CN103923976A - Method for detecting single nueleotide polymorphism of TERT - Google Patents
Method for detecting single nueleotide polymorphism of TERT Download PDFInfo
- Publication number
- CN103923976A CN103923976A CN201410042839.1A CN201410042839A CN103923976A CN 103923976 A CN103923976 A CN 103923976A CN 201410042839 A CN201410042839 A CN 201410042839A CN 103923976 A CN103923976 A CN 103923976A
- Authority
- CN
- China
- Prior art keywords
- primer
- mutational site
- sample
- tert
- urine
- 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.)
- Pending
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
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6858—Allele-specific amplification
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Health & Medical Sciences (AREA)
- Biophysics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Immunology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Analytical Chemistry (AREA)
- Physics & Mathematics (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention relates to a method for detecting the single nueleotide polymorphism of TERT, and belongs to the technical field of biotechnological molecular detection. The method comprises the following steps: 1, respectively extracting DNA from sample urine and normal tissues; 2, carrying out TaqMan Probe Real time PCR amplification against the difference of mutation sites to be detected by using the DNA extracted from the sample urine and normal tissues as templates and respectively treating a primer pair P1, P2 or P3 corresponding to mutation sites as primers; and 3, determining the size of deltaCtu and deltaCtn, wherein mutation sites exist in the urine sample if a result of deltaCtu - deltaCtn is lower than 0, and the mutation sites do not exist in the urine sample if the result of deltaCtu - deltaCtn is not lower than 0.
Description
Technical field
The invention belongs to biotechnology molecular detection technology field, be specifically related to a kind of method of the TERT of detection single nucleotide polymorphism.
Background technology
Bladder cancer is to come the modal tumour in the whole world of the 9th.2013, have and exceed 73000 patients and be diagnosed as bladder cancer, wherein death toll approximately 15000 people in the U.S..Wherein exceed 90% patient and be diagnosed as at first primary bladder cancer, about 75% patient is Superficial bladder cancer at present, 20% be patient with invasive bladder tumor, 5% remaining first visit is metastatic tumo(u)r.Previous research shows, bladder cancer has changeable clinical manifestation and genetic background.8 genes involveds (UTX, MLL-MLL3, CREBBP-EP300, NCOR1, ARID1A, CHD6) have been reported in nearest tumor of bladder research.
The tumor marker that detection " bladder cancer " adopts at present mainly contains following several, but each tumor markers all exists different shortcomings:
(1), bladder tumor antigen (BTA): BTA reagent is divided into two kinds of BTA stat and BTA test; First, these two kinds of reagent all can not independently be used for making a definite diagnosis bladder cancer; In addition, BTA reagent is expensive, is still difficult at present popularize in an all-round way use;
(2), Lewis X Detection of antigen: Lewis X is a kind of abo blood group related antigen, does not have this antigen in normal urothelium; And 5%～89% transitional cell carcinoma can detect Lewis X, but the classification of Lewis X and tumour is irrelevant;
(3), NMP-22 (nuclear matrix protein22, NMP22): NMP22 is nuclear mitotic apparatus protein, the susceptibility of diagnosing bladder cancer is 48%～90%, and specificity is 70%～92%, NMP22 is to senior, and high phase bladder cancer susceptibility is higher;
(4), scleroproein/fibrin degradation product (FDP) (fibrin degradation products, FDP): measuring the susceptibility of FDP diagnosing bladder cancer in urine by tachysynthesis detection method is 68%, to the susceptibility of T2～T4 phase bladder cancer especially up to 100%;
(5), hyaluronidase detects hyaluronidase, HAase: hyaluronidase is the hyaluronic endoglycosidase of a kind of extracellular matrix degradation, in tumour progression, play an important role, application gel technique detects G2, hyaluronidase activity in G3 level bladder cancer urine, susceptibility reaches 92%～100%;
(6), telomerase activation (telomerase): telomere is the protective structures that is positioned at end of chromosome; progressively shorten with cell fission; until necrocytosis; the effect of Telomerase is exactly to extend telomere; have now found that kinds of tumor cells Telomerase Activity strengthens; this method diagnosis comprises rudimentary, and lowstand tumour is in interior bladder cancer, and susceptibility can reach 91%.
