CN108546747A - The method and primer and probe used of Taqman probe in detecting APOE - Google Patents
The method and primer and probe used of Taqman probe in detecting APOE Download PDFInfo
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Abstract
The present invention provides a kind of methods carrying out APOE genetic tests using Taqman sonde methods, include the following steps：(1) normal population buccal swab genome extracts；(2) APOE primers and probe are designed；(3) primer and probe of APOE is used to carry out PCR (PCR) to genome；(4) fluorescence reading is carried out after PCR, and carries out Genotyping；(5) comprehensive 2 probe in detecting as a result, the APOE genes to individual carry out comprehensive assessment.The taqman primers and probe of the present invention has good parting effect, can accurately determine very much the genotype of individual.
Description
Technical field
The present invention relates to molecular biology more particularly to a kind of sides carrying out APOE genetic tests using Taqman sonde methods
Method and used primer and probe.
Background technology
APOE genes are located at No. 19 chromosomes, and encoding apolipoprotein E (APOE), is existed simultaneously in serum and nervous centralis
Lipid GAP-associated protein GAP in system, it participates in chylomicron and very low density lipoprotein as the ligand of LDL receptor
Removing, maintain body lipid-metabolism balance, concentration of the APOE in blood plasma determines the water of blood fat in blood plasma to a certain extent
It is flat.The albumen not only plays an important role in lipid metabolism, but also participates in other important biological functions, such as immunological regulation and
Nerve pathway is adjusted (neuron reparation and remodeling) etc..The result of APOE genetic tests at present can be assessed close with APOE protein functions
Relevant diseases of cardiovascular and cerebrovascular systems susceptible risk.
The main means of tumor susceptibility gene screening at present are generation sequencing, the sequencing of two generations and quantitative fluorescent PCR.A generation and two generations
Although it is relatively high that accuracy is sequenced, with high costs, take longer.Taqman sonde methods, which detect SNP site, has quick, standard
Really, the series of advantages such as at low cost.The probe of 2 kinds of different fluorescent markers is added in PCR reaction systems, they can be respectively with 2
A allele matches completely.Under normal circumstances, glimmering since probe 5 ' holds fluorophor and 3 ' end quenching groups close to together
Light is quenched.With effective progress of PCR, the probe matched completely with template is gradually circumscribed by Taq archaeal dna polymerases 5 ' → 3 '
Enzymatic activity is cut, and causes the fluorophor that probe 5 ' is held and the quenching group at 3 ' ends to detach, quenching effect releases, reporter fluorescence
Group is activated；And the probe that cannot be matched completely with template, show that another pair allele cannot effectively be cut, therefore detects
Less than fluorescence signal, SNP site detection can be realized in the variation that fluorescent value is detected by corresponding instrument.
Invention content
It is available based on the sites rs429358 and rs7412SNP on APOE genes in order to solve the problems, such as that this field exists
Come assess with the relevant cardiovascular and cerebrovascular disease susceptible risk of APOE protein functions, the present invention provides a kind of to be visited using Taqman
The method that the skill of handling needles carries out APOE genetic tests.
The purpose of the present invention is realized by following scheme：
A method of APOE genetic tests being carried out using Taqman sonde methods, are included the following steps：
(1) extraction of buccal swab genome；
(2) APOE primer and probes are determined；
(3) primer and probe of APOE is used to carry out PCR reactions to the genome of step (1)；
(4) fluorescence reading and Genotyping are carried out after PCR；
(5) comprehensive probe in detecting as a result, assessing APOE genes.
Preferably, the extracting method in the step (1) is to scrape Oral Mucosal Cells using buccal swab, uses 200 μ
L buccal swabs preserve liquid and preserve, and genomic DNA, rear selective precipitation removal are released through 400 μ L cell pyrolysis liquid lytic cells
Albumen, pure genomic DNA laid equal stress on by 400 μ L isopropanol precipitatings be dissolved in it is spare in 50 μ L DNA lysates.
Preferably, the primer in the step (2) includes the primer of the primer and the sites rs7412 in the sites rs429358.
Preferably, the nucleotide sequence of the primer in the sites rs429358 is as follows：
Forward primer：5'-CCGCGGTACTGCACCAG-3'
Reverse primer：5'-GGACGAGACCATGAAGGAGTT-3'
The nucleotide sequence of the probe in the sites rs429358 is as follows：
P1-C：5'-FAM-CCGCGCACGTCCT-MGB-3'
P1-T：5'-VIC-CGGCCGCACACGT-MGB-3'.
