CN107236805A - FOXP3 gene methylation detection methods based on bisulfite sequencing - Google Patents
FOXP3 gene methylation detection methods based on bisulfite sequencing Download PDFInfo
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Abstract
FOXP3 gene methylation detection methods based on bisulfite sequencing, including：With solidifying heparin tube collector venous blood 4mL, room temperature solidification 30min is promoted, 1000g × 15min is centrifuged；Extract blood clot genomic DNA；Preliminary integrality, concentration and the purity for confirming genomic DNA；Using the conversion reagent box modifier group DNA that methylates；Progress methylate PCR amplification, by 2% agarose gel electrophoresis identify PCR primer, gel imaging system observation, take pictures；18 μ L PCR primers are reclaimed using DNA purifying QIAquick Gel Extraction Kit purifying, is connected by carrier and the 2 μ L PCR primers purified after reclaiming is transformed into Trans1 T1 competence bacteriums, carry out blue white clone's spot screening, and identify positive colony；The thalline chosen in positive colony spot is inoculated in 50 μ g/mL kanamycins monoclonal antibody LB fluid nutrient mediums, after 37 DEG C of 12h of constant temperature 200rpm/min concussion and cultivates 8, takes the sample become cloudy to be sequenced.The present invention can successfully detect the degree that particular sequence 5 CpG sites in FOXP3 gene promoter areas methylate.
Description
Technical field
The present invention relates to biological field, more particularly to a kind of FOXP3 gene methylations based on bisulfite sequencing
Detection method.
Background technology
FOXP3 is regulatory T cells (regulatory T cell, Treg) characteristic mark, is maintaining immune tolerance
During play a significant role.Recent study is confirmed：The expression of FOXP3 genes and the performance of protein function are and epigenetic
Learn (such as DNA methylation, posttranslational modification) closely related.Bisulfite sequencing is the generally acknowledged deoxidation core of domestic and foreign scholars
Ribosomal ribonucleic acid (DNA) methylation sites detect goldstandard, can accurately and delicately detect that the different cytimidine birds of specific dna sequence are fast
The methylation state in nicotinamide adenine dinucleotide (CpG) site, but by factors in experimentation (such as Sample preservation condition, gene
Group DNA content and purity, conversion Post genome DNA content, PCR reaction systems, methylated primers sequence, blue white colony screening etc.
Experimental factor) influence success rate it is low.
The content of the invention
The present invention establishes a kind of FOXP3 (forkhead/winged helix based on bisulfite sequencing
Transcription factor, FOXP3) gene methylation detection method.
As one aspect of the present invention, there is provided a kind of FOXP3 gene methylations based on bisulfite sequencing
Detection method, including：
Step 1, with solidifying heparin tube collector venous blood 4mL, room temperature solidification 30min is promoted, 1000g × 15min is centrifuged, then
Packing 1mL blood clots enter 1.5mL Axygen PE pipes, are stored in -80 DEG C of refrigerators.
Step 2, blood clot genomic DNA is extracted using poba gene group DNA extraction kit.
Step 3, genomic DNA integrality, concentration and pure are primarily determined that by 0.8%-1.0% agarose gel electrophoresis
Degree, and it is standby to detect that qualified DNA sample is placed in -20 DEG C of refrigerators.
Step 4, using the conversion reagent box modifier group DNA that methylates, and the DNA sample modified is placed in -20 DEG C
Refrigerator is standby.
Step 5, it is determined that experiment condition under carry out methylate PCR amplification, by 2% agarose gel electrophoresis (130V,
PCR primer 40min) is identified, gel imaging system is observed, taken pictures, and the purpose PCR primer that recovery is finished is placed in -20 DEG C of ice
Case is standby；The experiment condition of the determination is：Pcr template amount is the genomic DNA after 10 μ L are converted, and PCR is reacted using 50 μ L
The 25mmol/L MgCl of system and 4 μ L2, amplified production length is 111bp.
Step 6, PCR primer is identified by 2% agarose gel electrophoresis (130V, 40min), gel imaging system observation,
Take pictures.
Step 7,18 μ L PCR primers in QIAquick Gel Extraction Kit purifying recycling step 5 are purified using DNA, connected by carrier
2 μ L PCR purifying recovery product is transformed into Trans1-T1 competence bacteriums, carries out blue white clone's spot screening, and with general
Primer (M13F and M13R) does PCR amplifications, and product is separated through 2% agarose gel electrophoresis (130V, 40min), gel imaging system
Overall view is examined, taken pictures, to identify positive colony.
