CN109207590A - The island TIMP3 gene promoter region CPG methylation detecting method based on pyrosequencing techniques - Google Patents
The island TIMP3 gene promoter region CPG methylation detecting method based on pyrosequencing techniques Download PDFInfo
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- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
- C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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- C12Q2600/00—Oligonucleotides characterized by their use
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Abstract
The invention discloses the methods of the island the TIMP3 gene promoter region CPG DNA methylation assay based on pyrosequencing techniques, and the quantitative detection of the island gene promoter region CPG TIMP3 methylation is realized with this.Specificity amplification primer sequence used in wherein for this section of gene carries out the sequencing primer of pyrosequencing as shown in TIMP3-S as shown in TIMP3-F and TIMP3-R, for amplimer.Primer and method shown in the present invention will be helpful to early diagnose tumour, be of great significance to screening for cancer.
Description
Technical field
The present invention relates to the detections of TIMP3 gene promoter region CPG island methylation level.
Background technique
Pyrosequencing techniques (pyrosequencing) are suitable for carrying out known short sequence high-throughput, accurate
And reproducible sequencing analysis, can be applied to single nucleotide polymorphism (single ucleotidepolymor-phism,
SNP), the multinomial research such as tumor methylation analysis.This method is known as " goldstandard " in methylation level detection.Its principle
After primer and template DNA annealing, in archaeal dna polymerase (DNA polymerase), ATP sulfurylase
(ATPsulfurylase), 4 kinds of enzymes of luciferase (luciferase) and apyrase (Apyrase) exist
Reaction system in, sequentially add one of 4 kinds of dNTP (dATP α S, dTTP, dCTP, dGTP), if occur base complementrity match
It is right, oxyluciferin is eventually generated by reaction of high order, optical signal is issued, so that it is determined that base at this.As the process is followed
Ring carries out, and complementary dna chain gradually synthesizes, and DNA sequence dna is determined by the signal peak of Pyrogram.By detect fluorescence release and
Intensity achievees the purpose that the real time measure DNA sequence dna.
Malignant tumour is to be formed the multifactor, multistage, and infiltration and transfer are also related to across extracellular matrix barrier, blood
Pipe basilar memebrane and the processes such as vascular wall are pierced by, necessary condition therein is basilar memebrane and extracellular degradation.In addition to because of DNA sequence dna
Change and generate mutation, cause tumour, the change on table science of heredity also will affect the generation of tumour.Tumor tissues D N
A it occur frequently that its promoter region abnormal methylation, including proto-oncogene hypomethylation and tumor suppressor gene hyper-methylation change
Become, on the one hand has activated oncogene, while also inactivate tumor suppressor gene.The hyper-methylation of usual Promoter CpG islands is to lead
Cause the important molecule biological mechanism of tumor development.
Tissue inhibitor of metalloproteinase (tissue inhibitor of matrix metalloproteinases,
TIMPs) family shares 4 members, they are successively named according to the sequence being found.Wherein TIMP3 and other 3 members
Difference, for its main positioning combination in the extracellular matrix layer of tissue, this also determines its action direction.TIMP3 gene position
In on human chromosomal 22q12.1-13.2, the protein for encoding generation by it is secreted into intracellular after extracellularly synthesizing
In matrix, the activity of energy antagonizing matrix metalloproteinases (matrix me tallopro te inases, MMPs), this is special
Anisotropic inhibiting effect maintains the balance of the degradation and synthesis of extracellular matrix, plays important work in inhibiting tumor invasion and metabasis
With.TIMP3 gene can inhibit tumour growth, angiogenesis, influence the invasion, infiltration and transfer of tumour, be a kind of suppression cancer base
Cause.It is also gene participating in apoptosis simultaneously, plays the role of promoting apoptosis.TIMP3 expression reduces or missing occurs with tumour
Closely related, promoter methylation also influences the prognosis situation of kinds of tumors, such as gastric cancer, kidney, glioma, colon cancer, cream
Gland cancer etc..The methylation level variation on the island TIMP3 gene promoter region CPG is present in Several Kinds of Malignancy, may
As the marker of lesion detection, facilitate the early screening of tumour, provides help for oncotherapy.
