CN108103163A - A kind of detection method of gene methylation - Google Patents
A kind of detection method of gene methylation Download PDFInfo
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- CN108103163A CN108103163A CN201810046429.2A CN201810046429A CN108103163A CN 108103163 A CN108103163 A CN 108103163A CN 201810046429 A CN201810046429 A CN 201810046429A CN 108103163 A CN108103163 A CN 108103163A
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Abstract
The invention belongs to gene engineering technology fields, and in particular to a kind of detection method of gene methylation.Detection method is to use restriction enzyme enzymatic treatment nucleic acid to be detected, and for the primed probe of region nucleotide sequence to be checked design specificity, carries out PCR amplification and fluorescence signal detects.The present invention has easy to operate, high specificity, the advantage of high sensitivity.
Description
Technical field
The invention belongs to gene engineering technology fields, and in particular to a kind of detection method of gene methylation.
Background technology
DNA methylation is one of apparent modification mode of the gene found earliest, and methylating in eucaryote occurs only at
Cytimidine, i.e., making the cytimidine at CpG dinucleotides 5 '-end under the action of DNA methylation transferase, to be changed into 5 '-methyl born of the same parents phonetic
Pyridine.
The usual inhibition of gene expression of DNA methylation, such as when tumour occurs, the CpG sequences beyond tumor suppressor gene CpG islands
Non- methylation increases, and the CpG in CpG islands then in high methylation state, causes the decline of expression of tumor suppressor gene.Therefore,
Methylating for gene of research has very important significance to the early screening of cancer.
At present, the detection of gene methylation is mainly by bisulf iotate-treated, then with the PCR of methylation-specific or
The method of sequencing distinguishes, such method easily occur bisulfite conversion it is incomplete caused by false positive results, and
Complex for operation step, required reagent type is more, and time-consuming.
The content of the invention
For existing technology there are the problem of, the present invention provides a kind of detection method of gene methylation, it is therefore an objective to by adopting
With restriction enzyme combination real-time fluorescence quantitative PCR, easy to operate, high specificity, the gene methylation of high sensitivity are realized
Detection.
Realize that the gene methylation detection method of the object of the invention follows the steps below：
(1) using the restriction enzyme enzymatic treatment sample to be tested nucleic acid DNA in restriction enzyme reaction liquid, digestion is obtained
Product；
(2) digestion products are added in into detection primer probe, the qPCR reaction solutions of formation carry out qPCR amplifications；
(3) FAN fluorescence channel detections are carried out according to qPCR results, if tested sample FAM fluorescence channels have amplification, for sun
Property sample, illustration purpose gene methylates, if FAM fluorescence channels do not expand, for negative sample, illustration purpose gene
Do not methylate.
Wherein, the sample to be tested includes：Whole blood sample, plasma sample, FFPE samples, saliva sample, the urine of the mankind
Liquid sample, fecal sample and plant and microorganism.
The restriction enzyme reaction liquid, concrete component include：10-100U restriction enzymes, 5 10 × limits of μ L
Property endonuclease digestion buffer solution processed.
The restriction enzyme is more than one or both of Second-Type restriction enzyme, according to target gene
It makes choice, specifically includes：AatII、AciI、AclI、AfeI、AgeI、AscI、AsiSI、AvaI、BceAI、BmgBI、
BsaAI、BsaHI、BsiEI、BsiWI、BsmBI、BspDI、BspEI、BsrFI、BssHII、BstBI、BstUI、BtgZI、
ClaI、EagI、FauI、FseI、FspI、HaeII、HgaI、HhaI、HinP1I、HpaII、Hpy99I、HpyCH4IV、KasI、
MluI、NaeI、NarI、NgoMIV、NotI、NruI、Nt.BsmAI、Nt.CviPII、PaeR7I、PluTI、PmlI、PvuI、
RsrII、SacII、SalI、SfoI、SgrAI、SmaI、SnaBI、TliI、TspMI、XhoI、XmaI、ZraI。
When the processing processing sample to be tested nucleic acid DNA time is 1~16 small.
The detection primer probe includes：Target gene primer pair and probe；Reference gene primer pair and probe.