Step 8, the thalline in the positives clone's spot of selecting step 7 is inoculated in the training of 50 μ g/mL kanamycins monoclonal antibody LB liquid
Support in base, after 37 DEG C of constant temperature 200rpm/min concussion and cultivates 8-12h, take the sample become cloudy to send Invitrogen (Shanghai) trade
Sequencing portion of Co., Ltd is sequenced.
Preferably, enter in the step 5 performing PCR amplification primer sequence be：
F:5 '-ATTTTTAGGATTTGAGGTTTTAATA-3 ',
R:5’-CAAAAAACCCACTAAAAAACTATAC-3’。
Preferably, in the step 5 carry out methylate PCR amplification when reaction condition be：
Step A, 94 DEG C of preheating 3min；
Step B, 94 DEG C of denaturation 30s, 58 DEG C of annealing 1min, 72 DEG C of extension 1min；
Circulation in step C, the B that repeats the above steps 35 times；
Step D, 72 DEG C keep after 7min, 4 DEG C of preservations.
Preferably, PCR experiment system is determined by the step 5：4 μ L 25mmol/L MgCl2。
Preferably, the blood collection method in the step 1 is with the solidifying heparin tube collector's venous blood of rush.
Preferably, the venous blood collected in the step 1 solidifies 30min in room temperature, centrifuges 1000g × 15min, then divides
Dress 1mL blood clots enter 1.5mL Axygen PE pipes.
Preferably, the blood clot preservation condition in the step 1 is：- 80 DEG C of refrigerators.
Preferably, the DNA extraction kit in the step 2 is：The biological Co., Ltd's poba gene group of Beijing's Tiangeng
DNA extraction kit (article No.：DP348).
Preferably, in the step 3 method of preliminary confirmation genomic DNA integrality, concentration and purity is：0.8%-
1.0% agarose gel electrophoresis method (130V, 40min).
Preferably, the genomic DNA concentration detected in the step 3 is：5-20ng/μL.
Preferably, genomic DNA preservation condition is in the step 3：- 20 DEG C of refrigerators.
Preferably, the Genome DNA content in the step 4 for converting is：100-400ng.
Preferably, the conversion reagent box that methylated in the step 4 is the unconcerned Products (article No. in the U.S. day：D5006).
Preferably, the genomic DNA preservation condition in the step 4 after conversion is：- 20 DEG C of refrigerators.
Preferably, the pcr template amount of the step 5 is the genomic DNA (full gene after converting after 10 μ L are converted
Group DNA).
Preferably, the PCR that methylates of the step 5 is using 50 μ L reaction systems.
Preferably, the amplified production length of the step 5 is 111bp.
Preferably, the PCR primer authentication method of the step 5：2.0% agarose gel electrophoresis method (130V,
40min)。
Preferably, the PCR primer volume for being used to purify recovery test of the step 6 is 18 μ L.
Preferably, the kit used in the white clone's spot screening test of the indigo plant of the step 6 is：The golden biotechnology of the full formula in Beijing
Co., Ltd TA Cloning Kit (article No.s：CT101-02).
Preferably, the volume of the PCR purifying recovery products being used in blue white clone's spot screening test of the step 6 is 2 μ
L。
Preferably, the antibiotic used in the white clone's spot screening test of the indigo plant of the step 6 is kanamycins.
Preferably, the step 6 identifies that the method for positive colony is：PCR expansions are done by universal primer (M13F and M13R)
Increase, product is identified through 2% agarose gel electrophoresis method (130V, 40min).
Preferably, the step 7 positive colony condition of culture is：In 50 μ g/mL kanamycins monoclonal antibody LB fluid nutrient mediums
In 37 DEG C of constant temperature 200rpm/min concussion and cultivates 8-12h.
The present invention is by improving gene after blood sample preservation condition, genomic DNA concentration and method for detecting purity, conversion
The final concentration of heterogeneity, methylated primers sequence-specific, blue white colony screening condition in group DNA content, PCR reaction systems
Deng experimental factor, the FOXP3 gene methylation detection methods based on bisulfite sequencing are successfully set up, can successfully be examined
The degree that particular sequence 5 CpG sites in FOXP3 gene promoter areas methylate is measured, the accuracy of experiment is improved on the whole
And repeatability.