Summary of the invention
It is located at the TIMP3 gene of human chromosomal chr22:32801395-32802281 involved in the present invention, passes through PCR
The DNA fragmentation for amplifying TIMP3 promoter region CPG island portion point detects each of which methylation position using pyrosequencing techniques
The methylation frequency of point, the methylation frequency range measured are 0%-100%, can accomplish quantitative analysis, sequencing result is accurate,
High sensitivity.
It is an object of that present invention to provide a kind of reagents for detecting the island TIMP3 gene promoter region CPG methylation
Box is related to detecting the sequencing primer of the island TIMP3 gene promoter region CPG methylation based on pyrosequencing techniques.Including spy
Specific amplification primers TIMP3-F and TIMP3-R, wherein 5 ' the end addition biotin labelings of TIMP3-R, and to amplifying
The sequencing primer TIMP3-S of segment progress pyrosequencing.Primer sequence are as follows:
TIMP3-F:5 '-AGTTGGAGTTTGGGGGATTG-3 '
TIMP3-R:5 '-AACATCTTCCCCTCTCAACT-3 '
TIMP3-S:5 '-AGTTTGGGGGATTGG-3 '
The reaction condition of PCR amplification are as follows: 95 DEG C 15 minutes；94 DEG C 30 seconds, 56 DEG C 30 seconds, 72 DEG C 30 seconds, 47 circulation；
72 DEG C 10 minutes.
The present invention relates to the reality sides based on the pyrosequencing techniques detection island TIMP3 gene promoter region CPG methylation
Method, method include:
(1) DNA in sample is extracted；
(2) base T is converted by base C unmethylated in sample DNA, the weight that it is 3M that processing mode, which is by concentration, is sub-
Metabisulfite solution mixes with the aqueous solution containing 2 μ g DNA and is placed in 98 DEG C, 10min, and then 64 DEG C, 2.5 hours, and to DNA
It is purified to obtain DNA profiling；
(3) using obtained DNA profiling, needed for being obtained using amplimer TIMP3-F and TIMP3-R through PCR amplification
DNA fragmentation；
(4) take 6 μ l pcr amplification products carry out agarose gel electrophoresis, according to product band whether naked eyes it is visible and
Whether between the slug band of 200 and 300bp judge whether amplification succeeds, and according to being under 100bp slug band
It is no to have band to determine whether there is primer dimer；
(5) for expanding successfully and the sample without obvious primer dimer, remaining amplified production is subjected to pyrophosphoric acid survey
Sequence, the sequencing primer used are TIMP3-S
(6) the methylation frequency of each methylation sites in sequencing sequence is obtained according to PyroMark sequencer address, is surveyed
Sequence are as follows: GGYGTYGAGG YGTGTATATG TTYGTTTAGT TATTTTTAGG AYGTTTTTTG TAATTTYGAT
ATYGGTAAGY GTTTTTGGTG TTTYGTTYGA GTTTTAYGTT GTAGTTAGGA TTGTAGYGTT GTTTAGGGAG
GTAGGGYGAG TTTTATTTTT TTTTTTTGTT TT。
The present invention provides a kind of kit for detecting the island TIMP3 gene promoter region CPG methylation level.Kit
Content include amplimer TIMP3-F and TIMP3-R, wherein TIMP3-R 5 ' end contains biotin labeling；Sequencing primer
TIMP3-S；The reagent of PCR amplification part, comprising: thermal starting archaeal dna polymerase (HotStar Taq DNAPolymerase),
PCR buffer (Tris-HCL buffer), dNTPs.
The part not being related to for kit: the PyroMark Q96 kit of QIAGEN company can be used to correspond to Q96's
Instrument uses.
