CN105400865A - TMEM176A gene promoter region DNA methylation detection - Google Patents
TMEM176A gene promoter region DNA methylation detection Download PDFInfo
- Publication number
- CN105400865A CN105400865A CN201510390230.8A CN201510390230A CN105400865A CN 105400865 A CN105400865 A CN 105400865A CN 201510390230 A CN201510390230 A CN 201510390230A CN 105400865 A CN105400865 A CN 105400865A
- Authority
- CN
- China
- Prior art keywords
- tmem176a
- methylated
- primers
- primer
- gene promoter
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- 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
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- 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
-
- 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
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/154—Methylation markers
Abstract
According to the present invention, TMEM176A gene is firstly adopted as a target gene, and the promoter region in esophageal cancer cells and colon cancer cells presents the high methylation state through a MSP technology; primers and a kit for detecting cell TMEM176A gene promoter region methylation state are provided, wherein the primers are a pair of methylation primers, or a pair of methylation primers and a pair of non-methylation primers; with the primers and the kit, the esophageal cancer detection specificity is good, and the sensitivity is high and achieves 0.5%, wherein the esophageal cancer detection specificity is 61.2%, the colorectal cancer detection specificity is 53.13%, and five cancer cells in 1000 cells can be detected; and the application of the kit to detect the TMEM176A gene promoter region DNA methylation state can be adopted as the powerful tool for digestive tumor diagnosis, treatment effect observation, prognosis determination, minimal residual disease detection and the like, and the advantages of easy operation, good stability, far-reaching clinical significance and promotion are provided.
Description
Technical field
The present invention relates to DNA methylation assay technology, relate in particular to
tMEM176A gene promoter area DNA methylation detects, particularly a kind of methylated primers pair for detecting TMEM176A gene promoter zone methylation state, comprises non-methylated primers pair further.Meanwhile, the invention still further relates to the test kit containing described primer pair.
Background technology
The esophageal carcinoma is one of malignant tumour that in worldwide, lethality rate is the highest, in kinds of tumor, ranked eighth position, in digestive tract tumor, ranked fourth position.Although along with making constant progress of Clinical Oncology, the esophageal carcinoma remains one of topmost reason of cancer related mortality rate.The esophageal carcinoma mainly comprises esophageal squamous cell carcinoma and adenocarcinoma of esophagus two kinds, and its sickness rate has very large regional disparity, and area occurred frequently mainly contains south, Africa and east, Central Asia and east, from Iran
northernin extending to Central Asia republic
state is northernbe called as with middle part " esophageal carcinoma band ", men and women falls ill indifference.According to statistics, only within 2012, just there is the new patient with esophageal carcinoma of 456000 example, have 400000 routine patient with esophageal carcinoma dead.China is the hotspot of the esophageal carcinoma, its M & M is respectively 22.4/100000 and 16.77/100000,90% is esophageal squamous cell carcinoma, main with smoking, drink, nutritional status is poor, vegetables and fruit intake is few and boiling hot food of taking food is relevant.Adenocarcinoma of esophagus is mainly flourishing in west
countrymore common, main take in less to gastroesophageal reflux disease, Barrett esophagus, smoking, obesity, fruits and vegetables and Helicobacter pylori infection history relevant.The overall prognosis of the esophageal carcinoma is poor, and within 5 years, survival rate is no more than 20%.The early stage esophageal carcinoma is generally asymptomatic, but, along with updating of endoscopic diagnosis technology, the gastroscope recall rate of the early stage esophageal carcinoma is improve.Dysphagia is the modal symptom of advanced esophageal carcinoma, can with weight loss.Although gastroscopy can observe mucous membrane of esophagus pathology more intuitively, finally make a definite diagnosis or depend on the pathological biopsy of stomach-tissue and the pathological diagnosis of Operated Specimens under gastroscope.The current treatment of the esophageal carcinoma mainly comprises simple surgical operation, surgical operation combined chemotherapy, radiotherapy or chemicotherapy, and its 5 years survival rates are followed successively by 16%-52%, 23%-36%, 18.6%-41.3%, 39%; For the unresectable esophageal carcinoma, based on simple chemicotherapy, but to maintaining remission rate, to improve the effect of long-term survival rate very limited, therefore, the prognosis mala of the esophageal carcinoma.
Colorectal cancer is the large malignant tumour in third place in the world, is number four in world's malignant tumour associated death cause of disease.Nearly 1,200,000 new cases are diagnosed as colorectal cancer every year, and 600,000 cases die from colorectal cancer.In male patient's kinds of tumor, colorectal cancer occupies the 3rd, and occupies second in female patient.According to American Cancer Society (AmericanCancerSociety, ACS) 2014 AUTHORITATIVE DATA announced show that colorectal cancer is the total the third-largest modal cancer of American male and women and the third-largest cancer mortality reason, and over nearly 20 years, the sickness rate of colorectal cancer and mortality ratio are obvious downtrending.And the latest data that China announces for 2014 shows that the M & M of male sex's colorectal cancer is after lung cancer, cancer of the stomach, liver cancer and the esophageal carcinoma, rank the 5th.The M & M of women's colorectal cancer is the 3rd after occuping mammary cancer, lung cancer.National register office tumour registration material is collected 2011 and 2013 by whole nation tumour Register, analyze morbidity and the Death Level of 2003-2007 and 2009 year national tumour registration coverage area cancer, result display 2003 ~ 2007 years Chinese colorectal cancer incidence rates are 28.08/10 ten thousand; Mortality of carcinoma is 13.41/10 ten thousand.Within 2009, Chinese colorectal cancer incidence rate is 29.44/10 ten thousand; Mortality of carcinoma is 14.23/10 ten thousand.The M & M of China's colorectal cancer rises year by year as can be seen here, should strengthen the integrated control work of colorectal cancer, and the morning of raising colorectal cancer examines morning and harnesses the river flat.