The qPCR reaction solutions, concrete component include：0.1-1 μM of target gene primer pair, 0.1-1 μM of target gene are visited
Pin, 0.1-1 μM of reference gene primer pair, 0.1-1 μM of reference gene probe, 2 × qPCR Master Mix.
The qPCR platforms include：Roche, ABI, Bio-Rad, Agilent, Hangzhou BIOER Technology Co., Ltd
(BIOER)。
The detection of the FAN fluorescence channels detection is limited to：Sample DNA content >=2ng, the frequency of mutation that methylates be low >=
1%
Compared with prior art, the features of the present invention and advantageous effect are：
Detection method is restriction enzyme enzymatic treatment nucleic acid to be detected, and methylated nucleic acid sequence is known for Restriction Enzyme
Other region, restriction enzyme identify simultaneously non-methylated nucleic acid sequence the nucleotide sequence identification to methylate but not digestion
Generation digestion activity.Therefore, the nucleotide sequence to methylate retains complete rather than methylated nucleic acid sequence and is digested into small fragment.
Based on above-mentioned principle, the present invention is for the specific primed probe of region nucleotide sequence to be checked design, if area to be measured
The modification that methylates is found in domain, not then being digested can be combined with specific primer probe, generate fluorescence signal；If in region to be measured
Do not methylate modification, then digestion is into small fragment and specific primer probe cannot combine, and does not generate fluorescence signal.
The present invention has easy to operate, high specificity, the advantage of high sensitivity.
Description of the drawings
Fig. 1 is 3 normal persons and 2 plasma of colorectal cancer sample Septin9 gene methylations inspections in the embodiment of the present invention 1
Mapping；
Fig. 2 is sample a1 in the embodiment of the present invention 2, a2, a3, a4, a5, the FAM passage amplification figures of a6；
Fig. 3 is sample a1 in the embodiment of the present invention 2, a2, a3, a4, a5, the Cy5 passage amplification figures of a6；
Fig. 4 is sample b1 in the embodiment of the present invention 2, b2, b3, b4, b5, the FAM passage amplification figures of b6；
Fig. 5 is sample b1 in the embodiment of the present invention 2, b2, b3, b4, b5, the Cy5 passage amplification figures of b6.
Specific embodiment
The invention will be further described with reference to embodiments.
Embodiment 1：
The present embodiment carries out DNA methylation assay to target gene Septin9 genes, and the material being related to has：
Test plasma sample, using methylating and non-standard items DNA (the Human Methylated＆Non- that methylate
Methylated DNA Set, Catalog#D5014, ZYMO RESEARCH) it is admixed, methylate DNA ratio is respectively
1% and 0%, concentration is 2ng/ μ L.
Instrument：Roche Lightcycler 480II real-time fluorescence quantitative PCRs instrument, regular-PCR instrument, spiral blending instrument, water
Bath, vortex oscillation instrument.
Specifically follow the steps below：
(1) sample to be tested is human normal plasma or colorectal cancer patients blood plasma in the present embodiment, using kit (The
MagMAXTM Cell-Free DNA Isolation Kit) extraction sample to be tested DNA, specific operation is referring to reagent kit product
Specification；After obtaining sample DNA, concentration is measured using Qubit3.0, qualified sample DNA solution is sample A, and -20 DEG C are protected
It deposits；
According to Septin9 gene methylation regional sequence features, the specific restriction enzyme A ciI that methylates is selected,
Sample to be tested A is handled using the AciI in AciI reaction solutions, digestion amount is 10U, and the digestion time is 2h, obtains the product after digestion
B；
(2) digestion products are added in into detection primer probe, the qPCR reaction solutions of formation carry out qPCR amplifications；Described
The purity of primer should reach electrophoresis grade (PAGE) or HPLC grades, without miscellaneous band；Septin9 gene probes 5 ' hold reporter fluorescence group