Detailed description of the invention
Fig. 1 is the primer sequence for including and sequencing sequence relevant information in the present invention, and the affiliated sequence of zone circle arrow is packet
Sequence containing biotin labeling.Due to incomplete, the attached complete sequencing sequence of the too long display of sequencing sequence, Y base shown in sequence
Site measures the site of methylation level after being；
Complete sequencing sequence: GGYGTYGAGG YGTGTATATG TTYGTTTAGT TATTTTTAGG AYGTTTTTTG
TAATTTYGAT ATYGGTAAGY GTTTTTGGTG TTTYGTTYGA GTTTTAYGTT GTAGTTAGGATTGTAGYGTT
GTTTAGGGAG GTAGGGYGAG TTTTATTTTT TTTTTTTGTT TT
Fig. 2 is the agarose gel electrophoresis figure for the island the TIMP3 promoter region CPG Partial Fragment that PCR amplification obtains；
Fig. 3 is pyrosequencing as a result, vertical bar region is the methylation sites of measurement, and percentages are above vertical bar
The methylation level.
Specific embodiment
The present invention is further elaborated below with reference to specific example.
The advantage of the invention is that all operations step and obtain a result and can be completed in one day, it is convenient and efficient, and be sequenced
As a result it is calculated by software, avoids error caused by subjective analysis, result credibility is high.
Example one: the island TIMP3 gene promoter region CPG DNA methylation assay in cell sample
1. extracting genomic DNA from cell sample.
It takes in 75cm2The A549 cell cultivated in culture bottle is digested after being cleaned twice using PBS by pancreatin, and cell is collected
Suspension is spare after 800r/min 2min is centrifuged and is cleaned by PBS in centrifuge tube.Using TIANGEN blood/cell/group
Genome DNA extracting reagent kit is knitted, according to specification operating procedure, extraction obtains genomic DNA, concentration 540.318ng/
ml。
2. converting and purifying DNA profiling
Base T, the heavy sulfurous acid that it is 3M that processing mode, which is by concentration, are converted by base C unmethylated in sample DNA
Sodium solution mixes with the aqueous solution containing 2 μ g DNA and is placed in 98 DEG C, 10min, and then 64 DEG C, 2.5 hours, and DNA is carried out
Purifying obtains DNA profiling.
3. carrying out PCR amplification using the DNA profiling converted
Using template obtained in the previous step, needed for being obtained using amplimer TIMP3-F and TIMP3-R through PCR amplification
DNA fragmentation, the sequence of amplimer are as follows:
Upstream primer TIMP3-F:5 '-AGTTGGAGTTTGGGGGATTG-3 '
Downstream primer TIMP3-R:5 '-AACATCTTCCCCTCTCAACT-3 ' containing biotin labeling
Used reaction condition are as follows: 95 DEG C 15 minutes；94 DEG C 30 seconds, 56 DEG C 30 seconds, 72 DEG C 30 seconds, 47 circulation；72
DEG C 10 minutes.
PCR reaction system are as follows:
| Hotstart | 12.5μl |
| PCR buffer (Tris-HCL) (100mM) | 7μl |
| dNTPs | 0.5μl |
| Primer F | 1μl |
| Primer R | 1μl |
| ddH2O | 1μl |
| Template DNA | 2μl |
| Total volume | 25μl |
4. amplified production is verified in gel electrophoresis
PCR product obtained by 6 μ l previous steps is taken, is judged whether to expand successfully using agarose gel electrophoresis, voltage setting
120V, the time 30 minutes.It is electrophoresis result figure in attached drawing 2, it is seen that purpose band is located at correct region, and limpid in sight.As institute
Band is darker, has more miscellaneous band or no band, then it is not recommended that carry out next step sequencing.
5 purifying are single-stranded and are sequenced with pyrosequencing instrument.
By required reagent be placed in advance equilibrium at room temperature it is good after, according to PyroMark Q96 specification carry out purification process,
It will be connected in system of the single-stranded addition containing sequencing primer TIMP3-S containing biotin labeling with beads, the sequence of TIMP3-S
It is classified as: 5 '-AGTTTGGGGGATTGG-3 '.According to needing the sequence measured to set program in software, it is sequenced.By
The methylation frequency of each methylation sites in sequencing sequence is calculated in software, as shown in the figure.In figure hundred above detection site
Divide than being methylation level.