In view of the Diagnosis and Treat means being applied to the clinical esophageal carcinoma and colorectal cancer at present, especially still very limited to the detection method of the early diagnosis of tumour, residual and recurrence, be problem in the urgent need to address at present.Therefore, find that the effective ways tool of the early diagnosis and therapy of the esophageal carcinoma and colorectal cancer is of great significance.
Along with the arrival of genome times afterwards comprehensively, epigenetics (Epigenetics) has become the study frontier of life science.Epigenetics is for genetics, refer to the science of heritable changes in gene expression that researching DNA sequence does not change, it can explain the inexplicable problem of some classical geneticses, such as: monovular twins brother has identical genotype, and grow up under same environment, but only wherein a people gets a cancer of the stomach, and an other person is healthy, and therefore epigenetic information provides the instruction of when, where going in which way to apply genetic information.In digestive system carcinoma field, epigenetics research is mainly concentrated on DNA methylation and acetylation of histone.It is mode main in genome epigenetic regulation that DNA methylation is modified, and generation, the development of human tumor are relevant with the exception of DNA methylation, and CpG island, gene promoter area hyper-methylation can cause expression of tumor suppressor gene silence and then cause tumour to occur.DNA methylation refers under the effect of methylated transferase, with s-adenosylmethionine for methyl donor, by Methyl transporters on No. 5 carbon atoms of CG dinucleotides cytosine(Cyt), forms 5 methylcysteins.DNA methylation be extremely early stage in cell carcinogenesis, take place frequently event, therefore the methylate molecular marker, therapy target and the judging prognosis means that can be used as early diagnosis of cancer of specific gene.Along with the progress of technology, detect methylated means and be also enriched, not only the distribution that methylates of certain segment DNA sequence detectable, and can also the whole genomic methylation of understanding of flux greatly.Current main method has two classes: one is sodium bisulfite method (SodiumBisulfite), comprises the mononucleotide TRAP (Ms-SNuE) etc. of the gene sequencing method of sodium bisulfite method dependence, sodium bisulfite associating restriction endonuclease analysis (COBRA), methylation status of PTEN promoter (MS-PCR), methyl-sensitive; Another kind of is the restriction enzyme enzyme process of methyl-sensitive, comprise Southern method, methyl-sensitive restricted
figurespectrum (MSRF), restriction labeling genome scanning technology (RLGS), otherness methylation hybridization analyze (DMH), methylated CpG island amplicon is analyzed (MCA) etc.It is worth mentioning that MS-PCR, after Herman in 1996 has invented technique, enormously simplify the detection method of DNA methylation, epigenetics research is advanced more rapidly, its outstanding advantage is the restriction of high specific, highly sensitive, easy and simple to handle, expense and all less, unrestricted restriction endonuclease consuming time, current MS-PCR has become the method be most widely used studying gene methylation state, especially for the research prefered method especially of large quantities of clinical samples methylation states.The ultimate principle of MS-PCR is: DNA is after sulphite phosphorothioate, cytosine(Cyt) CpG island not occurred in methylated CG dinucleotides is just converted into uridylic, finally be converted into thymus pyrimidine, there is methylated cytosine(Cyt) then to remain unchanged, therefore methylated primers and non-methylated primers is designed respectively for above-mentioned difference, carry out pcr amplification respectively, amplified production capable gel electrophoresis photograph observations, draw whether be methylated conclusion.
Transmembrane protein 176 (transmembraneprotein176, TMEM176) be and transmembrane protein 4A family (membrane-spanning4Afamilyofprotein, MS4A) relevant albumen, be present in Mammals and bone fish, comprise TMEM176A and 176B two kinds of hypotypes.Zuccolo etc. find the TMEM176 gene of a large amount of high conservatives in zebra fish.TMEM176A finds in the renal proximal tubules cell of mouse, is positioned at No. 6 karyomit(e)s.And mankind TMEM176A gene is positioned at 7q36.1, there are four membrane spaning domains, in hepatocellular carcinoma, screen tumor associated antigen the earliest find.Gehrau etc. find that the mRNA transcriptional level of TMEM176A obviously increases when liver-transplantation patients recurrence hepatitis C infection.The research such as Condamine finds that TMEM176A and 176B is interactional, and the two plays an important role in the crudity maintaining the inmature dendritic cell of mouse, and inflammatory stimulus makes TMEM176A and 176B down-regulated expression.Cuajungco etc. find that TMEM176A and 176B has obvious expression in the mankind much organize, and in lymphoma and lung cancer, the protein expression of TMEM176A obviously increases.Strelnikov etc. report the DNA methylation of the CpG island exception of the mankind TMEM176A and 176B and breast cancer related.In sum, also there is a lot of deficiencies at present to the functional study of TMEM176A, there is not been reported for the change of its epigenetics in the esophageal carcinoma and functional study.
