CN109266749A - A kind of primer and its detection method detecting RNASET2 gene promoter methylation - Google Patents
A kind of primer and its detection method detecting RNASET2 gene promoter methylation Download PDFInfo
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- CN109266749A CN109266749A CN201811405147.3A CN201811405147A CN109266749A CN 109266749 A CN109266749 A CN 109266749A CN 201811405147 A CN201811405147 A CN 201811405147A CN 109266749 A CN109266749 A CN 109266749A
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Abstract
The invention discloses a kind of primers and its detection method for detecting RNASET2 gene promoter methylation, it is related to technical field of bioengineering, the primer includes the methylation upstream primer MF and methylation downstream primer MR with the template matching to methylate, with the non-methylation upstream primer UF and non-methylation downstream primer UR of the template matching not methylated, the primer has many advantages, such as specific good, high sensitivity, realizes the accurate detection of detection RNASET2 promoter methylation.
Description
Technical field
The present invention relates to technical field of bioengineering, a kind of detection RNASET2 gene promoter methylation is particularly related to
Primer and its detection method.
Background technique
It proposes to capture cancer and plan from U.S.'s the 1970s, so far more than 40 year, a large amount of manpowers of world expenditure, object
Power is dedicated to the research of tumour.Preliminary understanding there has been to the mechanism of tumorigenesis now, but do not see clearly canceration really also
Essence.With the rapid development of Protocols in Molecular Biology, researcher has realized that the exception of oncogene and tumor suppressor gene
Variation play an important role in the generation, development of human malignancies, the especially forfeiture of tumor suppressor gene function, increasingly by
To the attention of people.Tumor suppressor gene has the function of inhibiting growth and proliferation of cell, under normal circumstances, they to the growth of cell,
Development and differentiation play important adjustment effect.These genes due to methylate, be mutated etc. a variety of causes cause gene inactivate or
When its product loses activity, the canceration of cell and the generation of tumour can lead to.
Cervical carcinoma is one of most common gynecologic malignant tumor, seriously threatens the health and life of women.Cervical carcinoma
Morbidity is a polygenes, multi-step as a result, wherein the forfeiture of tumor suppressor gene function and the expression of oncogene are its cell cancers
The molecular basis of change.DNA methylation is tumorigenic earliest events, therefore is carried out by the aberrant methylation of detection gene
The early diagnosis of tumour has important clinical meaning.Recent studies indicate that the generation of DNA methylation and cervical carcinoma, hair
It opens up closely related, can be used as effective molecular marker applied to cervical carcinoma screening.Methylation of tumor suppressor is in cervical carcinoma
Generally existing, the research of cervical carcinoma methylation of tumor suppressor will provide new think of for the early diagnosis, treatment and prognosis of cervical carcinoma
Road and method.It was found that more, better cervical cancer-related genes, find the methylation mark with high degree of specificity and sensibility
Object, and it is used to clinical practice, it is still a great problem and challenge.
Tumor suppressor gene RNASET2 is positioned at 6q27, is II class tumor suppressor gene.Its RNASET2 albumen encoded belongs to
Ribalgilase Rh/T2/S family is a kind of secreting type glycoprotein with catalytic activity, tetracycline-regulated gene expression and cell point
Change, changes tumor microenvironment, inhibit the progress of tumour.Previously research shows that in ovarian cancer cell line, malignant mela noma, forefront
RNASET2 transcriptional level is lowered in the malignant tumours such as gland cancer, prompts RNASET2 that may play important work in the occurrence and development of tumour
With.But it there is no the correlative study of the gene and cervical carcinoma at present.
Summary of the invention
In view of this, it is an object of the invention to propose it is a kind of detect RNASET2 gene promoter methylation primer and
Its detection method, for overcoming above-mentioned all or part of deficiency in the prior art.
Based on above-mentioned purpose, the present invention provides a kind of primer for detecting RNASET2 gene promoter methylation, the primer packet
Include with the methylation upstream primer MF of template matching to methylate and the downstream primer MR that methylates, and do not methylate
The non-methylation upstream primer UF of template matching and non-methylation downstream primer UR.
Optionally, the methylation upstream primer MF:5 '-TTTACGGGTGGTTTAGGTTAGTTC- 3 ', methylate downstream
Primer MR:5 '-TATCAAACGACTATATTCCTTCGC-3 ', wherein the DNA for being with being methylated of underscore mark
The site to match.
Optionally, the non-methylation upstream primer UF:5 '-TTTTATGGGTGGTTTAGGTTAGTTT- 3 ', non-methyl
Change downstream primer UR:5 '-ATCAAACAACTATATTCCTTCACC-3 ', wherein what underscore marked is and does not methylate
The site that DNA matches.