Brief description of the drawings
Fig. 1 schematically shows sample genomic dna electrophoretogram；
Fig. 2 schematically shows the pcr amplification product electrophoretogram that methylates；
Fig. 3 schematically shows blue white clone's spot screening figure；
Fig. 4 schematically shows negative recon and positive recombinant PCR primer electrophoretogram；
Fig. 5 schematically shows FOXP3 gene promoter areas particular sequence sequencer map.
Embodiment
Embodiments of the invention are described in detail below in conjunction with accompanying drawing, but the present invention can be defined by the claims
Implement with the multitude of different ways of covering.
FOXP3 is the characteristic mark of Treg cells, is played a significant role during immune tolerance is maintained.It has recently found that
The expression of FOXP3 genes and the performance of protein function show as the regulatory mechanism i.e. epigenetics beyond mutant dna sequence,
Including DNA methylation, histone modification and posttranslational modification etc..DNA methylation is with cytosine methylation occurrence frequency highest.Born of the same parents
Cytosine methylation refers to methyl is added in into cytimidine 5 ' using SAM as donor under the catalysis of dnmt rna
The reaction of 5-methylcytosine is formed on position.Moiety site be cytimidine with guanine with phosphate be connected formed by CpG
Site.The CpG sites of influence gene expression are predominantly located at promoter and the First Exon region of gene.When specific gene starts
Sub-district CpG sites hyper-methylation, directly can hinder transcription factor to be combined with promoter, so that water can not be transcribed or transcribed to gene
Pancake is low；On the contrary, the hypomethylation in CpG sites can then cause chromatin loose, so as to increase the accessible of target DNA sequence
Property so that idiosyncratic transcription factor is easier to be connected thereto.FOXP3 locus contains multiple CpG sites.Positioned at promoter and
The expression of the CpG sites methylation and FOXP3 genes of one extron is closely related, be in T cell atomization it is many because
The crucial target spot of son regulation.
The present invention gropes FOXP3 gene bisulfite sequencings, successfully detects people FOXP3 gene promoter areas given zone
The methylation state in domain (+2071 ,+2182) 5 CpG sites, specific experiment process is as follows：
1. materials and methods
1.1 materials and reagent
Promote solidifying heparin tube and be purchased from Guangzhou Jian Fu medical science and technologies Co., Ltd.
Healthy workers venous blood 4mL, room temperature solidification 30min are collected with solidifying heparin tube is promoted, 1000g × 15min is centrifuged, then
Packing 1mL blood clots enter 1.5mL Axygen PE pipes, are stored in -80 DEG C of refrigerators, to prevent the degraded of genomic DNA from causing
The failure of an experiment.
Poba gene group DNA extraction kit is purchased from the biological Co., Ltd of city of BeiJing, China Tiangeng, kanamycins and TA grams
Grand kit is purchased from Quan Shijin bio tech ltd of city of BeiJing, China；
Normal melting point agarose is purchased from Biowest companies of Spain；
Taq enzyme, DNA methylation conversion reagent box and Ago-Gel QIAquick Gel Extraction Kit are public by U.S. Fermentas respectively
Department, Tian Mo companies of the U.S. and Omega companies of the U.S. provide；
The PCR primer that methylates is synthesized by Invitrogen (Shanghai) Trading Co., Ltd.；
Remaining reagent is domestic analytical grade reagent.
1.2 methylated primers are designed
With reference to ncbi database (https://www.ncbi.nlm.nih.gov/pubmed)With ensembl databases
(http://asia.ensembl.org/index.html) data-gathering FOXP3 gene promoter sequences, use
MethPrimer on-line analyses software (http://www.urogene.org/methprimer/index1.html)Carry out primer
Design, then to bisearch databases (http://bisearch.enzim.hu/index.phpM=genompsearch) test
Demonstrate,prove primer.Primer sequence such as table 1, is synthesized by Invitrogen (Shanghai) Trading Co., Ltd..
The FOXP3 gene methylation primer information of table 1
The preparation of 1.3 genomic DNAs：
Blood clot genomic DNA is extracted using poba gene group DNA extraction kit, passes through 0.8%-1.0% agar
Sugared gel electrophoresis (130V, 40min) primarily determines that genomic DNA integrality, concentration and purity, and will detect qualified DNA samples
Originally -20 DEG C of refrigerators are placed in standby.
1.4 genomic DNA bisulfite conversions
Using the conversion reagent box modification 100-400ng genomic DNAs that methylate, and the DNA sample modified is placed in -20
DEG C refrigerator is standby.