But the key point that causes tumor of bladder to occur is illustrated not yet completely, simultaneously because the susceptibility of these individual marks is lower to such an extent as to be not applied in clinical practice the biomarker that this susceptibility that shows that needs are new is high and accordingly detection technique.
Summary of the invention
In the time of pcr amplification, the extension of primer, from its 3' end, requires the base of primer 3' end and template to need strict pairing, and only in this way primer could extend, the repressor gene of cutting TaqMan probe thereby amplification is just gone on, generation can be for the fluorescent signal detecting.If primer 3' end can not match with template, the extension of primer is blocked, and corresponding fluorescent signal can not be detected, based on the above fact, utilize 3' end to carry out TaqMan Probe Real time pcr amplification containing the primer of mutating alkali yl, can detect and in target DNA, have or not corresponding mutational site.Comprise two pcr amplification reactions for the reactive system in a specific mutational site, design respectively two couples of 3' and hold the primer there are differences, one is normal primer, the primer that another becomes for 3' distal process, normal primer strictly matches with normal template, the corresponding product that increases when PCR, and the primer of sudden change only goes out corresponding product with the template amplification of sudden change.By two pairs of primers increase sudden change and normal specimens simultaneously, can obtain different amplification cycles and count ratio △ Ct, compare judgement by the amplification cycles ratio to two pairs of primers, can determine in sample, whether there is mutational site.
In process of the test of the present invention, exceed in the research of 85% human tumor, find reverse transcriptase of telomere (TERT, TERT obtains the human genomic sequence of NCBI released version 37: Feb.2009 (GRCh37/hg19) by Human Genome Browser; >hg19_ensGene_ENST00000310581_0range=chr5: 1295163-12965625'pad=4003'pad=0strand=-repeatMasking=non is e) active, and we think a tumorigenic important mechanisms of the mankind.TERT is the catalysed partial of Telomerase, is positioned at the short arm of a chromosome No. 5, adjustable side telomerase activity in human tumor.People apply genome sequencing and in melanoma, find TERT promoter mutation recently.People have confirmed high-frequency TERT promoter mutation in some bladder cancer samples.Some study discovery, and the activated Telomerase of tool or TERT express to increase and be associated with pathological staging and the clinical stages of tumour.But people also do not obtain TERT gene copy, the expression of TERT or activity remain controversial to the clinical impact of bladder cancer.
The normal sequence of having pointed out TERT in application number is 201310512895.2,201310514526.7 and 201310514819.5 patent application as SEQ NO.ID14 of the present invention (the first in SEQ NO.ID14 is chr5,1,296,562 position, ends are chr5,1,295,163) shown in; The mutant nucleotide sequence of TERT is shown in SEQ NO.ID15 (in SEQ NO.ID15, first place is chr5, and 1,296,562 positions, end are chr5,1,295,163); Fig. 1 is seen in mutational site on TERT gene.
The object of the invention is to disclose a kind of method of the TERT of detection single nucleotide polymorphism.