Summary of the invention
The object of this invention is to provide the primer detecting cell TMEM176A gene promoter zone methylation state, namely for detecting the primer of TMEM176A gene promoter zone methylation state, meanwhile, another object of the present invention is to provide a kind of test kit containing described primer.Another object of the present invention is to provide a kind of method detecting TMEM176A gene promoter zone methylation state, carries out risk assessment with this to the esophageal carcinoma, colorectal cancer.
Through test repeatedly and deliberation, the present inventor select a pair good methylated primers of specificity to and non-methylated primers pair, and establish a stable reaction system, find out desirable reaction conditions, the higher acrylamide gel electrophoresis technology of application-aware degree as a result detect means, therefore draw reliable experimental result, result shows: the hyper-methylation state of TMEM176A gene promoter area has higher specificity in the esophageal carcinoma and colorectal cancer.Described primer pair and test kit is utilized to detect the highly sensitive of TMEM176A gene methylation state, solve that above-mentioned recall rate is low, result is difficult to analyze, only can show the series of technical such as genetics is substantially abnormal, therefore, primer of the present invention and test kit can be used for the early diagnosis, efficacy determination etc. of the esophageal carcinoma and colorectal cancer.
Technical scheme provided by the invention is: for detecting the primer pair of cell TMEM176A gene promoter zone methylation state, it is methylated primers, its upstream primer nucleotide sequence is as described in Primer-MF, that is: 5'GAAGAAAGACGTTTTGTGGATAGGAC3', downstream primer nucleotide sequence as described in Primer-MR, that is: 5'CTAATATCCGCTCTACTCGACCGCG3'.Utilize this methylated primers pair, with the DNA after phosphorothioate for template carries out MS-PCR amplification.Methylate if TMEM176A gene promoter area exists, then can obtain the fragment of 156bp size; If namely TMEM176A gene does not normally methylate, then do not produce amplified production.Based on this, this primer pair is called methylated primers by the application.Described methylated primers pair, the Tm59.17 of its upstream primer, GC content 42%, the Tm63.86 of downstream primer, GC content 56%.
The invention still further relates to following non-methylated primers pair, its upstream primer nucleotide sequence is as described in Primer-UF, that is: 5'GGAAGAAAGATGTTTTGTGGATAGGAT3', downstream primer nucleotide sequence as described in Primer-UR, that is: 5'CAACTAATATCCACTCTACTCAACCACA3'.Utilize this non-methylated primers pair, with the DNA after phosphorothioate for template carries out MS-PCR amplification, if namely TMEM176A gene does not normally methylate, then can obtain the fragment of 160bp size.Based on this, this primer pair is called non-methylated primers by the application.Described non-methylated primers pair, the Tm57.86 of its upstream primer, GC content 37%, the Tm59.58 of downstream primer, GC content 39%.
The present invention also provides a kind of method detecting TMEM176A gene promoter zone methylation state, with this, early diagnosis, efficacy determination etc. are carried out to the esophageal carcinoma, colorectal cancer, comprise the steps: first, utilize methylated primers pair, with the DNA after phosphorothioate for template carries out MS-PCR amplification; Then, methylation state is judged according to amplified production.Preferred version is, utilize simultaneously methylated primers to non-methylated primers pair, with the DNA after phosphorothioate for template carries out MS-PCR amplification; According to the amplified production of methylated primers and non-methylated primers, judge methylation state.Increasing with methylated primers, have amplified production, carry out increasing with non-methylated primers, is exhaustive methylation without amplified production; With to methylate and non-methylated primers increases, all having amplified production, is partial methylation; Increase with non-methylated primers, have amplified production, increase with methylated primers, without amplified production, for nothing methylates.Partial methylation and exhaustive methylation are all judged to methylate.
Further, primer pair of the present invention is utilized to carry out the method for MS-PCR, its desirable reaction conditions is as follows: denaturation 5min at 95 DEG C, then enter circulation, sex change 30s at 95 DEG C, anneal 30s at 60 DEG C, at 72 DEG C of downward-extension 40s, totally 35 circulations, afterwards, continue at 72 DEG C of downward-extension 5min.
Further, utilize primer pair of the present invention to detect the MS-PCR reaction system of digestive system carcinoma cell, in overall 25ul, it comprises:
(1) template: 2ul;
(2) concentration be 50pmol/ul described methylated primers to or non-methylated primers pair, wherein, upstream primer and downstream primer are respectively 0.5ul;
(3) 10 × MSPbuffer damping fluid: 2.5ul;
(4)20mMdNTP：1.25ul；
(5) hotstarttaq enzyme: 0.5ul;
(6) deionized water: 17.75ul;
Wherein, described template is the cell DNA to be measured after phosphorothioate, or the TMEM176A gene promoter area after phosphorothioate is the 100% methylated esophageal carcinoma, colorectal cancer cell DNA, or the TMEM176A gene promoter area after phosphorothioate is 100% non-methylated normal esophageal, Colon and rectum cell DNA.