Preferably, the nucleotide sequence of the primer in the sites rs7412 is as follows：
Forward primer：5'-CCCCGGCCTGGTACACT-3'
Reverse primer：5'-CTGCGCAAGCTGCGTAA-3'
The nucleotide sequence of the probe in the sites rs7412 is as follows：
P2-C：5'-FAM-CAGGCGCTTCTGCA-MGB-3'
P2-T：5'-VIC-CCAGGCACTTCTGCA-MGB-3.
Preferably, the PCR reaction systems of the step (3) are 10 μ l：
Preferably, the PCR conditions are as follows：
95 DEG C, 15min；
95 DEG C, 30s, 60 DEG C 1min, 35 cycles.
It is described to draw another aspect of the invention is the primer and probe for carrying out APOE genetic tests for Taqman sonde methods
Object includes the primer of the primer and the sites rs7412 in the sites rs429358；The probe include the sites rs429358 probe and
The probe in the sites rs7412.
Preferably, the nucleotide sequence of the primer in the sites rs429358 is as follows：
Forward primer：5'-CCGCGGTACTGCACCAG-3'
Reverse primer：5'-GGACGAGACCATGAAGGAGTT-3'
The nucleotide sequence of the probe in the sites rs429358 is as follows：
P1-C：5'-FAM-CCGCGCACGTCCT-MGB-3'
P1-T：5'-VIC-CGGCCGCACACGT-MGB-3'.
Preferably, the nucleotide sequence of the primer in the sites rs7412 is as follows：
Forward primer：5'-CCCCGGCCTGGTACACT-3'
Reverse primer：5'-CTGCGCAAGCTGCGTAA-3'
The nucleotide sequence of the probe in the sites rs7412 is as follows：
P2-C：5'-FAM-CAGGCGCTTCTGCA-MGB-3'
P2-T：5'-VIC-CCAGGCACTTCTGCA-MGB-3.
Signified extracting method is to scrape Oral Mucosal Cells using buccal swab in the present invention, is wiped using 200 oral cavities μ L
Son preserves liquid and preserves, and releases genomic DNA through 400 μ L cell pyrolysis liquid lytic cells, rear selective precipitation removes removing protein, pure
Net genomic DNA laid equal stress on by 400 μ L isopropanol precipitatings be dissolved in it is spare in 50 μ L DNA lysates.
Signified specific primer is to referring to for the sites rs429358 and rs7412SNP on APOE genes in the present invention
Primer pair, can specific amplification go out the primer pair of the DNA fragmentation comprising this 2 sites SNPs.Design these specific primers
To being completed by this laboratory technicians, by primer, Synesis Company synthesizes, and primer synthesis is using conventional primer synthesizing mean
It can.In the present invention signified specific taqman MGB probes to refer on APOE genes rs429358 and
The probe pair in the sites rs7412SNP can go out this 2 SNPs loci gene types by fluorescence quantifying PCR method specific detection
Taqman probes pair.It is that those skilled in the art can be unlabored to design this kind of taqman probes, by this lab assistant
Design, by primer, Synesis Company synthesizes, and primer synthesis is synthesized using conventional probe synthesizing mean.APOE
There are 3 genotype CC, CT and TT in the sites rs429358SNP；There are 3 genotype TT, CT, CC in the sites rs7412SNP.APOE genes
It is positioned at No. 19 chromosomes, encoding apolipoprotein E.
Method provided by the invention using Taqman MGB probe in detecting people's APOE gene pleiomorphisms, can high-throughput detection
As a result its SNP site is easy interpretation.Each veneer can detect 384 samples, and need not carry out follow-up point of PCR product
Analysis, while saving testing cost, substantially reduces detection cycle, improves detection efficiency.The primer that the present invention uses
It is strong with probe specificity, it will not be interfered by other genes, reduce the risk that PCR product pollution causes false positive, and
As a result interpretation is easier.High-throughput detection can be realized to the SNP site of APOE genes, and cardiovascular and cerebrovascular disease morbidity is being carried out to people
It is had potential application in probability analysis.
Description of the drawings
Fig. 1 is the testing result of 3 genotype CC, CT and TT of rs429358 SNP sites；
Fig. 2 is the testing result of 3 genotype TT, CT, CC of rs7412 SNP sites.
Specific implementation mode
Below in conjunction with the accompanying drawings, detailed explanation is carried out to the present invention.