The object of the invention is to be achieved through the following technical solutions:
A method that detects TERT single nucleotide polymorphism, comprises the steps:
(1), from the urine of sample and healthy tissues, extract respectively DNA;
(2) difference in the mutational site of, detecting for needs, taking the DNA that extracts from sample urine and healthy tissues as template, respectively taking primer pair P1, the P2 corresponding with mutational site or P3 as primer, carry out TaqMan Probe Real time pcr amplification, wherein with 1, primer pair P1 corresponding to 295,228G>A mutational site is:
P1Fm:CGGGTCCCCGGCCCAGCCCCT, (as SEQ NO.ID1)
P1Rm:GCGCCGCGAGGAGAGGGCG; (as SEQ NO.ID2)
P1Fn:CGGGTCCCCGGCCCAGCCCCC, (as SEQ NO.ID3)
P1Rn:GCGCCGCGAGGAGAGGGCG; (as SEQ NO.ID4)
The primer pair P2 corresponding with 1,295,250G>A mutational site is:
P2Fm:CGCCCCGTCCCGACCCCTT, (as SEQ NO.ID5)
P2Rm:CGCCGCGAGGAGAGGGCG; (as SEQ NO.ID6)
P2Fn:CGCCCCGTCCCGACCCCTC, (as SEQ NO.ID7)
P2Rn:CGCCGCGAGGAGAGGGCG; (as SEQ NO.ID8)
With 1,295,242-1, primer pair P3 corresponding to 295,243GG>AA mutational site is:
P3Fm:CGTCCCGACCCCTCCCGGGTTT, (as SEQ NO.ID9)
P3Rm:GCGCCGCGAGGAGAGGGCG; (as SEQ NO.ID10)
P3Fn:CGTCCCGACCCCTCCCGGGTCC, (as SEQ NO.ID11)
P3Rn:GCGCCGCGAGGAGAGGGCG; (as SEQ NO.ID12)
(3), determine the size between △ Ctu and △ Ctn, when having mutational site in △ Ctu-△ Ctn<0 urine sample; In the urine sample of △ Ctu-△ Ctn >=0, there is not mutational site; Wherein △ Ctu is the difference of cycle number when mutant primer and normal primer reach amplification threshold value in urine sample; △ Ctn is the difference of cycle number when mutant primer and normal primer reach amplification threshold value in healthy tissues sample.
A kind of method that detects TERT single nucleotide polymorphism described in technique scheme, wherein, the reaction conditions of described TaqManProbe Real time pcr amplification is:
The first step denaturation: 95 DEG C 30 seconds, repeat 1 time;
Second step PCR reaction: 95 DEG C of 5 seconds and 60 DEG C 30 seconds, repeat 40 times.
A kind of method that detects TERT single nucleotide polymorphism described in technique scheme, wherein, in described TaqMan Probe Real time pcr amplification system, contain: 12.5 μ l2 × Premix Ex Taq, 0.5 μ l PCR Forward Primer (10 μ M), 0.5 μ l PCR Reverse Primer (10 μ M), 1 μ l
probe, 2 μ l DNA profilings and 8.5 μ l d H
2o (sterile purified water).
A kind of method that detects TERT single nucleotide polymorphism described in technique scheme, wherein, the reaction TaqMan Probe sequence of described TaqManProbe Real time pcr amplification is:
5’-CTCCCAGCCCCTCCCCTTCCTTTCCGCGGC-3’。
The present invention has following beneficial effect:
1, the invention discloses the method that detects TERT single nucleotide polymorphism from urine sample that discloses, by designing specific PCR primer amplification fragment, detect the polymorphism of TERT mononucleotide by the detection mode of TapMan.The method is simple, quick, cost is low;
2, the present invention can be used as one of method of bladder cancer detection and detecting/monitoring;
3, practicality of the present invention is high, has higher clinical value.
Brief description of the drawings:
Fig. 1 is mutational site on TERT gene;
Fig. 2 is Real Time PCR reaction process.
Embodiment:
For making technical scheme of the present invention be convenient to understand, below in conjunction with concrete test example, the method for a kind of TERT of detection single nucleotide polymorphism of the present invention is further described.