Optionally, the concentration ratio of the upstream primer that methylates in the primer and downstream primer is 1:1.
Optionally, the concentration ratio of non-methylation upstream primer and downstream primer is 1:1 in the primer.
Optionally, the pcr amplification product length of the primer is 107bp.
A method of detection RNASET2 gene promoter methylation includes the following steps:
1) DNA sample is extracted；
2) modification of DNA；
3) modifying DNA methylation status of PTEN promoter.
Optionally, the modification of the DNA is to carry out bisulfites to DNA using EZ DNA methylation kit-Gold
Modification and purifying.
Optionally, the PCR amplification condition of the DNA methylation specific PCR: 94 DEG C of initial denaturation 2min；94 DEG C of denaturation
40s, 63 DEG C of annealing 40s, -0.5 DEG C of circulation, 72 DEG C of extension 45s, 10 circulations；95 DEG C of denaturation 40s, 58 DEG C of annealing 40s, 72 DEG C
Extend 45s, 35 circulations；Last 72 DEG C of extensions 10min.
From the above it can be seen that the present invention provides a kind of detection tumor suppressor gene RNASET2 promoter CpG island methylation
Primer and detection method, on the one hand target dna is detected by pairs of methylated primers and non-methylation, improves detection spirit
Sensitivity has specificity good, and the context of detection on the other hand used is simple to operation, realizes detection RNASET2 promoter first
The accurate detection of base.
Detailed description of the invention
Fig. 1 is RNASET2 of embodiment of the present invention gene M SP electrophoresis result schematic diagram.
MK-Marker, M- the methylation non-methylation of U-, the processed normal cervical tissues of MB-Sss I, B- is without methylation
Normal healthy controls peripheral blood, 1,2,3- patient's sample, 4- compare sample, Blank- blank control.
Specific embodiment
To make the objectives, technical solutions, and advantages of the present invention clearer, below in conjunction with specific embodiment, in conjunction with
Figure, the present invention is described in more detail.
In embodiments of the present invention, on the one hand, a kind of primer of detection RNASET2 gene promoter methylation of offer,
The primer include with the methylation upstream primer MF of template matching to methylate and the downstream primer MR that methylates, and do not occur
The non-methylation upstream primer UF of the template matching of methylation and non-methylation downstream primer UR.
In some optional embodiments, a kind of primer detecting RNASET2 gene promoter methylation provided by the invention,
Including the methylation upstream primer MF:5 '-TTTA with the template matching to methylateCGGGTGGTTTAGGTTAGTTC- 3 ',
Methylate downstream primer MR:5 '-TATCAAACGACTATATTCCTTCGC-3 ', wherein what underscore marked is and first has occurred
The site that the DNA of base matches, pcr amplification product length are 107bp, and PCR amplification is methylation upstream primer MF and downstream
The concentration ratio of primer MR is 1:1；Primer further includes the non-methylation upstream primer UF with the template matching not methylated:
5′-TTTTATGGGTGGTTTAGGTTAGTTT- 3 ', non-methylation downstream primer UR:5 '-
ATCAAACAACTATATTCCTTCACC-3 ', wherein the site for being with methylate DNA does not occur matching of underscore mark,
Pcr amplification product length is 107bp, and PCR amplification is that the concentration ratio of non-methylation upstream primer UF and downstream primer UR is 1:1.
On the other hand, a kind of method detecting RNASET2 gene promoter methylation, packet are also provided in the embodiment of the present invention
Include following steps:
1) DNA sample is extracted；
2) modification of DNA；
3) modifying DNA methylation status of PTEN promoter.
In some optional embodiments, a kind of detection RNASET2 gene promoter methyl is also provided in the embodiment of the present invention
The method of change, the specific steps are as follows:
1) DNA sample is extracted to include using blood, extract cell tissue, cell tissue genome DNA extracting reagent kit (goods
Number: DP304) extract DNA.
The genome DNA extracting reagent kit of use is purchased from Tiangeng biochemical technology Co., Ltd comprising buffer GA
(Buffer GA), buffer GB (Buffer GB), buffer GD (Buffer GD), rinsing liquid PW (Buffer PW), elution
Buffer TE (Buffer TE), Proteinase K, adsorption column CB3, collecting pipe.
