been carried out.
3rd step：The array digital pcr micro-fluid chip made using PDMS material is methylated to gene to be detected
Degree carries out absolute quantitation detection.The part A that will be treated in second step and part B sample, are separately added into 10uL Roches
480probs master mix, 0.5uL concentration are the preceding primers of 10uM and rear primer, and 0.5uL concentration is visited for the taqman of 10uM
Pin, and cumulative volume is added water to for 20uL.It is thus A, B two parts sample has prepared PCR reaction systems respectively.Using this hair
Bright digital pcr chip array formula digital pcr micro-fluid chip carries out absolute quantitation detection to A and B two parts sample respectively.Through
Cross after PCR processes, count the copy number in part A and part B sample, circular is：
If chip bright spot number is set to n, if always points are set to N to chip.Due to number x of the DNA molecular in reaction warehouse
Distribution meets Poisson distribution, then according to the distribution function of Poisson distribution：
The reaction warehouse of each unstressed configuration signal contains 0 target molecule, then：
The computing formula of the desired value λ of Poisson distribution can be released：
The molecular number x in reaction warehouse>At=1, it is bright spot that the reaction warehouse can all become, so there is following formula：
Draw：
λ is expectation in Poisson distribution, and what is referred to is exactly that all of reaction warehouse the inside the desired value of molecule amount occurs, so most
Latter step draws the total copy number copy_ for detecting aim sequence by only needing to be multiplied by with λ the number N in overall reaction storehouse
number：
Copy_number=λ × N
If the initial concentration of detected material to be drawn, only need to by total copy number copy_number divided by initial sample body
Product v：
So far, the copy number and concentration of aim sequence in sample to be detected have been accurately obtained.
Obtain methylating from the result of part A and copy sum with non-methylated alleles, from the quantitative knot of part B
In fruit, methylated alleles copy number in sample is obtained.Both are divided by, and just finally give and methylate in sample to be tested equipotential base
The methylation of the percentage of cause, i.e. sample.
The present invention is combined using PDMS array digital pcr chips with methylation-specific restriction enzyme, is designed
Go out a kind of gene methylation degree quantitative approach based on digital pcr chip.The sensitivity of DNA methylation copy detection can be reached
To 10 copies.Need not rely on standard items, it is not necessary to pre-rendered standard curve, can be to the methylation of genes of interest
Carry out absolute quantitation.The present invention can be changed with the small copy number of accurate quantification, accurately identify the such small concentrations difference of DNA molecular.From
And accurately distinguish the fine difference of methylation.
Beneficial effect
The present invention can reach 10 copies to the sensitivity of DNA methylation copy detection, and sensitivity is high；Entirely point liquid, expands
Increase, reading process is completed completely on same array digital pcr chip, it is not necessary to extra digital pcr special equipment；No
Need to rely on standard items, it is not necessary to pre-rendered standard curve, absolute quantitation can be carried out to the methylation of genes of interest；
Detection sensitivity is high；Detection process is simple；And detection time is short；The method disclosure satisfy that clinical demand, have in terms of medical science
Good application prospect.
Brief description of the drawings
Fig. 1 is testing process schematic diagram of the invention；
Fig. 2 is the real reaction occurred in digital pcr chip in embodiment 1；
Fig. 3 is the digital pcr result of sample 1 in embodiment 1.
Specific embodiment
With reference to specific embodiment, the present invention is expanded on further.It should be understood that these embodiments are merely to illustrate the present invention
Rather than limitation the scope of the present invention.In addition, it is to be understood that after the content for having read instruction of the present invention, people in the art
Member can make various changes or modifications to the present invention, and these equivalent form of values equally fall within the application appended claims and limited
Scope.
Embodiment 1
(1) design of primer and probe and preparation
First, the present embodiment selection and cancer early stage degree of correlation PCDHGB6 genes higher, the gene promoter region
Methylation it is high closely related with the generation of cancer.The methylation sensitive restriction enzyme used in the present embodiment is
HpaII, its cleavage site is CCGG sequences.So the promoter region in PCDHGB6 genes searches the region rich in CCGG,
For region design primer and taqman probes.