test example 1:a kind of method that detects TERT single nucleotide polymorphism:
One, healthy tissues sample and urine sample DNA extraction:
1, main agents:
TIANamp Micro DNA Kit micro-example genome DNA extracting reagent kit (TIANGEN BIOTECH (BEIJING) CO., LTD)
2, method: first get 50ml urine sample in aseptic centrifuge tube, 4000 leave heart 10min, obtains arena.Then operate according to the specification sheets in TIANamp Micro DNA Kit micro-example genome DNA extracting reagent kit:
(before using, please first in damping fluid GD and rinsing liquid PW, add dehydrated alcohol, add volume to please refer to test kit)
(1), get 100 μ l-200 μ l arenas in the centrifuge tube of 2ml, as less than 100 μ l, with damping fluid GA to 100 μ l final volume;
(2), add 20 μ l Proteinase K solution, vortex mixes;
(3), add the damping fluid GB of 200 μ l (as arena volume <50 μ l, can add 1 μ l Carrier RNA storage liquid, concentration be 1 μ g/ μ l), put upside down and mix gently, hatch 10min for 56 DEG C, and frequently shake sample.The drop of brief centrifugal removal cap wall;
While adding damping fluid GB in this step, may produce white precipitate, general 56 DEG C place time can disappear, can not affect subsequent experimental, as solution does not become limpid, illustrate that lysis is not thorough, may cause extracting DNA measure less and the DNA extracting impure;
The preparation of Carrier RNA storage liquid: in the time using Carrier RNA for the first time, please (310 μ g) are dissolved in 310 μ l RNase-Free ddH by Carrier RNA
2in O, solution packing is stored in to-20 DEG C, now the concentration of this solution is 1 μ g/ μ l; This storage liquid should be avoided multigelation, and number of freezing and thawing can not exceed 3 times;
(4), add the ethanol (96-100%) of 200 μ l.If room temperature exceedes 25 DEG C, please ethanol is put to precooling on ice.Put upside down gently and mix sample, room temperature is placed 5min, brief centrifugal to remove the drop of cap wall;
(5), previous step gained solution is added in an adsorption column CR2 (adsorption column is put into collection tube), (～13400 × g) centrifugal 30sec, abandons waste liquid to 12000rpm, and adsorption column CR2 is put back in collection tube.
(6), in adsorption column CR2, add 500 μ l damping fluid GD (please first check before use and whether added dehydrated alcohol), (～13,400 × g) centrifugal 30sec, abandons waste liquid to 12,000rpm, and adsorption column CR2 is put back in collection tube;
(7), in adsorption column CR2, add 600 μ l rinsing liquid PW (please first check before use and whether added dehydrated alcohol), (～13,400 × g) centrifugal 30sec, abandons waste liquid to 12,000rpm, and adsorption column CR2 is put back in collection tube;
(8), repetitive operation step (7);
(9), 12, (～13,400 × g) centrifugal 2min, outwells waste liquid to 000rpm; Adsorption column CR2 is placed in to room temperature and places 2-5min, thoroughly to dry rinsing liquid remaining in sorbing material; The object of this step is that rinsing liquid remaining in adsorption column is removed, residual can the impact follow-up enzyme reaction of ethanol in rinsing liquid (as enzyme is cut, PCR etc.) experiment.
(10), adsorption column CR2 is proceeded in a clean centrifuge tube,, to the unsettled dropping in adsorption film mid-way 20-50 μ l elution buffer TB, room temperature is placed 2-5min, 12, (～13,400 × g) centrifugal 2min, collects solution in centrifuge tube 000rpm; In this step, elution buffer volume should not be less than 20 μ l, the too small organic efficiency that affects of volume.For increasing the yield of genomic dna, the centrifugal solution obtaining can be added in adsorption column CR2 again, room temperature is placed 2min, 12,000rpm (～13,400 × g) centrifugal 2min.The pH value of elutriant has a significant impact for elution efficiency.Should ensure that its pH value is within the scope of 7.0-8.5 if water is cooked elutriant, pH value can reduce elution efficiency lower than 7.0; And DNA product should be kept at-20 DEG C, in case DNA degradation.
The same steps that the extraction of healthy tissues sample DNA is carried according to urine sample DNA is carried out.