The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention..Unless otherwise specified, embodiment
According to conventional laboratory conditions, as Sambrook equimoleculars Cloning: A Laboratory Manual (Sambrook J＆Russell DW,
Molecular cloning:A laboratory manual, 2001), or according to the condition of manufacturer's specification suggestion.
Embodiment 1
Utilize the method for Taqman MGB probe in detecting people's APOE rs429358 single nucleotide polymorphisms
1, experiment material
It is detection object to choose the healthy population by the sequencing of an APOE generation.
2, the extraction of genomic DNA scrapes Oral Mucosal Cells using buccal swab, uses buccal swab to preserve liquid first
It preserves, cell pyrolysis liquid lytic cell releases genomic DNA, and then albumen precipitation liquid selective precipitation removes removing protein, finally
Pure genomic DNA is laid equal stress on by isopropanol precipitating is dissolved in DNA lysates.The extraction of genomic DNA is not limited only to such
Method, the genomic DNA extracted using other methods is also in the research range of this experiment.
3, Taqman MGB sonde methods carry out Genotyping, using the sites rs429358 primer and probe to being detected.
The nucleotide sequence of the primer of the gene is as follows：
Forward primer：5'-CCGCGGTACTGCACCAG-3'
Reverse primer：5'-GGACGAGACCATGAAGGAGTT-3'；
The nucleotide sequence of the probe is as follows：
P1-C：5'-FAM-CCGCGCACGTCCT-MGB-3'
P1-T：5'-VIC-CGGCCGCACACGT-MGB-3'；
Primer and probe is synthesized by Qing Ke biotech firms, using the 2X GoldStar Taqman purchased from Kang Wei companies
Mixture, PCR instrument are LGC water-bath PCR instruments.
Reaction system：Using the genomic DNA of extraction as template, it is preferable that with the primer and probe of above-mentioned synthesis in LGC water
It is expanded (10 μ L reaction systems) shown according to the form below in bath PCR instrument：
Specific implementation step is as follows：
(1) consider that the error of pipettor and suction nozzle, general each 96 orifice plate (8 row * 12 row) press 110 Response calculation bodies
System, prepares mix.
(2) one blank control NTC of each 96 orifice plate setting, two known TT types APOE genomic controls of setting, one
Known CT types APOE genomic controls, a known CC types APOE genomic control.
(3) apply the volley of rifle fire in 1 μ L DNA samples of every Kong Zhongjia.
(5) it is sealed with sealed membrane after the completion of, plate centrifuge is centrifuged according to the conventional speeds of this field.
(6) it is uniformly mixed, is put into water-bath PCR instrument and carries out PCR amplification, arrange parameter, reaction condition is as follows：
95℃15min；
95 DEG C of 30sec, 60 DEG C of 1min, 35 cycles.
4, analysis result
Fluorescent value is read using Bio-tek after PCR, recycles Klustercaller software analysis of fluorescence data.Root
According to control amplification, parting generates three kinds of genotype, sees Fig. 1.
The first genotype：TT, with TT types to impinging upon on an axis；
Second of genotype：CT, with CT types to impinging upon on an axis；
The third genotype：CC, with CC types to impinging upon on an axis.
Embodiment 2
Utilize the method for Taqman MGB probe in detecting people's APOE rs7412 single nucleotide polymorphisms
1, experiment material
It is detection object to choose the healthy population by the sequencing of an APOE generation.
2, the extraction of genomic DNA scrapes Oral Mucosal Cells using buccal swab, uses buccal swab to preserve liquid first
It preserves, cell pyrolysis liquid lytic cell releases genomic DNA, and then albumen precipitation liquid selective precipitation removes removing protein, finally
Pure genomic DNA is laid equal stress on by isopropanol precipitating is dissolved in DNA lysates.The extraction of genomic DNA is not limited only to such
Method, the genomic DNA extracted using other methods is also in the research range of this experiment.
3, Taqman MGB sonde methods carry out Genotyping, using the sites rs7412 primer and probe to being detected.
The nucleotide sequence of the primer of the gene is as follows：
Forward primer：5'-CCCCGGCCTGGTACACT-3'
Reverse primer：5'-CTGCGCAAGCTGCGTAA-3'；
The nucleotide sequence of the probe is as follows：
P2-C：5'-FAM-CAGGCGCTTCTGCA-MGB-3'
P2-T：5'-VIC-CCAGGCACTTCTGCA-MGB-3；
Primer and probe is synthesized by Qing Ke biotech firms, using the 2X GoldStar Taqman purchased from Kang Wei companies
Mixture, PCR instrument are LGC water-bath PCR instruments.