In the present embodiment, for the ease of the result of observation reaction, FAM fluorescence molecules have been modified at taqman probes two ends respectively
With BHQ fluorescent quenching agent molecules.When extension increasing sequence is needed in PCR system, taqman probes can be with sequence to be detected by mutual
Mend pair principle to be combined, so as to during PCR, by taq enzyme hydrolysis, FAM fluoresceins are separated with BHQ fluorescent quenching agent molecules,
So as to discharging fluorescence.
Nucleic acid probe used and PCR primer synthesize by Shanghai Zhan Biao bio tech ltd.Sequence used is shown in Table
1。
The taqman probe sequences of table 1 and PCR primer sequence
Reagent and instrument：(Shanghai accounts for the mark limited public affairs of biotechnology for DEPC water (Sangon Biotech), primer and probe
Department), the Probes Master (Roche) of Lightcycler 480, BX51 fluorescence microscopes (OLYMPUS).
(2) in sample PCDHGB6 gene promoter regions methylation detection
The first step, restriction enzyme ferment treatment is carried out to sample.In the present embodiment, sample to be detected is from patients with lung cancer hand
The genomic DNA extracted in the cancerous tissue of art excision and cancer beside organism.Each sample to be detected is divided into equal two parts：A
Part and part B.According to the operation instruction of HpaII methylation sensitive restriction enzymes in part B, part B sample is entered
37 degrees Celsius of row restriction enzyme ferment treatment 12 hours, last 85 degrees Celsius inactivate HpaII in 15 minutes, in part B sample
The non-copy that methylates of PCDHGB6 gene promoters will be digested by restriction enzyme cleavage, only be left the gene promoter region
The copy that methylates.Part A does not use HpaII methylation sensitive restriction enzyme ferment treatments.Two parts sample is being carried out
Preserved after above-mentioned steps stand-by.
Second step：The array digital pcr micro-fluid chip made using PDMS material is methylated to gene to be detected
Degree carries out absolute quantitation detection.The part A that will be treated in the first step and part B sample, are separately added into 10uL Roches 480
Probs master mix, 0.5uL concentration are the preceding primers of 10uM and rear primer, and 0.5uL concentration is the taqman probes of 10uM, and
Cumulative volume is added water to for 20uL.It is definitely fixed that A and B two parts sample is carried out respectively using array digital pcr micro-fluid chip
Amount detection.After by PCR processes, the copy number in part A and part B sample is counted.
Obtain methylating and non-methylated alleles sum from the result of part A, from the quantitative result of part B
In, obtain the copy number that methylates in sample.Both are divided by, and just finally give the percentage of methylated alleles in sample to be tested
Than the i.e. methylation of sample.
Four samples have been carried out PCDHGB6 gene promoters by the method announced using the present invention in the present embodiment altogether
The methylation in region is determined, and four sample numbers are respectively sample 1, sample 2, sample 3, sample 4.Simultaneously to this four samples
This methylation has carried out pyrosequencing, and the method in the present invention has one with the testing result of pyrosequencing method
Cause property, it was demonstrated that the feasibility of detection method in the present invention.The Comparative result such as table 2 that two methods are determined：
Four PCDHGB6 promoter regions methylation digital pcr testing results of sample and burnt phosphorus in the embodiment of table 2
Sour sequencing assay result contrast
SEQUENCE LISTING
<110>Shanghai Inst. of Microsystem and Information Technology, Chinese Academy of Sci
<120>A kind of gene methylation degree quantitative approach based on digital pcr chip
<130> 1
<160> 3
<170> PatentIn version 3.3
<210> 1
<211> 20
<212> DNA
<213>Artificial sequence
<400> 1
gatgtacacc tgcattttcg 20
<210> 2
<211> 20
<212> DNA
<213>Artificial sequence
<400> 2
cgttcgctcg ggttctcgct 20
<210> 3
<211> 25
<212> DNA
<213>Artificial sequence
<400> 3
acgccgggga tccgtcagcc tctgg 25
Claims (5)
1. a kind of gene methylation degree quantitative approach based on digital pcr chip, including：
(1) for genes of interest to be detected, it is determined that the region rich in methylation sensitive restriction enzyme cleavage sites, and
To region design primer and taqman probes；
(2) sample to be detected is divided into two parts, through methylation sensitive restriction enzyme ferment treatment, another does not locate a copy of it
Reason；(3) quantitative determination is carried out to two parts of samples respectively using digital pcr chip, finally calculates the first of genes of interest in sample
Base degree.