Two, respectively for three place's mutantional hotspot design primers in TERT promotor:
For TERT core promoter primers to P, design primer with primer5.0, make mutating alkali yl be positioned at first (1,295 of primer 3' end, 228G>A and 1,295,250G>A), or first and second position (1,295,242-1,295,243GG>AA).For each mutantional hotspot design packet containing sudden change (under be designated as m) and normal base (under be designated as two pairs of primers n), the wherein primer pair P1 corresponding with 1,295,228G>A mutational site, primer sequence information is as follows:
P1Fm:CGGGTCCCCGGCCCAGCCCCT, (as SEQ NO.ID1)
P1Rm:GCGCCGCGAGGAGAGGGCG; (as SEQ NO.ID2)
P1Fn:CGGGTCCCCGGCCCAGCCCCC, (as SEQ NO.ID3)
P1Rn:GCGCCGCGAGGAGAGGGCG; (as SEQ NO.ID4)
The primer pair P2 corresponding with 1,295,250G>A mutational site, primer sequence information is as follows:
P2Fm:CGCCCCGTCCCGACCCCTT, (as SEQ NO.ID5)
P2Rm:CGCCGCGAGGAGAGGGCG; (as SEQ NO.ID6)
P2Fn:CGCCCCGTCCCGACCCCTC, (as SEQ NO.ID7)
P2Rn:CGCCGCGAGGAGAGGGCG; (as SEQ NO.ID8)
With 1,295,242-1, the primer pair P3 that 295,243GG>AA mutational site is corresponding, primer sequence information is as follows:
P3Fm:CGTCCCGACCCCTCCCGGGTTT, (as SEQ NO.ID9)
P3Rm:GCGCCGCGAGGAGAGGGCG; (as SEQ NO.ID10)
P3Fn:CGTCCCGACCCCTCCCGGGTCC, (as SEQ NO.ID11)
P3Rn:GCGCCGCGAGGAGAGGGCG; (as SEQ NO.ID12)
Three, the design of TaqMan Probe sequence and linking group (Hua Da genome company of Shenzhen provides):
TaqMan Probe sequence and linking group are:
5 '-FAM-CTCCCAGCCCCTCCCCTTCCTTTCCGCGGC-TAMRA-3 '; (as SEQ NO.ID13)
Four, TaqMan Probe Real time pcr amplification:
Main agents: TAKARA Premix Ex Taq
tM(Probe QPCR) (Hua Da genome company of Shenzhen provides)
The working method of application Thermal Cycler Dice Real Time System II
(1), by the component preparation PCR reaction system (carrying out the preparation of reaction system on ice) in table 1.
Table 1PCR reaction system
* 1: primer final concentration is that 0.2 μ M can obtain better result conventionally.When reactivity worth is poor, can within the scope of 0.1～1.0 μ M, adjust primer concentration; * 2: the concentration and probe concentration of use, relevant with the RealTime pcr amplification instrument, probe kind, the fluorescent mark material kind that use, reagent please refer to instrument specification sheets while use, or the concrete service requirements of each fluorescent probe is carried out.While using Thermal Cycler Dice Real Time System, probe final concentration is adjusted within the scope of 0.1～0.5 μ M conventionally; * 3: in 25 μ l reaction systems, the addition of DNA profiling is conventionally below 100ng.Because of the copy number difference of the target gene that contains in different types of DNA profiling, can carry out if desired gradient dilution, determine best DNA profiling addition.If the second step pcr amplification reaction that wish is used these goods to carry out 2Step RT-PCR reaction, the addition of the RT reaction solution of the first step during as DNA profiling do not exceed 10% of PCR reaction solution cumulative volume.
The difference in the mutational site of detecting for needs adds respectively the primer of corresponding mutant primer and normal base, for example need to detect whether have 1,295, when in 228G>A the mutational site, carry out respectively 4 PCR reactions according to the PCR reaction system in table 1, the DNA profiling of the 1st time is that urine sample DNA profiling, PCR Forward Primer are that P1Fm, PCR Reverse Primer are P1Rm; The DNA profiling of the 2nd time is that urine sample DNA profiling, PCR Forward Primer are that P1Fn, PCR Reverse Primer are P1Rn; The DNA profiling of the 3rd time is that healthy tissues sample DNA template, PCR Forward Primer are that P1Fm, PCR Reverse Primer are P1Rm; The 4th DNA profiling is that healthy tissues sample DNA template, PCR Forward Primer are that P1Fn, PCR Reverse Primer are P1Rn.
(2), Real Time PCR reaction:
Adopt the two-step approach PCR response procedures shown in Fig. 1; About the concrete reaction conditions of PCR please refer to TAKARAPremix Ex Taq
tM(Probe QPCR) (TIANGEN BIOTECH (BEIJING) CO., LTD) product description.
Two-step approach pcr amplification standard program (seeing Fig. 2):
The first step denaturation: 95 DEG C 30 seconds, repeat 1 time;
Second step PCR reaction: 95 DEG C of 5 seconds and 60 DEG C 30 seconds, repeat 40 times.