For FAM, the quenching group at 3 ' ends is BHQ1；GADPH gene probes 5 ' hold the quenching group that reporter fluorescence group is Cy5,3 ' ends
For BHQ1；Then all primer and probes are provided by research staff's autonomous Design of the present invention by supplier；
The primed probe of purpose Septin9 genes in the present embodiment：
Sense primer：5'-GCTGGATGGGATCATTTCGGA-3'
Anti-sense primer：5'-GGTCCTCTCCAGCACGTC-3'
Probe：5'-FAM-CATCATGTCGGACCCCGCGGTCAACG-Q-3'
Reference gene GADPH gene primer probes：
Sense primer：5'-CCTGCCGGTGACTAACCC-3'
Anti-sense primer：5'-GCCACACGCGACTCCAC-3'
Probe：5'-Cy5-TGCGCTCCTGCCTCGATGGGTGGAGTCGCG-Q-3'；
The qPCR amplification systems of the present embodiment are as shown in table 1：
Table 1
| Ingredient | Volume |
| Septin9 sense primers (10 μM) | 1μL |
| Septin9 anti-sense primers (10 μM) | 1μL |
| Septin9 probes (10 μM) | 0.5μL |
| GAPDH sense primers (10 μM) | 1μL |
| GAPDH anti-sense primers (10 μM) | 1μL |
| GAPDH probes (10 μM) | 0.5μL |
| 2x qPCR Master Mix | 25μL |
| Digestion products B | 10μL |
| dd H2O | Moisturizing is to 50 μ L |
Finger tip flicks reaction tube, and brief centrifugation is placed in progress qPCR detections, response procedures on Lightcycler 480II
As shown in table 2：
Table 2
(3) result judgement：
In order to monitor digestion system, using the Septin9 genes 1% of 2ng methylate mutation DNA as positive reference, 2ng
Septin9 genes 0% methylate mutation DNA as negative reference.
The Cy5 fluorescence channels of positive reference, negative reference and tested sample have amplification and Cp Zhi≤40, then illustrate to configure
QPCR reaction systems and qPCR detections program it is accurate, and then observe FAM fluorescence channels, have if positive with reference to FAM fluorescence channels
Amplification, negative reference FAM fluorescence channels then illustrate that digestion is complete, the result for being detected sample can be judged without amplification.If
Tested sample FAM fluorescence channels have amplification, then are positive sample, illustrate there are Septin9 gene methylations, if FAM fluorescence channels
It does not expand, is then negative sample, illustrates there is no Septin9 gene methylations.
The present embodiment takes the plasma sample and 2 plasma of colorectal cancer samples of 3 normal persons respectively, using commercial reagents
Box (MagMAXTM Cell-Free DNA Isolation Kit, A29319, ThermoFisher) extracts plasma DNA,
Digestion processing is carried out with restriction endonuclease AciI to the dissociative DNA of extraction, by the amplification program in the amplification system in table 1 and table 2 into
Row qPCR is detected.
Statistic mixed-state is as a result, the Cy5 fluorescence channels of positive reference, negative reference and 5 plasma samples have amplification and Cp
Value is less than 40, illustrates that the qPCR reaction systems of configuration and qPCR detections program are accurate, and then observes FAM fluorescence channels, detection knot
Fruit is as shown in Figure 1, curve 1.2.3 is respectively the Septin9 gene magnification curves of 3 normal persons, no amplification；Curve 4 is 2ng's
Septin9 genes 1% methylate the positive reference of mutation, have amplification, Cp values are 37.58；Curve 5 is 1 patients with bowel cancer sample,
There is amplification, Cp values are 36.84；Curve 6 is other 1 patients with bowel cancer sample, there is amplification, and Cp values are 34.37；Curve 7 is 2ng's
Septin9 genes 0% methylate the feminine gender reference of mutation, no amplification；Curve 8 be NTC, no amplification.
It can be seen that FAN fluorescence channels, 3 normal persons are without amplification, and 2 colorectal cancers have amplification, and amplification cycles number is small
In positive reference, illustrate that the method for the present invention can accurately detect the sample of Septin9 gene methylation mutation Shuai≤1% of 2ng.
Embodiment 2：
Referring to Fig. 2-5, the present embodiment is the experiment of the content of minimum nucleic acid and minimum detectability needed for the method for the present invention, still
So by taking Septin9 genes as an example.