The invention provides a kind of test kit for detecting digestive system tumor cell, this test kit comprises above-mentioned methylated primers pair.Preferred version is, test kit of the present invention also comprises above-mentioned non-methylated primers pair.
Further, test kit of the present invention also comprises methylation positive contrast pcr template, and this template is the TMEM176A gene promoter area after phosphorothioate is 100% methylated cell DNA.
Further, test kit of the present invention also comprises non-methylation positive contrast pcr template, and this template is the TMEM176A gene promoter area after phosphorothioate is 100% non-methylated normal tissue cell DNA.
Further, test kit of the present invention also comprises the following ingredients needed for MS-PCR reaction: 10 × MSPbuffer damping fluid, 20mMdNTP, hotstarttaq enzyme, deionized water.
The method of first passage MSP of the present invention is successfully authenticated the hyper-methylation of TMEM176A promoter region in digestive system tumor.Therefore infer that the promoter region hyper-methylation state of this gene may be a new digestive system tumor molecular marked compound, may to be that new digestive system tumor is relevant press down cancer candidate gene to TMEM176A gene.As solid theoretical foundation, the method that the present inventor applies MS-PCR detects digestive system tumor and normal specimen, with its TMEM176A gene promoter zone methylation state clear and definite.
Tool of the present invention has the following advantages: utilize primer of the present invention and test kit to detect the biopsy cells of oesophagus and Colon and rectum mucomembranous cell, in tumour cell, TMEM176A gene promoter area is hyper-methylation state, methyl rate is about 61.2%, 53.13% respectively in the esophageal carcinoma, colorectal cancer, and in normal tissue cell, TMEM176A gene promoter area is non-methylation state, shows that the methylation state of this gene promoter area has specificity for digestive system tumor cell; Apply primer of the present invention and test kit, reaction system and reaction conditions can make the sensitivity of detection digestive system tumor cell reach 0.5% simultaneously, namely have 5 cancer cells just can be detected in 1000 cells, this illustrates that TMEM176A gene promoter zone methylation state can be used as the new molecular marker of of digestive system tumor.This test kit selects TMEM176A gene as target gene first, has initiative.Therefore, apply primer of the present invention and test kit to detect TMEM176A gene promoter area hyper-methylation state and can be used as the powerful measure of digestive system tumor as the diagnosis, observation of curative effect, Index for diagnosis etc. of the esophageal carcinoma and colorectal cancer, and, easy and simple to handle, good stability, has far-reaching clinical meaning and promotional value.
Accompanying drawing explanation
fig. 1represent electrophoresis result
figure;
fig. 2represent electrophoresis result
figure; Wherein
With the normal esophageal after phosphorothioate, Colon and rectum cell DNA, the esophageal carcinoma after phosphorothioate, colorectal cancer cell DNA, deionized water (system contrast) are template, carry out MS-PCR with methylated primers and non-methylated primers respectively, amplified production Gel electrophoresis results respectively
as Fig. 1with
fig. 2shown in, and wherein:
EC1, EC2, EC3, EC4, EC5, EC6, EC7, EC8 represent human esophageal carcinoma sample, and CRC1, CRC2, CRC3, CRC4, CRC5, CRC6, CRC7, CRC8 represent Colorectal Carcinoma sample; EN1, EN2, EN3 and EN4 are normal esophageal tissue samples 4 example, and CN1, CN2, CN3, CN4, CN5, CN6, CN7, CN8 represent normal colorectal carcinoma sample 8 example;
DdH
2o is double-negative system contrast, whether there is the pollution of PCR primer, as ddH in order to evaluation system
2the result that O detects is double-negative (M and U is feminine gender), then system credible result;
What IVD marked is methylation positive contrast; NL is the human peripheral lymphocytes of the U positive, herein as negative control.
U represents the non-result that methylates; M represents the result that methylates; Marker is DL2000marker (molecular weight standard), upper and lower two bands generation respectively
table 200bp and 100bp; Body series amplified production size, U band is 156bp for 160bp, M are with.
fig. 3represent esophageal carcinoma cell line K410DNA (TMEM176A gene promoter area 100% methylates) and normal esophageal histocyte DNA (TMEM176A gene promoter area 100% is non-to methylate) to be mixed in proportion, carry out phosphorothioate, after this increase with methylated primers, amplified production electrophoresis result
figure, wherein:
1st group: 100% esophageal carcinoma cell line K410DNA+0% normal esophageal histocyte DNA;
2nd group: 50% esophageal carcinoma cell line K410DNA+50% normal esophageal histocyte DNA;
3rd group: 5% esophageal carcinoma cell line K410DNA+95% normal esophageal histocyte DNA;
4th group: 1% esophageal carcinoma cell line K410DNA+99% normal esophageal histocyte DNA;
5th group: 0.5% esophageal carcinoma cell line K410DNA+99.5% normal esophageal histocyte DNA;
6th group: 0% esophageal carcinoma cell line K410DNA+100% normal esophageal histocyte DNA.
Embodiment
For understanding the present invention in more detail, the invention will be further elaborated by the following examples.