0.2~0.5g of cervical cancer tissues or normal cervical tissues is taken, is rinsed with physiological saline, filter paper is dried, and weighing is put into
In mortar, tissue is shredded as early as possible with the small scissors of ophthalmology, the grinding of 1mL physiological saline is added, glass is poured into the tissue homogenate of grinding
In glass homogenizer, with 0.5mL normal saline flushing mortar, pour into homogenizer together；Grinding number 10 times is rotated upwardly and downwardly, tissue is made
Tissue after homogenate is transferred in centrifuge tube by homogenization, and 10000rpm is centrifuged 1min, is removed supernatant, is added into sediment
200 μ L buffer GA, concussion suspend to thorough；Then 20 μ L Proteinase Ks are successively added thereto, mix, 56 DEG C are placed to group
Dissolution is knitted, 200 μ L buffer GB are added, are sufficiently mixed by inversion, 70 DEG C of placement 10min, 200 μ L dehydrated alcohols are added, is overturned mixed
It is even, it then successively proceeds as follows, above-mentioned solution is added in adsorption column CB3 (adsorption column is put into collecting pipe),
12000rpm is centrifuged 30s, abandons waste liquid, adsorption column is put back in collecting pipe；500 μ L buffer GD are added into adsorption column,
12000rpm is centrifuged 30s, abandons waste liquid, adsorption column is put back in collecting pipe；600 μ L rinsing liquid PW are added into adsorption column (to use
Preceding addition dehydrated alcohol), 12000rpm is centrifuged 30s, abandons waste liquid, adsorption column is put back in collecting pipe, and repetitive operation is above-mentioned last
After centrifugally operated, adsorption column is put back into collecting pipe, 12000rpm is centrifuged 2min, abandons waste liquid, is placed at room temperature for a few minutes.It will inhale
Attached column is put into clean centrifuge tube, and 100 μ L TE of eluent is added into adsorption column, is placed at room temperature for 5min, 12000rpm centrifugation
2min obtains DNA extracting solution.
2) modification of DNA；Bisulfite is carried out to DNA using EZ DNA methylation kit-Gold (article No.: D5005)
Salt modification and purifying.Methylation reagent treatment is commercially available from Beijing Tian Mo Science and Technology Development Co., Ltd. comprising CT
Conversion Reagent、M-Binding Buffer、M-Wash Buffer、M-Desulphonation Buffer、M-
Elution Buffer, Zymo-Spin IC Column and Collection Tube.
The preparation of CT Conversion Reagent: by 900 μ L sterilizing distilled water, 50 μ L M-Dissolving
Buffer and 300 μ L M-Dilution Buffer to a pipe CT Conversion Reagent, shakes 10min at room temperature, and 4
DEG C overnight, with preceding concussion 2min.The CT Conversion Reagent of 130 μ L is added in PCR pipe, 20 μ L DNA samples are mixed
It is even.PCR pipe is put into PCR instrument, is operated according to the following steps: 98 DEG C of 10min；Then 600 μ L M- are added in 64 DEG C of 150min
Binding Buffer is put into the Collection Tube that kit provides into adsorption column, and by adsorption column.By PCR pipe
In sample be added in the adsorption column containing M-Binding Buffer, close the lid, be mixed by inversion for several times, 10000rpm from
Heart 30s abandons waste liquid；200 μ L M-Wash Buffer are added into adsorption column, 10000rpm is centrifuged 30s, abandons waste liquid；It is added 200
μ L M-Desulphonation Buffer is placed at room temperature for 20min into adsorption column, and 10000rpm is centrifuged 30s, abandons waste liquid；Add
Enter 200 μ L M-Wash Buffer into adsorption column, 10000rpm is centrifuged 30s, abandons waste liquid；Add 200 μ L M-Wash
For Buffer into adsorption column, 10000rpm is centrifuged 30s, abandons waste liquid；10 μ L M-Elution Buffer are added into base for post matter,
Adsorption column is placed in the EP pipe of new 1.5mL, stands 2min, 10000rpm is centrifuged 1min, the DNA modified.
3) modifying DNA methylation status of PTEN promoter: PCR kit is using Promega companyHot Start
Green Master Mix (article No. M5122), includingThermal starting enzyme, Reaction buffer (pH
8.5), 400uM dATP, 400uM dATP, 400uM dATP, 400uM dATP and 4mM MgCl2。
Methylation status of PTEN promoter (Methylation-specific PCR, MSP), MSP reaction system: 15-50 μ L, it is excellent
15 μ L of choosing, containHot Start Green Master Mix 7.5-25 μ L, preferred 7.5 μ L, EZ DNA first
DNA profiling DNA 2-4 μ L, preferred 2 μ L, upstream and downstream primer each 1-2 μ L, preferred 1 μ after base kit-Gold modification
L, sterilizing distilled water complement to 15-50 μ L, preferred 15 μ L.Using the PCR response procedures of Touch-down: 94 DEG C of initial denaturations
2min；94 DEG C of denaturation 40s, 63 DEG C of annealing 40s, -0.5 DEG C/circulation, 72 DEG C of extension 45s, 10 recycle；95 DEG C of denaturation 40s, 58
DEG C annealing 40s, 72 DEG C of extensions 45s, 35 recycle；Last 72 DEG C of extensions 10min, 4 DEG C of preservations.