Five, the interpretation of detected result
The difference in the mutational site of detecting for needs, use healthy tissues and the urine DNA sample of two pairs of same cases of primer pair to carry out QPCR amplification, the △ Ct value obtaining according to PCR instrument (Thermal Cycler Dice Real Time System) judges whether to exist mutational site.
If △ Ctu is in urine sample, the difference of cycle number when mutant primer and normal primer reach amplification threshold value; △ Ctn is in healthy tissues sample, the difference of cycle number when mutant primer and normal primer reach amplification threshold value.
△ Ctu-△ Ctn<0 can judge and in urine sample, has mutational site.
If judging in urine sample, △ Ctu-△ Ctn >=0 item there is not mutational site.
The above, it is only preferred embodiment of the present invention, not the present invention is done to any formal and substantial restriction, all those skilled in the art, do not departing within the scope of technical solution of the present invention, when utilizing disclosed above technology contents, and the equivalent variations of a little change of making, modification and differentiation is equivalent embodiment of the present invention; Meanwhile, the change of any equivalent variations that all foundations essence technology of the present invention is done above embodiment, modification and differentiation, all still belong in the scope of technical scheme of the present invention.
Claims (4)
1. a method that detects TERT single nucleotide polymorphism, comprises the steps:
(1), from the urine of sample and healthy tissues, extract respectively DNA;
(2) difference in the mutational site of, detecting for needs, taking the DNA that extracts from sample urine and healthy tissues as template, respectively taking primer pair P1, the P2 corresponding with mutational site or P3 as primer, carry out TaqMan Probe Real time pcr amplification, wherein with 1, primer pair P1 corresponding to 295,228G>A mutational site is:
P1Fm:CGGGTCCCCGGCCCAGCCCCT，
P1Rm:GCGCCGCGAGGAGAGGGCG；
P1Fn:CGGGTCCCCGGCCCAGCCCCC，
P1Rn:GCGCCGCGAGGAGAGGGCG；
The primer pair P2 corresponding with 1,295,250G>A mutational site is:
P2Fm:CGCCCCGTCCCGACCCCTT，
P2Rm:CGCCGCGAGGAGAGGGCG；
P2Fn:CGCCCCGTCCCGACCCCTC，
P2Rn:CGCCGCGAGGAGAGGGCG；
With 1,295,242-1, primer pair P3 corresponding to 295,243GG>AA mutational site is:
P3Fm:CGTCCCGACCCCTCCCGGGTTT，
P3Rm:GCGCCGCGAGGAGAGGGCG；
P3Fn:CGTCCCGACCCCTCCCGGGTCC，
P3Rn:GCGCCGCGAGGAGAGGGCG；
(3), determine the size between △ Ctu and △ Ctn, when having mutational site in △ Ctu-△ Ctn<0 urine sample; In the urine sample of △ Ctu-△ Ctn >=0, there is not mutational site; Wherein △ Ctu is the difference of cycle number when mutant primer and normal primer reach amplification threshold value in urine sample; △ Ctn is the difference of cycle number when mutant primer and normal primer reach amplification threshold value in healthy tissues sample.
2. a kind of method that detects TERT single nucleotide polymorphism according to claim 1, is characterized in that, the reaction conditions of described TaqMan Probe Real time pcr amplification is:
The first step denaturation: 95 DEG C 30 seconds, repeat 1 time;
Second step PCR reaction: 95 DEG C of 5 seconds and 60 DEG C 30 seconds, repeat 40 times.
3. a kind of method that detects TERT single nucleotide polymorphism according to claim 1, it is characterized in that, in described TaqMan Probe Real time pcr amplification system, contain: 12.5 μ l2 × Premix Ex Taq, 0.5 μ l PCR Forward Primer (10 μ M), 0.5 μ l PCR Reverse Primer (10 μ M), 1 μ l
probe, 2 μ l DNA profilings and 8.5 μ l d H
2o (sterile purified water).