The present embodiment use methylates and non-standard items DNA (the Human Methylated＆Non-Methylated that methylate
DNA Set, Catalog#D5014, ZYMO RESEARCH) it carries out admixing different sample initial amounts, methylate DNA ratio is
1%, as shown in table 3：
Table 3
| Sample number | Sample DNA content | The frequency of mutation | Preparation method |
| a1 | 1ng | 1% methylate DNA | The non-methylate DNAs of 0.01ng methylate DNAs+0.99ng |
| a2 | 2ng | 1% methylate DNA | The non-methylate DNAs of 0.02ng methylate DNAs+1.98ng |
| a3 | 5ng | 1% methylate DNA | The non-methylate DNAs of 0.05ng methylate DNAs+4.95ng |
| a4 | 10ng | 1% methylate DNA | The non-methylate DNAs of 0.1ng methylate DNAs+9.9ng |
| a5 | 20ng | 1% methylate DNA | The non-methylate DNAs of 0.2ng methylate DNAs+19.8ng |
| a6 | 50ng | 1% methylate DNA | The non-methylate DNAs of 0.5ng methylate DNAs+49.5ng |
Using methylating and the non-standard items DNA that methylates (Human Methylated＆Non-Methylated DNA
Set, Catalog#D5014, ZYMO RESEARCH) blending different proportion methylate DNA is carried out, initial amount is 2ng, such as table 4
It is shown：
Table 4
| Sample number | Sample DNA content | The frequency of mutation | Preparation method |
| b1 | 2ng | 10% methylate DNA | The non-methylate DNAs of 0.2ng methylate DNAs+1.8ng |
| b2 | 2ng | 7.5% methylate DNA | The non-methylate DNAs of 0.15ng methylate DNAs+1.85ng |
| b3 | 2ng | 5% methylate DNA | The non-methylate DNAs of 0.1ng methylate DNAs+1.9ng |
| b4 | 2ng | 2.5% methylate DNA | The non-methylate DNAs of 0.05ng methylate DNAs+1.95ng |
| b5 | 2ng | 1% methylate DNA | The non-methylate DNAs of 0.02ng methylate DNAs+1.98ng |
| b6 | 2ng | 0% methylate DNA | The non-methylate DNAs of 0ng methylate DNAs+2ng |
The restriction enzyme A ciI of 10U is taken to the sample a1, a2, a3, a4, a5, a6 that prepare above；B1, b2, b3, b4,
B5, b6 distinguish digestion 2h, and preparing reaction system progress qPCR detections, (reaction system, amplification program, primer and probe sequence are shown in reality
Apply example 1).
The results are shown in Figure 3, and the reference gene GADPH of detection sample a1, a2, a3, a4, a5, a6 have amplification and Cp values
Respectively less than 40, it was demonstrated that the experiment accurate and effective.And then target gene passage is observed, the results are shown in Figure 2 for statistic mixed-state, according to figure
2 statistical result draws table 5.
Table 5
| Sample number | Cp values |
| a1 | -- |
| a2 | 45.63 |
| a3 | 42.89 |
| a4 | 41.59 |
| a5 | 39.25 |
| a6 | 38.65 |
The results are shown in Figure 5, and the reference gene GADPH of detection sample b1, b2, b3, b4, b5, b6 have amplification and Cp values
Respectively less than 40, it was demonstrated that the experiment accurate and effective.And then target gene passage is observed, the results are shown in Figure 4 for statistic mixed-state, according to figure
4 statistical result draws table 6.
Table 6
| Sample number | Cp values |
| b1 | 37.6 |
| b2 | 39.25 |
| b3 | 40.84 |
| b4 | 42.10 |
| b5 | 44.24 |
| b6 | -- |
Analytical table 5,6 result of table can obtain, and the method for the present invention can be in DNA content down to 2ng, and Septin9 gene methylations are dashed forward
Variability detects methylating for Septin9, can greatly reduce the DNA nucleic acid amount needed for DNA methylation assay and carry down to 1%
The sensitivity of hyper-methylation detection.
Claims (9)
1. a kind of gene methylation detection method, it is characterised in that follow the steps below：
(1) using the restriction enzyme enzymatic treatment sample to be tested nucleic acid DNA in restriction enzyme reaction liquid, digestion production is obtained
Object；
(2) digestion products are added in into detection primer probe, the qPCR reaction solutions of formation carry out qPCR amplifications；
(3) FAN fluorescence channel detections are carried out according to qPCR results, if tested sample FAM fluorescence channels have amplification, for positive sample
This, illustration purpose gene methylates, if FAM fluorescence channels do not expand, for negative sample, illustration purpose gene does not have
It methylates.