Divided with human peripheral lymphocytes DNA through the processed cervical cancer tissues DNA of methylase Sss I and water
Zuo Wei it is non-methylation, methylation and blank control carry out gel analysis, take 5 μ L MSP reaction products, 2.5% Ago-Gel
Electrophoresis detection amplification, the result is shown in Figure 1.The corresponding sequence of DNA sequence dna and primer of amplification is as follows:
Gel analysis is as follows: if only U primer can amplify band, and pillar location is correct (107bp), is judged as methylation
It is negative；If only purpose band (107bp) occurs in M primer, it is judged as exhaustive methylation；If there is purpose band in M and U primer,
Then it is judged as partial methylation；Exhaustive methylation and partial methylation are methylation positive.Testing result shows that the primer has
There is specific good, high sensitivity, realizes the accurate detection of detection RNASET2 promoter methylation.
It should be understood by those ordinary skilled in the art that: the discussion of any of the above embodiment is exemplary only, not
It is intended to imply that the scope of the present disclosure (including claim) is limited to these examples；Under thinking of the invention, above embodiments
Or can also be combined between the technical characteristic in different embodiments, step can be realized with random order, and be existed such as
Many other variations of the upper different aspect of the invention, for simplicity, they are not provided in details.
The embodiment of the present invention be intended to cover fall into all such replacements within the broad range of appended claims,
Modifications and variations.Therefore, all within the spirits and principles of the present invention, any omission, modification, equivalent replacement, the improvement made
Deng should all be included in the protection scope of the present invention.
Claims (9)
1. a kind of primer for detecting RNASET2 gene promoter methylation, which is characterized in that the primer includes and methylates
Template matching methylation upstream primer MF and methylation downstream primer MR, the non-first with the template matching not methylated
Base upstream primer UF and non-methylation downstream primer UR.
2. the primer of detection RNASET2 gene promoter methylation according to claim 1, which is characterized in that the first
Base upstream primer MF:5 '-TTTACGGGTGGTTTAGGTTAGTTC- 3 ', methylate downstream primer MR:5 '-
TATCAAACGACTATATTCCTTCGC-3 ', wherein the position that the DNA for being with being methylated of underscore mark matches
Point.
3. the primer of detection RNASET2 gene promoter methylation according to claim 1, which is characterized in that described non-
Methylate upstream primer UF:5 '-TTTTATGGGTGGTTTAGGTTAGTTT- 3 ', non-methylation downstream primer UR:5 '-
ATCAAACAACTATATTCCTTCACC-3 ', wherein the site for being with methylate DNA does not occur matching of underscore mark.
4. the primer of detection RNASET2 gene promoter methylation according to claim 1, which is characterized in that described to draw
The concentration ratio of the upstream primer that methylates in object and downstream primer is 1:1.
5. the primer of detection RNASET2 gene promoter methylation according to claim 1, which is characterized in that described to draw
The concentration ratio of non-methylation upstream primer and downstream primer is 1:1 in object.
6. the primer of detection RNASET2 gene promoter methylation according to claim 1, which is characterized in that described to draw
The pcr amplification product length of object is 107bp.
7. a kind of method for detecting RNASET2 gene promoter methylation, which comprises the steps of:
1) DNA sample is extracted；
2) modification of DNA；
3) modifying DNA methylation status of PTEN promoter.
8. the method for detection RNASET2 gene promoter methylation according to claim 7, which is characterized in that the DNA
Modification be using EZ DNA methylation kit-Gold to DNA carry out it is bisulphite modified and purifying.
9. the method for detection RNASET2 gene promoter methylation according to claim 7, which is characterized in that the DNA
The PCR amplification condition of methylation status of PTEN promoter: 94 DEG C of initial denaturation 2min；94 DEG C of denaturation 40s, 63 DEG C of annealing 40s, -0.5 DEG C is followed
Ring, 72 DEG C of extension 45s, 10 circulations；95 DEG C of denaturation 40s, 58 DEG C of annealing 40s, 72 DEG C of extension 45s, 35 recycle；Last 72 DEG C
Extend 10min.
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