Embodiment 1
1, the preparation (extraction of genomic dna and phosphorothioate process) of template
The preparation of DNA: obtain the esophageal carcinoma, colorectal cancer sample and normal above-mentioned tissue sample.Tie the esophageal carcinoma 8 example (EC1-EC8), colorectal cancer 8 example (CRC1-CRC8), 4 routine normal esophageal (EN1-EN4), the normal Colon and rectum (CN1-CN8) of 8 example in the present embodiment, the method of application phenol-chloroform extracting extracts genomic dna respectively, and UV detector measures absorbancy (A) value and determines its content and purity.
Sulphite is modified: with reference to herman (J.G.Herman, J.R.Graff, S.Myohanen, B.D.NelkinandS.B.Baylin, Methylation-specificPCR:anovelPCRassayformethylationstat usofCpGislands, Proc.Natl.Acad.Sci.USA93 (1996), 9821 – 9826.) etc. report method.Get the genomic dna of above-mentioned preparation, after dilution, accurately get 2ugDNA, add deionized water to final volume 50ul, add the NaOH5ul of freshly prepared 3M, hatch 25min for 37 DEG C.Add the quinhydrones 30ul of freshly prepared 10mM and 3M sodium bisulfite 520ul afterwards to mix and (in it, added the NaOH of freshly prepared 3M, method of calculation: the NaOH740ul adding 3M in 10ml3M sodium bisulfite), hatch 16h for 50 DEG C, after this (Promega is public to apply DNA purification kit, department's product) purifying reclaim DNA, application 50ul deionized water eluted dna, the NaOH of 5ul3M is added to stop phosphorothioate in it, with the dehydrated alcohol precipitation DNA of 3 times of volumes, DNA after final phosphorothioate is dissolved in 20ul deionized water again, obtain DNA profiling, immediately use or-20 DEG C save backup.
2, MS-PCR amplification
Carry out the PCR reaction system increased with methylated primers, in cumulative volume 25ul, comprising:
Template (DNA after phosphorothioate): 2ul;
Methylated primers (50pmol/ul):
Upstream primer (5'GAAGAAAGACGTTTTGTGGATAGGAC3'): 0.5ul,
Downstream primer (5'CTAATATCCGCTCTACTCGACCGCG3'): 0.5ul;
10 × MSPbuffer (damping fluid) 2.5ul;
20mMdNTP：1.25ul；
Hotstarttaq enzyme: 0.15ul;
Deionized water: 18.1ul
Carry out the PCR reaction system increased with non-methylated primers, in cumulative volume 25ul, comprising:
The PCR reaction system difference of carrying out increasing from methylated primers is: primer is different, is non-methylated primers (50pmol/ul) in this system:
Upstream primer (5'GGAAGAAAGATGTTTTGTGGATAGGAT3'): 0.5ul,
Downstream primer (5'CAACTAATATCCACTCTACTCAACCACA3'): 0.5ul;
All the other all carry out in the PCR reaction system increased with methylated primers identical.
Apply above-mentioned MSP reaction system to the DNA profiling prepared (8 esophageal carcinoma cancerous tissue sample EC1 ~ EC8,8 Colorectal Carcinoma sample CRC1 ~ CRC8,4 normal esophageal tissue samples EN1 ~ EN4,8 normal colorectal carcinoma sample CN1 ~ CN8) increase respectively, and with ddH
2o is as negative control, and amplification program is: 95 DEG C of denaturation 5min, then enter circulation, sex change 30s at 95 DEG C, and anneal 30s at 60 DEG C, and at 72 DEG C of downward-extension 40s, totally 35 circulations, afterwards, continue at 72 DEG C of downward-extension 7min.
The detection of 3.PCR reaction product
PCR primer carries out 2% agarose gel electrophoresis, and ultraviolet transmission analyser detects and takes a picture.
4. result
Result interpretation method is as follows:
(1) apply methylated primers (
in figurerepresent with M) increase, have amplified production, and apply non-methylated primers (
in figurerepresent with U) increase, without amplified production sample then interpretation be exhaustive methylation result;
(2) application methylated primers and non-methylated primers increase, and all have the sample interpretation of amplified production to be partial methylation result;
(3) apply non-methylated primers to increase, have amplified production, and increase with methylated primers, without amplified production sample then interpretation be the non-result that methylates.
(4) the sample standard deviation interpretation of partial methylation and exhaustive methylation is the result that methylates.
as Fig. 1shown in, in human esophageal carcinoma, EC2 and EC7 is the non-result that methylates, and EC1, EC3, EC4, EC5, EC6, EC8 are the result that methylates; In Colorectal Carcinoma, CRC3, CRC5, CRC6, CRC7, CRC8 are the non-result that methylates, and CRC1, CRC2, CRC4 are the result that methylates.
as Fig. 2shown in, normal esophageal tissue (EN1, EN2, EN3 and EN4), normal colorectal carcinoma (CN1, CN2, CN3, CN4, CN5, CN6, CN7, CN8) are the non-result that methylates.Control systems reaction is normal, credible result.