2. a kind of gene methylation degree quantitative approach based on digital pcr chip according to claim 1, its feature exists
In：FAM fluorescence molecules and BHQ fluorescent quenching agent molecules are modified in taqman probes two ends in the step (1) respectively.
3. a kind of gene methylation degree quantitative approach based on digital pcr chip according to claim 1, its feature exists
In：Digital pcr chip in the step (3) is the array digital pcr micro-fluid chip made using PDMS.
4. a kind of gene methylation degree quantitative approach based on digital pcr chip according to claim 1, its feature exists
In：Pcr chip reaction system in the step (3) is：10 μ L Roche 480probs master mix, 0.5 μ L concentration is 10
μM preceding primer and rear primer, 0.5uL concentration are 10 μM of taqman probes, and add water to cumulative volume for 20 μ L.
5. a kind of gene methylation degree quantitative approach based on digital pcr chip according to claim 1, its feature exists
In：Computational methods in the step (3) are：Copy number in two parts of samples of statistics, from the quantitative knot of undressed sample
Fruit obtains methylating and non-methylated alleles sum, from the quantitative result of the sample through processing obtain sample in methylate
Copy number, both are divided by, and finally give the percentage of methylated alleles in sample, the i.e. methylation of sample.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109022556A (en) * | 2018-08-29 | 2018-12-18 | 天津智鱼生物科技有限公司 | A kind of quantitative approach and application of DNA methylation degree |
| CN113249487A (en) * | 2021-06-29 | 2021-08-13 | 北京求臻医学检验实验室有限公司 | Biomarker combination, detection method and kit for early screening of liver cancer |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101855363A (en) * | 2007-08-03 | 2010-10-06 | 香港中文大学 | Analysis of nucleic acids by digital pcr |
| CN102625854A (en) * | 2009-08-11 | 2012-08-01 | 香港中文大学 | Method for detecting chromosomal aneuploidy |
| CN104762301A (en) * | 2015-04-24 | 2015-07-08 | 武汉大学 | Kit and method for detecting methylation level of liver cancer risk gene TSPYL5 |
-
2017
- 2017-03-24 CN CN201710183073.2A patent/CN106916893A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101855363A (en) * | 2007-08-03 | 2010-10-06 | 香港中文大学 | Analysis of nucleic acids by digital pcr |
| CN102625854A (en) * | 2009-08-11 | 2012-08-01 | 香港中文大学 | Method for detecting chromosomal aneuploidy |
| CN104762301A (en) * | 2015-04-24 | 2015-07-08 | 武汉大学 | Kit and method for detecting methylation level of liver cancer risk gene TSPYL5 |
Non-Patent Citations (4)
| Title |
|---|
| ANDREA SARTORE-BIANCHI等: "Digital PCR assessment of MGMT promoter methylation coupled with reduced protein expression optimises prediction of response to alkylating agents in metastatic colorectal cancer patients", 《EUROPEAN JOURNAL OF CANCER》 * |
| JIANGYAN ZHANG等: "Associated Analysis of DNA Methylation for Cancer Detection Using CCP-Based FRET Technique", 《AANALYTICAL CHEMISTRY》 * |
| 周云龙: "《转基因生物标准物质研制与应用》", 13 August 2014, 中国质检出版社 * |
| 许海燕 王琛: "《纳米生物医学技术》", 30 June 2009, 中国协和医科大学出版社 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109022556A (en) * | 2018-08-29 | 2018-12-18 | 天津智鱼生物科技有限公司 | A kind of quantitative approach and application of DNA methylation degree |
| CN113249487A (en) * | 2021-06-29 | 2021-08-13 | 北京求臻医学检验实验室有限公司 | Biomarker combination, detection method and kit for early screening of liver cancer |
| CN113249487B (en) * | 2021-06-29 | 2021-10-01 | 北京求臻医学检验实验室有限公司 | Biomarker combination, detection method and kit for early screening of liver cancer |
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Application publication date: 20170704 |