CN108060229A - A kind of TERT genes rs10069690 sites SNP nucleic acid Mass Spectrometry detection methods - Google Patents
A kind of TERT genes rs10069690 sites SNP nucleic acid Mass Spectrometry detection methods Download PDFInfo
- Publication number
- CN108060229A CN108060229A CN201711446671.0A CN201711446671A CN108060229A CN 108060229 A CN108060229 A CN 108060229A CN 201711446671 A CN201711446671 A CN 201711446671A CN 108060229 A CN108060229 A CN 108060229A
- Authority
- CN
- China
- Prior art keywords
- sample
- sites
- nucleic acid
- mass spectrometry
- detection methods
- 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
- C12Q1/6876—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
- 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
-
- 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
- C12Q2600/156—Polymorphic or mutational markers
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Immunology (AREA)
- Analytical Chemistry (AREA)
- Genetics & Genomics (AREA)
- Physics & Mathematics (AREA)
- Pathology (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- General Health & Medical Sciences (AREA)
- General Engineering & Computer Science (AREA)
- Biophysics (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Oncology (AREA)
- Hospice & Palliative Care (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The present invention provides a kind of TERT genes rs10069690 sites SNP nucleic acid Mass Spectrometry detection methods, the segment containing SNP site is amplified by first step PCR reactions first, and non-individual amplification SNP site, to improve the precision of the operability of sample and subsequent operation；Dephosphorylation process is carried out to the amplified production that step S1 is obtained by SAP, DNA molecular 5' ends is prevented to be connected with 3' ends, DNA molecular are allowed to keep linear condition before subsequent step is ready to, so as to ensure the accuracy of subsequent experimental；Single base extension is carried out to rs10069690 sites finally by step S3, makes the abundant uncoiling of DNA double chain, separation using cycle in denaturation annealing, so as to fulfill the accurately typing of different SNP.By the above method, the SNP parting information in the rs10069690 sites of TERT genes can be quickly and accurately obtained, it is easy to operate, highly reliable, reliable reference information can be provided for subsequent correlative study.
Description
Technical field
The invention belongs to SNP detection technique fields, more particularly to a kind of TERT genes rs10069690 sites SNP nucleic acid matter
Spectrum detection method.
Background technology
Single nucleotide polymorphism (single nucleotide polymorphism, SNP), is primarily referred to as in genome water
Flat DNA sequence polymorphism caused by a single nucleotide variation.It is most common one in human heritable mutation
Kind.Account for more than the 90% of all known polymorphisms.SNP is widely present in human genome, average every 500~1000 bases
Centering just has 1, estimates that its sum is even more up to 3,000,000.
TERT genes are located on No. 5 chromosomes, for instructing the synthesis of reverse transcriptase of telomere (TRET).Telomere
(telomere) it is a kind of special construction of end of chromosome, on the one hand can reduces end shortening, on the other hand can also avoid adjacent
End of chromosome merges, and key effect is played in the processes such as cell Proliferation and aging；Telomerase is responsible for telomerase and repeats sequence
Column-generation avoids telomere from shortening.Reverse transcriptase of telomere is the catalytic subunit of Telomerase, can effectively keep telomere structural integrity
Property, it is the key factor for determining telomere length.Research shows that one of tumorigenic feature is the expression quantity increase of Telomerase,
And the expression of reverse transcriptase of telomere and the expression of Telomerase are in high-positive correlation relation.At present studies have pointed out that melanin
The diseases such as knurl, cutaneum carcinoma, oophoroma and carcinoma of urinary bladder have positive correlation with the expression of reverse transcriptase of telomere.
More SNP sites in existing on TERT genes, the different expression-forms of each SNP site may produce above-mentioned illness
Raw different influence.Therefore it provides a kind of efficient, accurate SNP detection method, it will to study shadows of the TERT to above-mentioned disease
The mechanism of sound provides important reference information and theories integration.
The content of the invention
In order to solve the above technical problem, the present invention provides a kind of TERT genes rs10069690 sites SNP nucleic acid matter
Spectrum detection method.
Specific technical solution of the present invention is as follows：
One aspect of the present invention provides a kind of TERT genes rs10069690 sites SNP nucleic acid Mass Spectrometer Method primer sets, bag
Include following primer：
Sense primer rs10069690-F：5`-ACGTTGGATGTCAGTTGCCTGGGCTTA TTG-3` are (such as
Shown in SEQ.ID.No.1)；
Anti-sense primer rs10069690-R：5`-ACGTTGGATGCTGAGCACTACCCATGA TAG-3` are (such as
Shown in SEQ.ID.No.2)；
Extension primer rs10069690-E：5`-GGAAGGGAGATTTTGACAG-3` (as shown in SEQ.ID.No.3)；
TERT gene rs10069690 sites wild-type base sequence is as shown in SEQ.ID.No.4.
Another aspect of the present invention provides a kind of rs10069690 sites SNP nucleic acid of the TERT genes of the application primer sets
Mass Spectrometry detection method includes the following steps：
S1：First round PCR is carried out with sense primer rs10069690-F and anti-sense primer rs10069690-R, to template
DNA is expanded, and obtains the segment containing the rs10069690 sites；
S2：Dephosphorylation process is carried out to the segment that step S1 is obtained, obtains dephosphorization acid fragment；
S3：Single base extension is carried out to the dephosphorization acid fragment with extension primer rs10069690-E, different SNP are carried out
Parting；
S4：Desalting processing is carried out to the sample that step S3 is obtained, is detected with mass spectrograph.
Further, in step S1, the reaction system of first round PCR includes following component：
Template DNA 2ng/ μ l, 0.05 μM of rs10069690-F primers, 0.05 μM of rs10069690-R primers, Mg2+4mM,
1 × PCR Buffer, dNTP 10 μM and Taq archaeal dna polymerases 1U.
Further, in step S1, the reaction condition of first round PCR is as follows：
(1) pre-degeneration：95℃2min；
(2) expand：95 DEG C of 30s, 56 DEG C of 30s, 72 DEG C of 60s, totally 45 cycle；
(3) extend：72 DEG C of 5min, 4 DEG C of ∞.
The purpose of step S1 is to amplify the segment containing SNP site, and non-individual amplification SNP site, to improve sample
Operability and subsequent operation precision；" 4 DEG C of ∞ " is to be maintained at 4 DEG C, to prevent ambient temperature acute variation pair
Amplified production impacts.
Further, in step S2, dephosphorylized reaction system further includes on the basis of first round pcr amplification product
Following ingredient：
0.24 × SAP Buffer and SAP enzymes 0.5U.
Further, in step S2, dephosphorylized reaction condition is as follows：
(1) dephosphorylation：37℃40min；
(2) SAP enzyme-deactivatings：85 DEG C of 5min, 4 DEG C of ∞.
The purpose of step S2 is to carry out dephosphorylation process to the amplified production that step S1 is obtained by SAP, prevents DNA points
Sub- 5' ends are connected with 3' ends, DNA molecular are allowed to keep linear condition before subsequent step is ready to, so as to ensure subsequent experimental
Accuracy.
Further, in step S3, the reaction system of Single base extension further included on the basis of dephosphorylation product as
Lower ingredient：
0.222 × iPLEX GOLD Buffer, 1 × iPLEX Termination mix, 0.15 μM of iPLEX extension primers
And 1 × iPLEX Enzyme.
Further, in step S3, the reaction condition of Single base extension is as follows：
(1) pre-degeneration：94℃30s；
(2) Xun Huan in amplification：94 DEG C of 5s, 52 DEG C of 5s, totally 5 cycle；
(3) outer circulation is expanded：94 DEG C of 5s, 52 DEG C of 5s, 80 DEG C of 5s, totally 40 cycle；
(4) extend：72 DEG C of 4min, 4 DEG C of ∞.
The purpose of step S3 is to carry out Single base extension to the sequence in rs10069690 sites, so as to fulfill different SNP's
Parting.
In the present solution, Xun Huan in also being set among the outer circulation of denaturation-annealing-extension to denaturation-annealing, so as to abundant
The double-strand of DNA is untwisted, is separated, so as to improve the accuracy of the single-stranded separation degrees of DNA and Single base extension
Further, the step S4 includes the following steps：
S4.1：It is for use that clean resin is completed on resin plate, per hole 6mg resins, is air-dried 10~20 minutes；
S4.2：Sample to be tested is added in into the sample sky of sample plane, each sample aperture adds 15~20 μ L ddH2O again, with envelope
Membrana oralis seals, and carries out brief centrifugation；
S4.3：The sealed membrane for covering the sample aperture is opened, the sample plane is tipped upside down on the resin plate and is fixed, it will
The sample plane and resin plate fixed is integrally overturn, and resin plate is touched, so that the resin in resin plate is fallen completely in sample plane
Respective sample hole, is sealed with sealed membrane, carries out brief centrifugation；
S4.4：Sample resin compound is shaken up, after rotating 15min at a slow speed on circulator, under the conditions of 4000rpm from
Heart 5min, machine on point sample after centrifugation are detected sample with mass spectrograph.
The purpose of step S4 is to remove the salt dissolved in sample, prevents salt from being polluted on mass spectrometer system, influences letter
Number generation, so as to ensure the reliability of measurement result.
Beneficial effects of the present invention are as follows：The present invention provides a kind of TERT genes rs10069690 sites SNP nucleic acid matter
Spectrum detection method amplifies the segment containing SNP site by first step PCR reactions first, and non-individual expands SNP site,
To improve the precision of the operability of sample and subsequent operation；Dephosphorization is carried out to the amplified production that step S1 is obtained by SAP
Acidification, prevents DNA molecular 5' ends to be connected with 3' ends, and DNA molecular is allowed to keep linear condition before subsequent step is ready to,
So as to ensure the accuracy of subsequent experimental；Single base extension is carried out to rs10069690 sites finally by step S3, utilizes change
Property-annealing in cycle make the abundant uncoiling of DNA double chain, separation, so as to fulfill the accurately typing of different SNP.By the above method,
The SNP parting information in the rs10069690 sites of TERT genes can be quickly and accurately obtained, it is easy to operate, highly reliable, it can
To provide reliable reference information for subsequent correlative study.
Description of the drawings
Fig. 1 is that the rs10069690 sites SNP genotyping results of distinct methods in experimental example 1 compare；
Fig. 2 is the rs10069690 sites SNP parting schematic diagrames of all samples in experimental example 2；
Fig. 3 is the mass spectrogram of the CT heterozygous in sample rs10069690 sites in experimental example 2；
Fig. 4 is the homozygous mass spectrograms of the TT in sample rs10069690 sites in experimental example 2；
Fig. 5 is the homozygous mass spectrograms of the CC in sample rs10069690 sites in experimental example 2.
Specific embodiment
Embodiment
A kind of TERT genes rs10069690 sites SNP nucleic acid Mass Spectrometry detection methods, include the following steps：
S1：First round PCR is carried out with sense primer rs10069690-F and anti-sense primer rs10069690-R, to template
DNA is expanded, and the base sequence of primer is as follows：
Sense primer rs10069690-F：5`-ACGTTGGATGTCAGTTGCCTGGGCTTA TTG-3`；
Anti-sense primer rs10069690-R：5`-ACGTTGGATGCTGAGCACTACCCATGA TAG-3`；
Reaction system is as follows：
Reaction condition is as follows：
(1) pre-degeneration：95℃2min；
(2) expand：95 DEG C of 30s, 56 DEG C of 30s, 72 DEG C of 60s, totally 45 cycle；
(3) extend：72 DEG C of 5min, 4 DEG C of ∞；
S2：Dephosphorylation process is carried out to the amplified production that the step S1 is obtained, obtains dephosphorization acid fragment, dephosphorylation
The SAP mixture systems operated with are as follows：
| Concentration | Addition (μ L) | |
| Nanopure Water,Autoclaved | N/A | 1.53 |
| SAP Buffer | 0.24× | 0.17 |
| SAP Enzyme(1.7U/μL) | 0.5U | 0.30 |
| Total volume [μ L] | 2/rnx |
Above-mentioned SAP mixed liquors are added in the amplified production that step S1 is obtained, and are gone according to following reaction condition
Phosphatizing treatment：
(1) dephosphorylation：37℃40min；
(2) SAP enzyme-deactivatings：85 DEG C of 5min, 4 DEG C of ∞；
S3：Single base extension is carried out to the dephosphorization acid fragment with extension primer rs10069690-E, different SNP are carried out
Parting, the base sequence of extension primer are as follows：
rs10069690-E：5`-GGAAGGGAGATTTTGACAG-3`；
Reaction system is as follows：
| Concentration | Addition (μ L) | |
| Nanopure water | N/A | 0.619 |
| iPLEX GOLD Buffer | 0.222× | 0.200 |
| iPLEX Termination mix | 1× | 0.200 |
| IPLEX extension primers | 0.11 | 0.940 |
| iPLEX Enzyme | 1× | 0.041 |
| Volume(μL) | 2.000 | |
| SAP+PCR reaction | 7.000 | |
| Total volume (μ L) | 9.000 |
Reaction condition is as follows：
(1) pre-degeneration：94℃30s；
(2) Xun Huan in amplification：94 DEG C of 5s, 52 DEG C of 5s, totally 5 cycle；
(3) outer circulation is expanded：94 DEG C of 5s, 52 DEG C of 5s, 80 DEG C of 5s, totally 40 cycle；
(4) extend：72 DEG C of 4min, 4 DEG C of ∞；
S4：Desalting processing is carried out to the sample that the step S3 is obtained, is detected with mass spectrograph, specifically includes following step
Suddenly：
S4.1：It is for use that clean resin is completed on resin plate, per hole 6mg resins, is air-dried 10~20 minutes；
S4.2：Sample to be tested is added in into the sample sky of sample plane, each sample aperture adds 15~20 μ L ddH2O again, with envelope
Membrana oralis seals, and carries out brief centrifugation；
S4.3：The sealed membrane for covering the sample aperture is opened, the sample plane is tipped upside down on the resin plate and is fixed, it will
The sample plane and resin plate fixed is integrally overturn, and resin plate is touched, so that the resin in resin plate is fallen completely in sample plane
Respective sample hole, is sealed with sealed membrane, carries out brief centrifugation；
S4.4：Sample resin compound is shaken up, after rotating 15min at a slow speed on circulator, under the conditions of 4000rpm from
Heart 5min, machine on point sample after centrifugation are detected sample with mass spectrograph.
Reference examples
A kind of reaction item of TERT genes rs10069690 sites SNP nucleic acid Mass Spectrometry detection methods, wherein single base amplification
Part is as follows：
(1) pre-degeneration：94℃30s；
(2) Xun Huan in amplification：52 DEG C of 5s, 80 DEG C of 5s, totally 5 cycle；
(3) outer circulation is expanded：94 DEG C of 5s, 52 DEG C of 5s, 80 DEG C of 5s, totally 40 cycle；
(4) extend：72 DEG C of 4min, 4 DEG C of ∞；
Remaining step and reaction system are identical with experimental example.
Experimental example 1
Using method provided by the invention as experimental group, the method as a control group 1 that is provided with reference examples, with conventional
Taqman methods as a control group 2, it is (miscellaneous including 5ng CT to the TERT gene rs10069690 sites standard items of 15ng respectively
Close, 5ng CC are homozygous and 5ng TT are homozygous) it is detected, and the parting quantitative result of each group experiment is compared.
As shown in Figure 1, experimental group and the genotyping result of control group 1 are better than control group 2, and the quantitative result of experimental group
Higher than control group 1, it is more nearly the contents of standard items.Show method provided by the invention to TERT gene rs10069690 sites
The detection of SNP partings is more accurate than existing methods, effect is more preferable, while illustrate that this method is set during Single base extension
Denaturation-annealing in cycle, there is better SNP separation and extension effect than cycle in conventional annealing-extension.
Experimental example 2
26 healthy adult (18~60 one full year of life) volunteers are randomly selected, gather blood sample respectively and extract whole blood gene
Group carries out 26 samples rs10069690 site SNP detections according to the method provided in embodiment, and to above-mentioned multiple samples
Mass Spectrometer Method result analyzed.
Experimental result as shown in Figure 2-5, shares 22 samples and has obtained analyzable as a result, wherein 15 samples are CT
Heterozygosis, 5 samples are homozygous for TT, and 2 samples are homozygous for CC.Show that the heterozygote of C-T in the SNP site is more, the homozygosis of TT
Son and CC homozygotes also have detection.The sample spot of different genotype is separated from each other in Fig. 2, nothing is intersecting mutually, while Fig. 3~5
It the peak type of middle difference parting and separates all right, shows that primer specificity provided by the invention is good, high sensitivity, parting knot
Fruit is accurate.
Embodiment described above only expresses the several embodiments of the present invention, and description is more specific and detailed, but simultaneously
Cannot the limitation to the scope of the claims of the present invention therefore be interpreted as.It should be pointed out that for those of ordinary skill in the art
For, without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to the guarantor of the present invention
Protect scope.Therefore, the protection domain of patent of the present invention should be determined by the appended claims.
Sequence table
<110>Watson Crick（Beijing）Bio tech ltd
<120>A kind of TERT genes rs10069690 sites SNP nucleic acid Mass Spectrometry detection methods
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 30
<212> DNA
<213>Artificial synthesized ()
<400> 1
acgttggatg tcagttgcct gggcttattg 30
<210> 2
<211> 30
<212> DNA
<213>Artificial synthesized ()
<400> 2
acgttggatg ctgagcacta cccatgatag 30
<210> 3
<211> 19
<212> DNA
<213>Artificial synthesized ()
<400> 3
ggaagggaga ttttgacag 19
<210> 4
<211> 201
<212> DNA
<213> Homo sapiens
<400> 4
ccacccagac ccgggaccta gaacccctcc cagcttcctc agaccctgtt tgaaacgggt 60
tcctggccgc atgtgtgttg cacacgggat cctcatgcca cacctctgtc cacctcaccc 120
cacactctcc tcagatgacg gggtcaccgc agccaccgca gccacagggg tggggtgcag 180
gagccgtggg gcaaggtcca g 201
Claims (9)
1. the rs10069690 sites SNP nucleic acid Mass Spectrometer Method primer sets of a kind of TERT genes, which is characterized in that including drawing as follows
Object：
Sense primer rs10069690-F：5`-ACGTTGGATGTCAGTTGCCTGGGCTTATTG-3`；
Anti-sense primer rs10069690-R：5`-ACGTTGGATGCTGAGCACTACCCATGATAG-3`；
Extension primer rs10069690-E：5`-GGAAGGGAGATTTTGACAG-3`.
2. a kind of TERT gene rs10069690 sites SNP nucleic acid Mass Spectrometry detection methods using primer sets described in claim 1,
It is characterised in that it includes following steps：
S1：Carry out first round PCR with sense primer rs10069690-F and anti-sense primer rs10069690-R, to template DNA into
Row amplification, obtains the segment containing the rs10069690 sites；
S2：Dephosphorylation process is carried out to the segment that step S1 is obtained, obtains dephosphorization acid fragment；
S3：Single base extension is carried out to the dephosphorization acid fragment with extension primer rs10069690-E, SNP base sequences are carried out
Amplification, so as to fulfill the parting of different SNP；
S4：Desalting processing is carried out to the sample that step S3 is obtained, is detected with mass spectrograph.
3. TERT genes rs10069690 sites SNP nucleic acid Mass Spectrometry detection methods as claimed in claim 2, which is characterized in that
In step S1, the reaction system of first round PCR includes following component：
Template DNA 2ng/ μ l, 0.05 μM of rs10069690-F primers, 0.05 μM of rs10069690-R primers, Mg2+4mM、1×
PCR Buffer, dNTP 10 μM and Taq archaeal dna polymerases 1U.
4. TERT genes rs10069690 sites SNP nucleic acid Mass Spectrometry detection methods as claimed in claim 3, which is characterized in that
In step S1, the reaction condition of first round PCR is as follows：
(1) pre-degeneration：95℃ 2min；
(2) expand：95 DEG C of 30s, 56 DEG C of 30s, 72 DEG C of 60s, totally 45 cycle；
(3) extend：72 DEG C of 5min, 4 DEG C of ∞.
5. TERT genes rs10069690 sites SNP nucleic acid Mass Spectrometry detection methods as claimed in claim 2, which is characterized in that
In step S2, dephosphorylized reaction system further includes following ingredient on the basis of first round pcr amplification product：
0.24 × SAP Buffer and SAP enzymes 0.5U.
6. TERT genes rs10069690 sites SNP nucleic acid Mass Spectrometry detection methods as claimed in claim 5, which is characterized in that
In step S2, dephosphorylized reaction condition is as follows：
(1) dephosphorylation：37℃ 40min；
(2) SAP enzyme-deactivatings：85 DEG C of 5min, 4 DEG C of ∞.
7. TERT genes rs10069690 sites SNP nucleic acid Mass Spectrometry detection methods as claimed in claim 2, which is characterized in that
In step S3, the reaction system of Single base extension further includes following ingredient on the basis of dephosphorylation product：
0.222 × iPLEX GOLD Buffer, 1 × iPLEX Termination mix, iPLEX extension primers mixture 0.84
~1.57 μM and 1 × iPLEX Enzyme.
8. TERT genes rs10069690 sites SNP nucleic acid Mass Spectrometry detection methods as claimed in claim 7, which is characterized in that
In step S3, the reaction condition of Single base extension is as follows：
(1) pre-degeneration：94℃ 30s；
(2) Xun Huan in amplification：94 DEG C of 5s, 52 DEG C of 5s, totally 5 cycle；
(3) outer circulation is expanded：94 DEG C of 5s, 52 DEG C of 5s, 80 DEG C of 5s, totally 40 cycle；
(4) extend：72 DEG C of 4min, 4 DEG C of ∞.
9. the rs10069690 sites SNP nucleic acid Mass Spectrometry detection methods of TERT genes as claimed in claim 2, feature exist
In the step S4 includes the following steps：
S4.1：It is for use that clean resin is completed on resin plate, per hole 6mg resins, is air-dried 10~20 minutes；
S4.2：Sample to be tested is added in into the sample sky of sample plane, each sample aperture adds 15~20 μ L ddH again2O uses sealed membrane
Sealing carries out brief centrifugation；
S4.3：The sealed membrane for covering the sample aperture is opened, the sample plane is tipped upside down on the resin plate and is fixed, by fixation
Good sample plane and resin plate is integrally overturn, and touches resin plate, so that the resin in resin plate is fallen completely in sample plane accordingly
Sample aperture is sealed with sealed membrane, carries out brief centrifugation；
S4.4：Sample resin compound is shaken up, after rotating 15min at a slow speed on circulator, is centrifuged under the conditions of 4000rpm
5min, machine on point sample after centrifugation are detected sample with mass spectrograph.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711446671.0A CN108060229A (en) | 2017-12-27 | 2017-12-27 | A kind of TERT genes rs10069690 sites SNP nucleic acid Mass Spectrometry detection methods |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711446671.0A CN108060229A (en) | 2017-12-27 | 2017-12-27 | A kind of TERT genes rs10069690 sites SNP nucleic acid Mass Spectrometry detection methods |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN108060229A true CN108060229A (en) | 2018-05-22 |
Family
ID=62140419
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201711446671.0A Pending CN108060229A (en) | 2017-12-27 | 2017-12-27 | A kind of TERT genes rs10069690 sites SNP nucleic acid Mass Spectrometry detection methods |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108060229A (en) |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101200729A (en) * | 2007-11-09 | 2008-06-18 | 南开大学 | Tumor susceptibility related mononucleotide polymorphism site and uses thereof |
| US20140038181A1 (en) * | 2011-01-05 | 2014-02-06 | Trilink Biotechnologies | Chemically substituted thermosensitive probes and cofactors for hot start ligation |
| CN103757104A (en) * | 2012-12-28 | 2014-04-30 | 中山大学肿瘤防治中心 | Kit and gene chip for predicting nasopharynx cancer onset risk |
| CN107523646A (en) * | 2017-10-20 | 2017-12-29 | 武汉赛云博生物科技有限公司 | Method based on nucleic acid mass-spectrometric technique detection early diagnosing mammary cancer related gene |
| US10683549B2 (en) * | 2014-09-30 | 2020-06-16 | Genetic Technologies Limited | Methods for assessing risk of developing breast cancer |
-
2017
- 2017-12-27 CN CN201711446671.0A patent/CN108060229A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101200729A (en) * | 2007-11-09 | 2008-06-18 | 南开大学 | Tumor susceptibility related mononucleotide polymorphism site and uses thereof |
| US20140038181A1 (en) * | 2011-01-05 | 2014-02-06 | Trilink Biotechnologies | Chemically substituted thermosensitive probes and cofactors for hot start ligation |
| CN103757104A (en) * | 2012-12-28 | 2014-04-30 | 中山大学肿瘤防治中心 | Kit and gene chip for predicting nasopharynx cancer onset risk |
| US10683549B2 (en) * | 2014-09-30 | 2020-06-16 | Genetic Technologies Limited | Methods for assessing risk of developing breast cancer |
| CN107523646A (en) * | 2017-10-20 | 2017-12-29 | 武汉赛云博生物科技有限公司 | Method based on nucleic acid mass-spectrometric technique detection early diagnosing mammary cancer related gene |
Non-Patent Citations (2)
| Title |
|---|
| STIG E BOJESEN等: "Multiple independent variants at the TERT locus are associated with telomere length and risks of breast and ovarian cancer", 《NATURE GENETICS》 * |
| 高雷等: "TERT基因rs10069690多态性与中国汉族人肺癌易感性研究", 《中华医学会呼吸病学年会——2013第十四次全国呼吸病学学术会议论文汇编》 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN111020031A (en) | Method for detecting tumor gene mutation by combining sequence specific blocker with specific PCR (polymerase chain reaction) program | |
| WO2016165591A1 (en) | Mgmt gene promoter methylation detection based on pyrosequencing technology | |
| CN108103160A (en) | A kind of XPC genes rs2228001 sites SNP nucleic acid Mass Spectrometry detection methods | |
| CN105886606A (en) | Kit for rapid detection of polymorphism of Warfarin metabolic enzyme gene by virtue of pyrosequencing method and application of kit | |
| CN108018335A (en) | A kind of KIF1B genes rs17401966 sites SNP nucleic acid Mass Spectrometry detection methods | |
| EP2721171A1 (en) | Discrimination of blood type variants | |
| US7914996B2 (en) | Polynucleotide associated with a colon cancer comprising single nucleotide polymorphism, microarray and diagnostic kit comprising the same and method for diagnosing a colon cancer using the polynucleotide | |
| CN111041079A (en) | Flight mass spectrum genotyping detection method | |
| KR101138862B1 (en) | A polynucleotide associated with a breast cancer comprising single nucleotide polymorphism, microarray and diagnostic kit comprising the same and method for diagnosing a breast cancer using the same | |
| CN107058486B (en) | Primer group and kit for detecting glycolytic related genotyping of nasopharyngeal carcinoma | |
| CN102199668B (en) | Nano-particle molecule haplotype typing technique | |
| CN108060229A (en) | A kind of TERT genes rs10069690 sites SNP nucleic acid Mass Spectrometry detection methods | |
| US11248261B2 (en) | RhD gene allele associated with a weak D phenotype and its uses | |
| CN108410966B (en) | Method for detecting insertion and deletion of ACE gene | |
| CN108070660A (en) | A kind of rs13181 sites SNP nucleic acid Mass Spectrometry detection methods of ERCC2 genes | |
| CN108103161A (en) | A kind of PLCE1 genes rs2274223 sites SNP nucleic acid Mass Spectrometry detection methods | |
| US11124840B2 (en) | Methods and compositions for classifying DLBCL | |
| CN104928349B (en) | A kind of method for detecting single nucleotide polymorphism based on nanogold particle | |
| FR2914316A1 (en) | METHODS AND TOOLS FOR DETERMINING THE ORIGIN OF A SUBJECT. | |
| CN108949964A (en) | For detecting the primed probe group and its application of rs12041331 | |
| KR102575912B1 (en) | Single nucleotide polymorphism marker for discriminating cabbage having low content of sinigrin and uses thereof | |
| KR100647304B1 (en) | 2 A polynucleotide associated with a type II diabetes mellitus comprising single nucleotide polymorphism microarray and diagnostic kit comprising the same and method for analyzing polynucleotide using the same | |
| KR102461769B1 (en) | Novel marker for selecting a lemon variety and the use thereof | |
| CN117230184B (en) | Nucleic acid combination for detecting Alzheimer disease gene based on time-of-flight nucleic acid mass spectrometry technology and application | |
| RU2580005C1 (en) | Test system for detection of expression of nat2 genes of human being |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20180522 |