Embodiment 2 clinical samples detects
Get esophageal carcinoma clinical samples 103 example, colorectal cancer clinical samples 96 example, normal esophageal tissue sample 4 example, normal colorectal carcinoma sample 8 example.Carry out MS-PCR amplification, the detection of Template preparation, PCR amplification system and condition, amplified production is all with to implement in one identical, and detected result is asked for an interview
following table:
| Classification | Number of cases | M (methylating) number of cases | U (without methylating) number of cases | Methylation positive rate |
| The esophageal carcinoma | 103 | 63 | 40 | 61.2％ |
| Normal esophageal tissue | 4 | 0 | 4 | 0 |
| Colorectal cancer | 96 | 51 | 45 | 53.13％ |
| Normal colorectal carcinoma | 8 | 0 | 8 | 0 |
Embodiment 3 susceptibility is tested
The DNA (TMEM176A gene promoter area 100% methylates) of esophageal carcinoma cell line K410 and the histiocytic DNA of normal esophageal (TMEM176A gene promoter area 100% is non-to methylate) are mixed in proportion, carry out phosphorothioate (method is with embodiment one), then carry out MS-PCR.PCR primer carries out 2% agarose gel electrophoresis, and ultraviolet transmission analysis-e/or determining is also taken a picture.
Grouping: the 1st group: 100% esophageal carcinoma cell line K410DNA+0% normal esophageal histocyte DNA
2nd group: 50% esophageal carcinoma cell line K410DNA+50% normal esophageal histocyte DNA
3rd group: 5% esophageal carcinoma cell line K410DNA+95% normal esophageal histocyte DNA
4th group: 1% esophageal carcinoma cell line K410DNA+99% normal esophageal histocyte DNA
5th group: 0.5% esophageal carcinoma cell line K410DNA+99.5% normal esophageal histocyte DNA
6th group: 0% esophageal carcinoma cell line K410DNA+100% normal esophageal histocyte DNA
Result is asked for an interview
fig. 3: in 1000 normal cells, there are 5 cancer cells just can be detected, namely 0.5% can be reached with its sensitivity of methylation state that the primer pair of the TMEM176A gene specific in the present invention and reaction system, condition detect esophageal cancer cell TMEM176A gene promoter area, sensitivity is higher, can be used to monitor early carcinomatous change.
Embodiment 4 detects the test kit composition of digestive system carcinoma cell
The test kit detecting digestive system carcinoma cell comprises following composition, and wherein, the consumption carrying out 1 MS-PCR is:
1, concentration is the methylated primers of 50pmol/ul: upstream primer nucleotides sequence is classified as Primer-MF, and downstream primer nucleotides sequence is classified as Primer-MR, is respectively 0.5ul.
2, concentration is the non-methylated primers of 50pmol/ul: upstream primer nucleotides sequence is classified as Primer-UF, and downstream primer nucleotides sequence is classified as Primer-UR, is respectively 0.5ul.
3, reaction solution 2 parts, coordinate methylated primers and non-methylated primers to try out respectively, every part of reaction system comprises:
(1) 10xMSPbuffer (damping fluid) 2.5ul;
(2)20mMdNTP：1.25ul；
(3) Hotstarttaq enzyme: 0.15ul;
(4)ddH
2O：18.1ul。
A test kit can comprise the consumption that above-mentioned each composition carries out repeatedly MS-PCR, as 25 times, 50 times, 100 inferior, the Specific amounts of each composition is depending on the circumstances or the needs of the situation determined.
For preventing false positive and the false negative of MS-PCR amplified production, test kit preferably also comprises:
(1) positive control pcr template: the TMEM176A gene promoter area after phosphorothioate is 100% methylated esophageal carcinoma cell line K410 system DNA, and the consumption carrying out 1 MSP is: 2ul.
(2) negative control pcr template: the TMEM176A gene promoter area after phosphorothioate is 100% non-methylated normal peripheral blood lymphocyte DNA, and the consumption carrying out 1 MSP is: 2ul.
(3) double-negative system contrast: whether ddH2O, exist the pollution of PCR primer in order to evaluation system, the result detected as ddH2O is double-negative (M and U is feminine gender), then system credible result.
Claims (10)
1. one kind for detecting the primer pair of cell TMEM176A gene promoter zone methylation state, it is methylated primers, its upstream primer nucleotide sequence is as described in Primer-MF, that is: 5'GAAGAAAGACGTTTTGTGGATAGGAC3', downstream primer nucleotide sequence as described in Primer-MR, that is: 5'CTAATATCCGCTCTACTCGACCGCG3'.
2. one kind for detecting the primer pair of cell TMEM176A gene promoter zone methylation state, comprise methylated primers to non-methylated primers pair, described methylated primers is to as claimed in claim 1, the right upstream primer nucleotide sequence of described non-methylated primers is as described in Primer-UF, that is: 5'GGAAGAAAGATGTTTTGTGGATAGGAT3', downstream primer nucleotide sequence as described in Primer-UR, that is: 5'CAACTAATATCCACTCTACTCAACCACA3'.
3. detect a method for TMEM176A gene promoter zone methylation state, comprise the steps: first, to utilize methylated primers pair, with the DNA after phosphorothioate for template carries out MS-PCR amplification; Then, methylation state is judged according to amplified production; Wherein, described methylated primers is to as claimed in claim 1; Described template is the cell DNA to be measured after phosphorothioate.