2. a kind of gene methylation detection method according to claim 1, it is characterised in that the sample to be tested includes：
Whole blood sample, plasma sample, FFPE samples, saliva sample, urine specimen, fecal sample and the plant of the mankind and microorganism.
A kind of 3. gene methylation detection method according to claim 1, it is characterised in that the restriction enzyme
Reaction solution, concrete component include：10-100U restriction enzymes, 5 μ L 10 × digestion with restriction enzyme buffer solutions.
A kind of 4. gene methylation detection method according to claim 1, it is characterised in that the restriction enzyme
It more than one or both of Second-Type restriction enzyme, is made choice, specifically included according to target gene：AatII、
AciI、AclI、AfeI、AgeI、AscI、AsiSI、AvaI、BceAI、BmgBI、BsaAI、BsaHI、BsiEI、BsiWI、
BsmBI、BspDI、BspEI、BsrFI、BssHII、BstBI、BstUI、BtgZI、ClaI、EagI、FauI、FseI、FspI、
HaeII、HgaI、HhaI、HinP1I、HpaII、Hpy99I、HpyCH4IV、KasI、MluI、NaeI、NarI、NgoMIV、NotI、
NruI、Nt.BsmAI、Nt.CviPII、PaeR7I、PluTI、PmlI、PvuI、RsrII、SacII、SalI、SfoI、SgrAI、
SmaI、SnaBI、TliI、TspMI、XhoI、XmaI、ZraI。
5. a kind of gene methylation detection method according to claim 1, it is characterised in that the processing processing is to be measured
When the sample nucleic acid DNA times are 1~16 small.
A kind of 6. gene methylation detection method according to claim 1, it is characterised in that the detection primer probe
Including：Target gene primer pair and probe；Reference gene primer pair and probe.
7. a kind of gene methylation detection method according to claim 1, it is characterised in that the qPCR reaction solutions, tool
Body component includes：0.1-1 μM of target gene primer pair, 0.1-1 μM of target gene probe, 0.1-1 μM of reference gene primer pair,
0.1-1 μM of reference gene probe, 2 × qPCR Master Mix.
8. a kind of gene methylation detection method according to claim 1, it is characterised in that the qPCR platforms include：
Roche, ABI, Bio-Rad, Agilent, Hangzhou BIOER Technology Co., Ltd (BIOER).
A kind of 9. gene methylation detection method according to claim 1, it is characterised in that the FAN fluorescence channels inspection
The detection of survey is limited to：Sample DNA content >=2ng, the frequency of mutation that methylates be low >=and 1%.
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| CN110283889A (en) * | 2019-07-22 | 2019-09-27 | 中南大学 | The dual-gene monitoring reaction system of one kind, kit and its application |
| CN110643677A (en) * | 2019-09-30 | 2020-01-03 | 国家烟草质量监督检验中心 | G-quadruplex-based fluorescence sensor for detecting activity of Dnmt1 |
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Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108998529A (en) * | 2018-08-30 | 2018-12-14 | 上海交通大学医学院附属上海儿童医学中心 | Adenocarcinoma of lung detection kit and the method for detecting TULP2 gene methylation level |
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| CN110643677A (en) * | 2019-09-30 | 2020-01-03 | 国家烟草质量监督检验中心 | G-quadruplex-based fluorescence sensor for detecting activity of Dnmt1 |
| CN110643677B (en) * | 2019-09-30 | 2022-08-16 | 国家烟草质量监督检验中心 | G-quadruplex-based fluorescence sensor for detecting activity of Dnmt1 |
| CN110923300A (en) * | 2019-11-18 | 2020-03-27 | 人和未来生物科技（长沙）有限公司 | Gene methylation detection method and application |
| CN111876472A (en) * | 2020-06-17 | 2020-11-03 | 李凯 | Method for detecting trace nucleic acid in multiple mixed nucleic acids |
| CN111876472B (en) * | 2020-06-17 | 2023-12-01 | 江门市灿明生物科技有限公司 | Method for detecting trace nucleic acid in multiple mixed nucleic acids |
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