1.5 methylate PCR amplification
Pcr template amount is the genomic DNA (the full gene group DNA after converting) after 10 μ L are converted, and PCR uses 50 μ L
Reaction system, amplified production length is 111bp, and primer sequence and other component final concentrations are constant, refer to table 2.
PCR reaction conditions：94 DEG C of preheating 3min；94 DEG C of denaturation 30s, 58 DEG C of annealing 1min, 72 DEG C of extension 1min；Repeat
Above-mentioned circulation 35 times.Then, 72 DEG C keep after 7min, 4 DEG C of preservations.
PCR extensions product is separated using 2% agarose gel electrophoresis (130V, 40min), gel imaging system observation,
Take pictures.Also the PCR primer of 18 μ L mesh can be reclaimed according to the operating procedure of Ago-Gel QIAquick Gel Extraction Kit specification.It will reclaim
It is stand-by that complete purpose PCR primer is placed in -20 DEG C of refrigerators.
Table 2 methylates PCR reaction systems
The clone of 1.6 target gene and screening
2 μ L (about 50ng) PCR purifying recovery product is connected with carrier, is transformed into Trans1-T1 competence bacteriums, is used
Kalamycin resistance, carries out blue white clone's spot screening test；Colony PCR amplification, product warp are done with universal primer M13F and M13R
2% agarose gel electrophoresis (130V, 40min) is separated, and gel imaging system is observed, taken pictures, to identify positive colony.
1.7 sequencing
The thalline chosen in positive colony spot is inoculated in 50 μ g/mL kanamycins monoclonal antibody LB fluid nutrient mediums, 37 DEG C of perseverances
After warm 200rpm/min concussion and cultivates 8-12h, the sample become cloudy is taken to send sequencing portion of Invitrogen (Shanghai) Trading Co., Ltd.
It is sequenced.
2. result
The agarose gel electrophoresis result of 2.1 genomic DNA 1%
As shown in figure 1, top visible bright purpose bar of most of sample path in DNA marker 15000bp bands
Band, this band is genomic DNA, and its size is consistent with expected results, the visible hangover of part band.
Wherein：
M：DNA marker；
1：Sample genomic dna；
2.2 methylate the agarose gel electrophoresis results of PCR 2%
Blank control path has no band, sample path occur between DNA marker 100bp-250bp bands it is bright and
Clearly purpose band, its length matches with FOXP3 gene methylation PCR primer length 111bp, sees Fig. 2.
Wherein：
M：DNA marker；
1：Blank control；
2：Sample methylates PCR primer；
2.3 blue white clone's spot results
Negative group has no locus coeruleus or hickie, sample group visible white or the smooth single bacterium colony of blue circle.Wherein, white bacterium
Fall the plasmid conversion bacterium colony (positive recombinant) to there is FOXP3 gene methylations PCR primer to insert, blue colonies turn for empty plasmid
Change bacterium colony (negative recon), specifically refer to Fig. 3.
2.4PCR identification positive colony analyses
The PCR primer length of positive recombinant (i.e. positive colony) should be the FOXP3 gene methylation PCR primers of insertion
(111bp) and part carrier T (199bp) sequence sum, length should be 310bp；The PCR productions of negative recon (i.e. negative clone)
Thing length should be part carrier T sequence length 199bp.2% agarose gel electrophoresis result shows that white colony path is in DNA
Occur bright and clearly purpose band between label 250bp and 500bp band, and blue colonies path is in DNA marker
There is purpose band between 100bp and 250bp bands, see Fig. 4.This confirms that white colony is positive recombinant, and blue colonies
I.e. negative recon.
Wherein：
M：DNA marker；
1：Negative recon；
2：Positive recombinant；
3：Blank control.
The sequencing result of 2.5 clones
Sequencing result and people FOXP3 gene promoter areas specific dna sequence (+2071 ,+2182) are compared, purpose fragment without
Mutation, the cytimidine in 5 CpG sites is not bisulphite modified, is finally shown as cytimidine, shows the sample peripheral blood
Hyper-methylation is presented in all CpG sites in FOXP3 gene promoters specific region, sees Fig. 5.
5 CpG sites hyper-methylations of people FOXP3 gene promoter areas particular sequence are shown after Fig. 5 sequencings.
By above-mentioned checking example, research and design methylated primers of the present invention determine every bar of whole experiment process
Part, establishes the FOXP3 gene methylation detection methods based on bisulfite sequencing.
Test of many times has been repeated using the method in the present invention in inventor, can obtain preferable amplified production.Survey
Sequence result shows that bisulfite conversion is complete, 5 CpG sites of sample peripheral blood FOXP3 gene promoter areas specific region
It is hyper-methylation.