4. detection method according to claim 3, utilize simultaneously methylated primers to non-methylated primers pair, with the DNA after phosphorothioate for template carries out MS-PCR amplification; Wherein said non-methylated primers is to as claimed in claim 2.
5. the detection method according to claim 3 or 4, primer pair is wherein utilized to carry out in MS-PCR amplification, reaction conditions is as follows: denaturation 5min at 95 DEG C, then enter circulation, sex change 30s at 95 DEG C, anneal 30s at 60 DEG C, at 72 DEG C of downward-extension 40s, totally 35 circulations, afterwards, continue at 72 DEG C of downward-extension 5min.
6. detect a test kit for cell TMEM176A gene promoter zone methylation state, it is characterized in that: comprise the primer pair described in claim 1 or 2.
7. test kit according to claim 6, is characterized in that: also comprise methylation positive contrast pcr template, this template is the methylated esophageal carcinoma in TMEM176A gene promoter area 100%, colorectal cancer cell DNA after phosphorothioate.
8. test kit according to claim 7, is characterized in that: also comprise non-methylation positive contrast pcr template, this template is the non-methylated normal esophageal in TMEM176A gene promoter area 100%, Colon and rectum cell DNA after phosphorothioate.
9. the test kit according to any one of claim 6 to 8, is characterized in that: also comprise the following ingredients needed for MS-PCR reaction: 10 × MSPbuffer damping fluid, 20mMdNTP, hotstartaq enzyme, deionized water.
10., for detecting a MS-PCR reaction system for cell TMEM176A gene promoter zone methylation state, in cumulative volume 25ul, it comprises:
(1) template: 2ul;
(2) the concentration methylated primers according to claim 1 that is 50pmol/ul to the non-methylated primers pair described in claim 2, wherein, upstream primer and downstream primer are respectively 0.5ul;
(3) 10 × MSPbuffer damping fluid: 2.5ul;
(4)20mMdNTP：1.25ul；
(5) hotstarttaq enzyme: 0.5ul;
(6) deionized water: 17.75ul;
Wherein, described template is the cell DNA to be measured after phosphorothioate, or the TMEM176A gene promoter area after phosphorothioate is the 100% methylated esophageal carcinoma, colorectal cancer cell DNA, or the TMEM176A gene promoter area after phosphorothioate is 100% non-methylated normal esophageal, Colon and rectum cell DNA.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510390230.8A CN105400865B (en) | 2015-07-06 | 2015-07-06 | The DNA methylation detection of the gene promoter areas TMEM176A |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510390230.8A CN105400865B (en) | 2015-07-06 | 2015-07-06 | The DNA methylation detection of the gene promoter areas TMEM176A |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN105400865A true CN105400865A (en) | 2016-03-16 |
| CN105400865B CN105400865B (en) | 2018-10-23 |
Family
ID=55466621
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510390230.8A Active CN105400865B (en) | 2015-07-06 | 2015-07-06 | The DNA methylation detection of the gene promoter areas TMEM176A |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105400865B (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106676168A (en) * | 2016-11-08 | 2017-05-17 | 北京交通大学 | Apparent modification detection primer pair based on ATP4B genosome DNA and kit thereof |
| CN107245519A (en) * | 2017-06-06 | 2017-10-13 | 俞晓敏 | A kind of PWS and AS quick determination method |
| CN112646881A (en) * | 2019-10-11 | 2021-04-13 | 中国人民解放军总医院 | Primer probe, PCR method and kit for detecting methylation state of CCL28 gene promoter region of cell |
Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2009105533A2 (en) * | 2008-02-19 | 2009-08-27 | The Johns Hopkins University | Methods for predicting esophageal adenocarcinoma (eac) |
| WO2010042228A2 (en) * | 2008-10-10 | 2010-04-15 | Cornell University | Methods for predicting disease outcome in patients with colon cancer |
| US20120004127A1 (en) * | 2010-05-12 | 2012-01-05 | Christopher Sears | Gene expression markers for colorectal cancer prognosis |
| CN102311953A (en) * | 2011-09-23 | 2012-01-11 | 上海市肿瘤研究所 | Method and kit for diagnosing bladder cancer with urine |
| CN102373286A (en) * | 2011-11-28 | 2012-03-14 | 中国科学院化学研究所 | Primer capable of detecting methylation of promoter of gene related to colonic adenocarcinoma |
| WO2012167145A2 (en) * | 2011-06-01 | 2012-12-06 | University Of Southern California | Genome-scale analysis of aberrant dna methylation in colorectal cancer |
| CN103417983A (en) * | 2012-05-18 | 2013-12-04 | 北京大学 | Novel apoptosis-related molecule TMEM106A and applications thereof |
-
2015
- 2015-07-06 CN CN201510390230.8A patent/CN105400865B/en active Active
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2009105533A2 (en) * | 2008-02-19 | 2009-08-27 | The Johns Hopkins University | Methods for predicting esophageal adenocarcinoma (eac) |
| WO2010042228A2 (en) * | 2008-10-10 | 2010-04-15 | Cornell University | Methods for predicting disease outcome in patients with colon cancer |