4. a kind of method that detects TERT single nucleotide polymorphism according to claim 1, is characterized in that, the reaction TaqMan Probe sequence of described TaqMan Probe Real time pcr amplification is:
5’-CTCCCAGCCCCTCCCCTTCCTTTCCGCGGC-3’。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410042839.1A CN103923976A (en) | 2014-01-28 | 2014-01-28 | Method for detecting single nueleotide polymorphism of TERT |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410042839.1A CN103923976A (en) | 2014-01-28 | 2014-01-28 | Method for detecting single nueleotide polymorphism of TERT |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN103923976A true CN103923976A (en) | 2014-07-16 |
Family
ID=51142371
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410042839.1A Pending CN103923976A (en) | 2014-01-28 | 2014-01-28 | Method for detecting single nueleotide polymorphism of TERT |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103923976A (en) |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104805206A (en) * | 2015-04-29 | 2015-07-29 | 苏州工业园区为真生物医药科技有限公司 | Kit for detecting TERT (telomerase reverse transcriptase) gene promoter mutation, and detection method of kit |
| CN105063234A (en) * | 2015-09-24 | 2015-11-18 | 河南省职业病防治研究院 | Human TERT gene rs2853669-locus polymorphism investigation technology |
| CN105087817A (en) * | 2015-09-24 | 2015-11-25 | 郑州大学 | Method for detecting polymorphism of site rs2853669 of TERT human gene and kit |
| CN105200134A (en) * | 2015-09-18 | 2015-12-30 | 杭州泛生子医学检验所有限公司 | Method and reagent kit for detecting mutation of human TERT gene promoter |
| CN105331699A (en) * | 2015-11-05 | 2016-02-17 | 北京泛生子基因科技有限公司 | Probe method detecting human TERT gene promoter mutation and reagent kit thereof |
| WO2016032947A1 (en) | 2014-08-25 | 2016-03-03 | Duke University | Methods for rapid and sensitive detection of hotspot mutations |
| CN110240999A (en) * | 2018-03-09 | 2019-09-17 | 林云富 | A kind of detection device and method improving Circulating tumor DNA recall rate |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101235415A (en) * | 2007-01-30 | 2008-08-06 | 中山大学达安基因股份有限公司 | Method for detecting gene simple point mutation by using TaqMan probe quantitative polymerase chain reaction technique |
-
2014
- 2014-01-28 CN CN201410042839.1A patent/CN103923976A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101235415A (en) * | 2007-01-30 | 2008-08-06 | 中山大学达安基因股份有限公司 | Method for detecting gene simple point mutation by using TaqMan probe quantitative polymerase chain reaction technique |
Non-Patent Citations (3)
| Title |
|---|
| CAROLYN D.HURST ET AL: "Comprehensive Mutation Analysis of the TERT Promoter in Bladder Cancer and Detecion of Mutations in Voided Urine", 《EUROPEAN UROLOGY》, vol. 65, 3 September 2013 (2013-09-03), pages 367 - 369 * |
| XIAOLI LIU ET AL: "Highly prevalent TERT promoter mutations in bladder cancer and glioblastoma", 《CELL CYCLE》, vol. 12, no. 10, 15 May 2013 (2013-05-15), pages 1637 - 1638, XP008168404, DOI: 10.4161/cc.24662 * |
| YVES ALLORY ET AL: "Telomerase Reverse Transvriptase Promoter Mutations in Bladder Cancer：High Frequency Across Stages，Detection in Urine，and Lack of Association with Outcome", 《EUROPEAN UROLOGY》, vol. 65, 7 September 2013 (2013-09-07), pages 360 - 366 * |
Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2016032947A1 (en) | 2014-08-25 | 2016-03-03 | Duke University | Methods for rapid and sensitive detection of hotspot mutations |
| JP2017525375A (en) * | 2014-08-25 | 2017-09-07 | デューク ユニバーシティー | A method for rapid and sensitive detection of hot spot mutations |
| EP3186399A4 (en) * | 2014-08-25 | 2018-07-18 | Duke University | Methods for rapid and sensitive detection of hotspot mutations |
| US10633711B2 (en) | 2014-08-25 | 2020-04-28 | Duke University | Methods for rapid and sensitive detection of hotspot mutations |
| CN104805206A (en) * | 2015-04-29 | 2015-07-29 | 苏州工业园区为真生物医药科技有限公司 | Kit for detecting TERT (telomerase reverse transcriptase) gene promoter mutation, and detection method of kit |