It can be seen that, the present invention establishes the FOXP3 gene methylation detection methods of bisulfite sequencing.
The preferred embodiments of the present invention are the foregoing is only, are not intended to limit the invention, for the skill of this area
For art personnel, the present invention can have various modifications and variations.Within the spirit and principles of the invention, that is made any repaiies
Change, equivalent substitution, improvement etc., should be included in the scope of the protection.
Claims (7)
1. a kind of FOXP3 gene methylation detection methods based on bisulfite sequencing, it is characterised in that including：
Step 1, with solidifying heparin tube collector venous blood 4mL, room temperature solidification 30min is promoted, 1000g × 15min is centrifuged, then will
1mL blood clots are dispensed into 1.5mL Axygen PE pipes, are stored in -80 DEG C of refrigerators；
Step 2, using blood clot genomic DNA in poba gene group DNA extraction kit extraction step 1；
Step 3, by the integrality of the genomic DNA in the 0.8%-1.0% preliminary verification step 2 of agarose gel electrophoresis,
Concentration and purity, and it is standby to detect that qualified DNA sample is placed in -20 DEG C of refrigerators；
Step 4, using the conversion reagent box modifier group DNA that methylates, and the DNA sample modified is placed in -20 DEG C of refrigerators
It is standby；
Step 5, the PCR amplifications that methylate are carried out, PCR primer is identified by 2% agarose gel electrophoresis, gel imaging system is seen
Examine, take pictures；
Step 6,18 μ L PCR primers in QIAquick Gel Extraction Kit purifying recycling step 5 are purified using DNA, connected by carrier by 2 μ
PCR primer after L purifying is reclaimed is transformed into Trans1-T1 competence bacteriums, carries out blue white clone's spot screening, and by general
Primer (M13F and M13R) does PCR amplifications, and product is separated through 2% agarose gel electrophoresis, and gel imaging system is observed, taken pictures,
To identify positive colony；
Step 7, the thalline in the positives clone's spot of selecting step 6 is inoculated in 50 μ g/mL kanamycins monoclonal antibody LB fluid nutrient mediums
In, after 37 DEG C of constant temperature 200rpm/min concussion and cultivates 8-12h, take the sample become cloudy to be sequenced.
2. the FOXP3 gene methylation detection methods according to claim 1 based on bisulfite sequencing, its feature
It is, the primer sequence that performing PCR amplification is entered in the step 5 is：
F:5 '-ATTTTTAGGATTTGAGGTTTTAATA-3 ',
R:5’-CAAAAAACCCACTAAAAAACTATAC-3’。
3. the FOXP3 gene methylation detection methods based on bisulfite sequencing according to claim 1 and 2, its
Be characterised by, in the step 5 carry out methylate PCR amplification when reaction condition be：
Step A, 94 DEG C of preheating 3min；
Step B, 94 DEG C of denaturation 30s, 58 DEG C of annealing 1min, 72 DEG C of extension 1min；
Circulation in step C, the B that repeats the above steps 35 times；
Step D, 72 DEG C keep after 7min, 4 DEG C of preservations.
4. the FOXP3 gene methylation detection methods according to claim 1 based on bisulfite sequencing, its feature
It is, the method for preliminary confirmation genomic DNA integrality, concentration and purity in the step 3 is：0.8%-1.0% fine jade
Sepharose electrophoresis (130V, 40min).
5. the FOXP3 gene methylation detection methods according to claim 1 based on bisulfite sequencing, its feature
It is, the PCR primer authentication method of the step 5：2.0% agarose gel electrophoresis method (130V, 40min).
6. the FOXP3 gene methylation detection methods according to claim 1 based on bisulfite sequencing, its feature
It is, the step 6 identifies that the method for positive colony is：PCR amplifications, product warp are done by universal primer (M13F and M13R)
2% agarose gel electrophoresis method (130V, 40min) is identified.
7. the FOXP3 gene methylation detection methods according to claim 1 based on bisulfite sequencing, its feature
It is, the step 7 positive colony condition of culture is：37 DEG C of constant temperature in 50 μ g/mL kanamycins monoclonal antibody LB fluid nutrient mediums
200rpm/min concussion and cultivates 8-12h.
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| CN112779320A (en) * | 2020-12-04 | 2021-05-11 | 深圳市易基因科技有限公司 | Multi-region DNA methylation detection probe design and detection method thereof |
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