Example two: the island TIMP3 gene promoter region CPG DNA methylation assay in blood sample
200 μ l of patient blood to be measured is taken, erythrocyte cracked liquid and blood/cell/tissue base of TIANGEN company are used
Because a group DNA extraction kit obtains genomic DNA according to specification operating procedure.Other steps are the same as example one.
The purpose stated above is that operating procedure is described in detail, and can be carried out according to actual conditions to step when practical operation
Change.
Claims (4)
1. the specificity amplification primer based on the pyrosequencing techniques detection island TIMP3 gene promoter region CPG methylation
TIMP3-F and TIMP3-R, wherein biotin labelings are added at the 5 ' ends of TIMP3-R；TIMP3 is detected based on pyrosequencing techniques
The sequencing primer TIMP3-S of the island gene promoter region CPG methylation；Primer sequence are as follows:
TIMP3-F:5 '-AGTTGGAGTTTGGGGGATTG-3 '
TIMP3-R:5 '-AACATCTTCCCCTCTCAACT-3 '
TIMP3-S:5 '-AGTTTGGGGGATTGG-3 '.
2. application primer as described in claim 1 method, which is characterized in that the reaction condition of amplification are as follows: 95 DEG C 15 minutes；94
DEG C 30 seconds, 56 DEG C 30 seconds, 72 DEG C 30 seconds, 47 circulations；72 DEG C 10 minutes.
3. based on the real method of the pyrosequencing techniques detection island TIMP3 gene promoter region CPG methylation, feature exists
In step includes:
One, the DNA in sample is extracted；
Two, base T is converted by base C unmethylated in sample DNA, the weight that it is 3M that processing mode, which is by 180 μ l concentration, is sub-
Metabisulfite solution mixes with the 20 μ l aqueous solutions containing 2 μ g DNA and is placed in 98 DEG C, 10min, and then 64 DEG C, 2.5 hours；
Three, using processed DNA as template, PCR amplification is carried out using amplimer TIMP3-F and TIMP3-R and obtains required DNA
Segment, amplification program is as shown in right 2；
Four, take 6 μ l pcr amplification products carry out agarose gel electrophoresis, according to product band whether naked eyes it is visible and whether position
Judge whether amplification succeeds between the slug band of 200 and 300bp, and according to whether having item under 100bp slug band
It brings and judges whether there is primer dimer；
Five, for expanding successfully and the sample of primer free dimer, remaining amplified production is subjected to pyrosequencing, the survey used
Sequence primer is TIMP3-S；
Six, the report according to the output of PyroMark software obtains the methylation frequency of each methylation sites in sequencing sequence.
4. being related to based on the kit of the pyrosequencing techniques detection island TIMP3 gene promoter region CPG methylation level
Reagent include 250units thermal starting polymerase, 200 μ l concentration be 10mMdNTPs, 2ml buffer solution, 1.5mldd H2O、1ml
Concentration is the MgCl of 15mM2, upstream amplification primer TIMP3-F, downstream amplification primer TIMP3-R and sequencing primer TIMP3-S.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2005085196A2 (en) * | 2004-03-08 | 2005-09-15 | Dkfz | Inhibitors of dna methylation in tumor cells |
| KR20140077446A (en) * | 2012-12-14 | 2014-06-24 | 서울대학교산학협력단 | Methods and Methylation Markers for detecting or diagnosing cholangiocarcinoma |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2005085196A2 (en) * | 2004-03-08 | 2005-09-15 | Dkfz | Inhibitors of dna methylation in tumor cells |
| KR20140077446A (en) * | 2012-12-14 | 2014-06-24 | 서울대학교산학협력단 | Methods and Methylation Markers for detecting or diagnosing cholangiocarcinoma |
Non-Patent Citations (1)
| Title |
|---|
| ERIN M. SIEGEL等: "Quantitative DNA Methylation Analysis of Candidate Genes in Cervical Cancer", PLOS ONE, vol. 10, no. 3, pages 1 * |
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