| CN104805206B (en) * | 2015-04-29 | 2017-06-20 | 苏州工业园区为真生物医药科技有限公司 | The kit and its detection method of detection TERT gene promoter mutation |
| CN105200134A (en) * | 2015-09-18 | 2015-12-30 | 杭州泛生子医学检验所有限公司 | Method and reagent kit for detecting mutation of human TERT gene promoter |
| CN105063234A (en) * | 2015-09-24 | 2015-11-18 | 河南省职业病防治研究院 | Human TERT gene rs2853669-locus polymorphism investigation technology |
| CN105087817A (en) * | 2015-09-24 | 2015-11-25 | 郑州大学 | Method for detecting polymorphism of site rs2853669 of TERT human gene and kit |
| CN105331699A (en) * | 2015-11-05 | 2016-02-17 | 北京泛生子基因科技有限公司 | Probe method detecting human TERT gene promoter mutation and reagent kit thereof |
| CN110240999A (en) * | 2018-03-09 | 2019-09-17 | 林云富 | A kind of detection device and method improving Circulating tumor DNA recall rate |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103923976A (en) | Method for detecting single nueleotide polymorphism of TERT | |
| CN104673896B (en) | SDC2 for detecting colorectal cancer methylates | |
| CN114736968B (en) | Application of plasma free DNA methylation marker in lung cancer early screening and lung cancer early screening device | |
| JP5750710B2 (en) | Cancer marker, cancer evaluation method and evaluation reagent using the same | |
| EP2891720B1 (en) | Method for screening cancer | |
| Crocitto et al. | Prostate cancer molecular markers GSTP1 and hTERT in expressed prostatic secretions as predictors of biopsy results | |
| KR102605749B1 (en) | Circulating rna signatures specific to preeclamsia | |
| BR122020016370B1 (en) | methods for predicting a breast cancer outcome in an estrogen receptor-positive and her2-negative breast tumor from a breast cancer patient | |
| CN104805206B (en) | The kit and its detection method of detection TERT gene promoter mutation | |
| Joo et al. | Variable promoter methylation contributes to differential expression of key genes in human placenta-derived venous and arterial endothelial cells | |
| CN110616229A (en) | Fusion gene of FGFR1 translocation blood disease and detection primer and application thereof | |
| CN105296659A (en) | Gene marker related to ischemic stroke | |
| CN114891886B (en) | Nucleic acid product, kit and application for diagnosing bladder cancer | |
| CN106868124A (en) | One group of methylated genes and its detection method | |
| Feng et al. | TET1-mediated different transcriptional regulation in prostate cancer | |
| Cao et al. | Genetic alterations in cfDNA of benign and malignant thyroid nodules based on amplicon-based next-generation sequencing | |
| Niu et al. | Promoter methylation of protocadherin8 is an independent prognostic factor for biochemical recurrence of early-stage prostate cancer | |
| CN108624693A (en) | Applications of the miR-577 in preparing diagnosis of nephropathy marker | |
| CN107058579A (en) | Adenocarcinoma of lung related miRNA, composition and its application | |
| JP2011211955A (en) | Composition and method for prognostic prediction of hepatic cancer patient | |
| Li et al. | nm23 gene polymorphisms are associated with survival of patients with epithelial ovarian cancer but not with susceptibility to disease | |
| Manderson et al. | Molecular genetic analysis of a cell adhesion molecule with homology to L1CAM, contactin 6, and contactin 4 candidate chromosome 3p26pter tumor suppressor genes in ovarian cancer | |
| CN109825595A (en) | LncRNA marker relevant to breast cancer and its detection primer and application | |
| CN105567797A (en) | PCR primer, method and kit for detecting CpG island methylation of DACT2 gene promoter area | |
| CN109913481B (en) | PIK3CA gene g.179224821G & gtA mutation and application thereof in breast cancer auxiliary diagnosis |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C12 | Rejection of a patent application after its publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20140716 |