| US20120004127A1 (en) * | 2010-05-12 | 2012-01-05 | Christopher Sears | Gene expression markers for colorectal cancer prognosis |
| WO2012167145A2 (en) * | 2011-06-01 | 2012-12-06 | University Of Southern California | Genome-scale analysis of aberrant dna methylation in colorectal cancer |
| CN102311953A (en) * | 2011-09-23 | 2012-01-11 | 上海市肿瘤研究所 | Method and kit for diagnosing bladder cancer with urine |
| CN102373286A (en) * | 2011-11-28 | 2012-03-14 | 中国科学院化学研究所 | Primer capable of detecting methylation of promoter of gene related to colonic adenocarcinoma |
| CN103417983A (en) * | 2012-05-18 | 2013-12-04 | 北京大学 | Novel apoptosis-related molecule TMEM106A and applications thereof |
Non-Patent Citations (5)
| Title |
|---|
| JAMES W. SCHROEDER等: "Neonatal DNA methylation patterns associate with gestational age", 《EPIGENETICS》 * |
| MATH P. CUAJUNGCO等: "Abnormal accumulation of human transmembrane (TMEM)-176A and 176B proteins is associated with cancer pathology", 《ACTA HISTOCHEMICA》 * |
| TSUGUTERU OTSUBO等: "Identification of novel targets for antiangiogenic therapy by comparing the gene expressions of tumor and normal endothelial cells", 《CANCER SCI.》 * |
| VLADIMIR STRELNIKOV等: "NON-MICROARRAY DNA DIFFERENTIAL METHYLATION SCREENING IN BREAST CANCER", 《CANCER GENETICS AND CYTOGENETICS》 * |
| 张游: "TMEM176A基因启动子区在食管癌中的异常甲基化改变", 《中国博士学位论文全文数据库 医药卫生科技辑》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106676168A (en) * | 2016-11-08 | 2017-05-17 | 北京交通大学 | Apparent modification detection primer pair based on ATP4B genosome DNA and kit thereof |
| CN107245519A (en) * | 2017-06-06 | 2017-10-13 | 俞晓敏 | A kind of PWS and AS quick determination method |
| CN112646881A (en) * | 2019-10-11 | 2021-04-13 | 中国人民解放军总医院 | Primer probe, PCR method and kit for detecting methylation state of CCL28 gene promoter region of cell |
Also Published As
| Publication number | Publication date |
|---|---|
| CN105400865B (en) | 2018-10-23 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103436606B (en) | Kit for auxiliary diagnosis and/or prognosis judgment of esophageal carcinoma | |
| US20190136330A1 (en) | Method for screening cancer | |
| WO2013038737A1 (en) | Method for detecting bladder cancer cells, primer used in method for detecting bladder cancer cells, and bladder cancer marker | |
| CN111235238A (en) | DNA methylation detection method and related application | |
| CN105400865A (en) | TMEM176A gene promoter region DNA methylation detection | |
| Wu et al. | Ovarian cancer detection by DNA methylation in cervical scrapings | |
| US20230383356A1 (en) | Composition for diagnosing colorectal cancer, rectal cancer or colorectal adenoma by using cpg methylation change of linc01798 gene, and use thereof | |
| TW202028473A (en) | Purpose of HOXA9 methylation detection reagent in preparation of lung cancer diagnosis reagent | |
| CN111808963A (en) | Composition for noninvasive screening of early lung cancer, application and kit and sample processing method | |
| CN115315530A (en) | Composition for diagnosing bladder cancer using CPG methylation change in specific gene and use thereof | |
| CN109439750B (en) | Molecular marker for detecting susceptibility of cervical cancer, kit and application | |
| CN112575082A (en) | Fluorescent quantitative PCR detection kit for target gene methylation detection | |
| CN114107498B (en) | Colorectal cancer blood detection marker and application thereof | |
| CN102851354B (en) | Primer pair and kit for detecting methylation state of DNA at cell DACH1 gene promoter region | |
| TW202028464A (en) | Application of HOXA7 methylation detection reagent to preparation of lung cancer diagnosis reagent | |
| CN110029165A (en) | A kind of kit marked for detecting the susceptible correlated inheritance of carcinoma of endometrium | |
| CN115433778B (en) | Detection reagent and kit for detecting colorectal cancer or colorectal adenoma related gene methylation and application of detection reagent and kit | |
| CN108796078A (en) | A kind of primer and probe group for digestive system cancer diagnosis, detection or screening | |
| CN109321658B (en) | Kit for detecting susceptibility of cervical cancer | |
| Tomita et al. | Development of canine X-chromosome inactivation pattern analysis for the detection of cell clonality by incorporating the examination of the SLIT and NTRK-like family member 4 (SLITRK4) gene | |
| TW201514308A (en) | Novel epigenetic biomarkers for bladder cancer detection and method thereof | |
| CN113430272A (en) | Reagent and kit for diagnosis or auxiliary diagnosis of esophageal cancer or precancerous lesion and application of reagent and kit | |
| US20180179597A1 (en) | Cancer markers | |
| CN116200499B (en) | Gene combination for liver cancer detection, related reagent and application | |
| CN107287286A (en) | Detect the primer pair and kit of cell RAI2 gene promoter areas methylation state of DNA |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |