CN108070660A - A kind of rs13181 sites SNP nucleic acid Mass Spectrometry detection methods of ERCC2 genes - Google Patents
A kind of rs13181 sites SNP nucleic acid Mass Spectrometry detection methods of ERCC2 genes Download PDFInfo
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Abstract
The present invention provides a kind of ERCC2 genes rs13181 sites SNP nucleic acid Mass Spectrometry detection methods, amplify 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；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 rs13181 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 ERCC2 gene rs13181 sites 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, the rs13181 sites SNP nucleic acid matter of more particularly to a kind of ERCC2 genes
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.
ERCC2 genes are also known as DNA Excision Repair Genes, are nucleotide excisions at No. 19 chromosomes 13.2~13.3
Important gene in repair pathways.ERCC2 genes have multiple functions, on the one hand participate in Nucleotide Sequence Analysis approach, and
Platinum-DNA adduct can be taken out during platinum-based chemotherapy is used；On the other hand the albumen of its coding also participates in composition II
Transcription factor complex and the apoptotic response of p53 mediations, while also participate in basal transcription.
More SNP sites in existing on ERCC2 genes, the different expression-forms of each SNP site may be to above-mentioned illness
Generate different influences.Wherein the wild type in rs13181 sites is TT, while also there are the saltant types of two kinds of forms of GT and GG.
At present some researches show that, both saltant types can increase the risk of cutaneous melanoma, at the same GG types be also possible to
There is correlation in oophoroma.Therefore it provides a kind of efficient, accurate SNP detection method, it will for ERCC2 pairs of research
The influencing mechanism of above-mentioned disease 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 rs13181 sites SNP nucleic acid matter of ERCC2 genes
Spectrum detection method.
Specific technical solution of the present invention is as follows：
One aspect of the present invention provides a kind of rs13181 sites SNP nucleic acid Mass Spectrometer Method primer sets of ERCC2 genes, bag
Include following primer：
Sense primer rs13181-F：5`-ACGTTGGATGTAAGACCTTCTAGCACC ACC-3` (such as SEQ.ID.No.1
It is shown)；
Anti-sense primer rs13181-R：5`-ACGTTGGATGAGCAGCTAGAATCAGAG GAG-3` (such as SEQ.ID.No.2
It is shown)；
Extension primer rs13181-E：5`-CAATCTGCTCTATCCTCT-3` (as shown in SEQ.ID.No.3)；
The rs13181 sites wild-type base sequence of ERCC2 genes is as shown in SEQ.ID.No.4.
Another aspect of the present invention provides a kind of ERCC2 gene rs13181 sites SNP nucleic acid mass spectrums of application primer sets
Detection method includes the following steps：
S1：First round PCR is carried out with sense primer rs13181-F and anti-sense primer rs13181-R, template DNA is carried out
Amplification, obtains the segment containing the rs13181 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 rs13181-E, different SNP are divided
Type；
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 rs13181-F primers, 0.05 μM of rs13181-R primers, Mg2+4mM, 1 × PCR
10 μM of Buffer, dNTP 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 rs13181 sites, so as to fulfill point of different SNP
Type.
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 ERCC2 genes rs13181 sites SNP nucleic acid mass spectrums
Detection method amplifies the segment containing SNP site by first step PCR reactions first, and non-individual expands SNP site, with
Improve the operability of sample and the precision of subsequent operation；Phosphoric acid is carried out to the amplified production that step S1 is obtained by SAP
Change is handled, and DNA molecular 5' ends is prevented to be connected with 3' ends, and DNA molecular is allowed to keep linear condition before subsequent step is ready to, from
And ensure the accuracy of subsequent experimental；Single base extension is carried out to rs13181 sites finally by step S3, using being denatured-move back
Xun Huan makes the abundant uncoiling of DNA double chain, separation in fiery, so as to fulfill the accurately typing of different SNP.It, can be with by the above method
The SNP parting information in ERCC2 gene rs13181 sites is quickly and accurately obtained, it is easy to operate, highly reliable, can be follow-up
Correlative study reliable reference information is provided.
Description of the drawings
Fig. 1 is that the rs13181 sites SNP genotyping results of distinct methods in experimental example 1 compare；
Fig. 2 is the rs13181 sites SNP parting schematic diagrames of all samples in experimental example 2；
Fig. 3 is the homozygous mass spectrograms of the TT in sample rs13181 sites in experimental example 2；
Fig. 4 is the mass spectrogram of the GT heterozygous in sample rs13181 sites in experimental example 2；
Fig. 5 is the homozygous mass spectrograms of the GG in sample rs13181 sites in experimental example 2.
Specific embodiment
Embodiment
A kind of rs13181 sites SNP nucleic acid Mass Spectrometry detection methods of ERCC2 genes, include the following steps：
S1：First round PCR is carried out with sense primer rs13181-F and anti-sense primer rs13181-R, template DNA is carried out
Amplification, the base sequence of primer are as follows：
Sense primer rs13181-F：5`-ACGTTGGATGTAAGACCTTCTAGCACC ACC-3`；
Anti-sense primer rs13181-R：5`-ACGTTGGATGAGCAGCTAGAATCAGAG GAG-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 rs13181-E, different SNP are divided
Type, the base sequence of extension primer are as follows：
rs13181-E：5`-CAATCTGCTCTATCCTCT-3`；
Reaction system is as follows：
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 condition of ERCC2 genes rs13181 sites SNP nucleic acid Mass Spectrometry detection methods, wherein single base amplification
It 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 GT to the rs13181 sites standard items of the ERCC2 genes of 15ng respectively
Close, 5ng GG 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 rs13181 site of the method provided by the invention to ERCC2 genes
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
24 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 24 samples rs13181 site SNP detections according to the method provided in embodiment, and to above-mentioned multiple samples
Mass Spectrometer Method result is analyzed.
As shown in Figure 2-5, all samples have obtained analyzable as a result, wherein 19 samples are TT homozygosis, 3 samples
Product are homozygous for GG, and 2 samples are homozygous for GT.Show that the homozygote of TT in the SNP site is more, the homozygote and G-T of GG is miscellaneous
Zygote also has detection.The sample spot of different genotype is separated from each other in Fig. 2, nothing is intersecting mutually, while different partings in Fig. 3~5
Peak type and separate all right, show that primer specificity provided by the invention is good, high sensitivity, genotyping result 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 rs13181 sites SNP nucleic acid Mass Spectrometry detection methods of ERCC2 genes
<160> 4
<170> SIPOSequenceListing 1.0
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<211> 30
<212> DNA
<213>Artificial synthesized ()
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acgttggatg taagaccttc tagcaccacc 30
<210> 2
<211> 30
<212> DNA
<213>Artificial synthesized ()
<400> 2
acgttggatg agcagctaga atcagaggag 30
<210> 3
<211> 18
<212> DNA
<213>Artificial synthesized ()
<400> 3
caatctgctc tatcctct 18
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<211> 201
<212> DNA
<213> Homo sapiens
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cgctgggaac cagggccagg caagactcag gagtcaccag gaaccgttta tggccccacc 60
cgccccactc agagctgctg agcaatctgc tctatcctct tcagcgtctc ctctgattct 120
agctgctcca ggctgagcag ggacaggccc agctgatcct cctgcagaga acagaggaaa 180
gggagagggg ggcactgttg g 201
Claims (9)
1. a kind of ERCC2 genes rs13181 sites SNP nucleic acid Mass Spectrometer Method primer sets, which is characterized in that including following primer：
Sense primer rs13181-F：5`-ACGTTGGATGTAAGACCTTCTAGCACCACC-3`；
Anti-sense primer rs13181-R：5`-ACGTTGGATGAGCAGCTAGAATCAGAGGAG-3`；
Extension primer rs13181-E：5`-CAATCTGCTCTATCCTCT-3`.
2. a kind of ERCC2 gene rs13181 sites SNP nucleic acid Mass Spectrometry detection methods using primer sets described in claim 1,
It is characterized in that, includes the following steps：
S1：First round PCR is carried out with sense primer rs13181-F and anti-sense primer rs13181-R, template DNA is expanded,
Obtain the segment containing the rs13181 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 rs13181-E, SNP base sequences are expanded
Increase, 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. ERCC2 genes rs13181 sites SNP nucleic acid Mass Spectrometry detection methods as claimed in claim 2, which is characterized in that step
In rapid S1, the reaction system of first round PCR includes following component：
Template DNA 2ng/ μ l, 0.05 μM of rs13181-F primers, 0.05 μM of rs13181-R primers, Mg2+4mM、1×PCR
10 μM of Buffer, dNTP and Taq archaeal dna polymerases 1U.
4. ERCC2 genes rs13181 sites SNP nucleic acid Mass Spectrometry detection methods as claimed in claim 3, which is characterized in that step
In rapid 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. ERCC2 genes rs13181 sites SNP nucleic acid Mass Spectrometry detection methods as claimed in claim 2, which is characterized in that step
In rapid 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. ERCC2 genes rs13181 sites SNP nucleic acid Mass Spectrometry detection methods as claimed in claim 5, which is characterized in that step
In rapid 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. ERCC2 genes rs13181 sites SNP nucleic acid Mass Spectrometry detection methods as claimed in claim 2, which is characterized in that step
In rapid 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. ERCC2 genes rs13181 sites SNP nucleic acid Mass Spectrometry detection methods as claimed in claim 7, which is characterized in that step
In rapid 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. ERCC2 genes rs13181 sites SNP nucleic acid Mass Spectrometry detection methods as claimed in claim 2, which is characterized in that step
Rapid 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 LddH again2O, it is close with sealed membrane
Envelope 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.
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| US20040259100A1 (en) * | 2003-06-20 | 2004-12-23 | Illumina, Inc. | Methods and compositions for whole genome amplification and genotyping |
| CN105505766A (en) * | 2016-01-28 | 2016-04-20 | 上海美吉逾华生物医药科技有限公司 | Automation micro-flow workstation and method for carrying out solid phase PCR reaction |
| CN106755390A (en) * | 2016-12-15 | 2017-05-31 | 上海东方杰玛基因生物科技有限公司 | A kind of Primer composition and detection method for skin anti-aging ability genetic test |
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2017
- 2017-12-27 CN CN201711450591.2A patent/CN108070660A/en active Pending
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20040259100A1 (en) * | 2003-06-20 | 2004-12-23 | Illumina, Inc. | Methods and compositions for whole genome amplification and genotyping |
| CN105505766A (en) * | 2016-01-28 | 2016-04-20 | 上海美吉逾华生物医药科技有限公司 | Automation micro-flow workstation and method for carrying out solid phase PCR reaction |
| CN106755390A (en) * | 2016-12-15 | 2017-05-31 | 上海东方杰玛基因生物科技有限公司 | A kind of Primer composition and detection method for skin anti-aging ability genetic test |
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Application publication date: 20180525 |