Reaction system：Using the genomic DNA of extraction as template, it is preferable that with the primer and probe of above-mentioned synthesis in LGC water
It is expanded (10 μ L reaction systems) shown according to the form below in bath PCR instrument：
Specific implementation step is as follows：
(1) consider that the error of pipettor and suction nozzle, general each 96 orifice plate are pressed 110 Response calculation systems, prepared
mix。
(2) one blank control NTC of each 96 orifice plate setting, two known CC types APOE genomic controls of setting, one
Known CT types APOE genomic controls.
(3) apply the volley of rifle fire in 1 μ L DNA samples of every Kong Zhongjia.
(5) it is sealed with sealed membrane after the completion of, plate centrifuge centrifugation.
(6) it is uniformly mixed, is put into water-bath PCR instrument and carries out PCR amplification, arrange parameter, reaction condition is as follows：
95 DEG C of 15min, 95 DEG C of 30sec, 60 DEG C of 1min, 35 cycles.
4, analysis result
Fluorescent value is read using Bio-tek after PCR, recycles Klustercaller software analysis of fluorescence data.Root
According to control amplification, parting generates two kinds of genotype, sees Fig. 2.
The first genotype：CT, with CT types to impinging upon on an axis；
Second of genotype：CC, with CC types to impinging upon on an axis.
Taqman MGB sonde methods carry out SNP partings, are suitble to large sample size, can accomplish that single makees 5000 samples, lead to
Amount is high.
The foregoing is only a preferred embodiment of the present invention, but scope of protection of the present invention is not limited thereto,
Any one skilled in the art is in the technical scope of present disclosure, the change or replacement that can be readily occurred in,
It should all be included within the scope of the present invention.Therefore, protection scope of the present invention should be with the protection of claims
Subject to range.
Sequence table
<110>U.S. is because of healthy science and technology（Beijing）Co., Ltd
<120>The method and the primer and probe of Taqman probe in detecting APOE
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<213>Artificial sequence (Artificial Sequence)
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ggacgagacc atgaaggagt t 21
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<212> DNA
<213>Artificial sequence (Artificial Sequence)
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ccgcgcacgt cct 13
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ccccggcctg gtacact 17
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ctgcgcaagc tgcgtaa 17
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ccaggcactt ctgca 15
Claims (10)
1. a kind of method carrying out APOE genetic tests using Taqman sonde methods, which is characterized in that include the following steps：
(1) extraction of buccal swab genome；
(2) APOE primer and probes are determined；
(3) primer and probe of APOE is used to carry out PCR reactions to the genome of step (1)；
(4) fluorescence reading and Genotyping are carried out after PCR；
(5) comprehensive probe in detecting as a result, assessing APOE genes.
2. the method according to claim 1 for carrying out APOE genetic tests using Taqman sonde methods, which is characterized in that institute
It is to scrape Oral Mucosal Cells using buccal swab to state the extracting method in step (1), and preserving liquid using 200 μ L buccal swabs protects
It deposits, releases genomic DNA through 400 μ L cell pyrolysis liquid lytic cells, rear selective precipitation removes removing protein, pure genome
DNA laid equal stress on by 400 μ L isopropanol precipitatings be dissolved in it is spare in 50 μ L DNA lysates.
3. the method according to claim 1 for carrying out APOE genetic tests using Taqman sonde methods, which is characterized in that institute
State the primer that the primer in step (2) includes the primer and the sites rs7412 in the sites rs429358.
4. the method according to claim 3 for carrying out APOE genetic tests using Taqman sonde methods, which is characterized in that institute
The nucleotide sequence for stating the primer in the sites rs429358 is as follows：
Forward primer：5'-CCGCGGTACTGCACCAG-3'
Reverse primer：5'-GGACGAGACCATGAAGGAGTT-3'
The nucleotide sequence of the probe in the sites rs429358 is as follows：
P1-C：5'-FAM-CCGCGCACGTCCT-MGB-3'
P1-T：5'-VIC-CGGCCGCACACGT-MGB-3'.
5. the method according to claim 3 for carrying out APOE genetic tests using Taqman sonde methods, which is characterized in that institute
The nucleotide sequence for stating the primer in the sites rs7412 is as follows：
Forward primer：5'-CCCCGGCCTGGTACACT-3'
Reverse primer：5'-CTGCGCAAGCTGCGTAA-3'
The nucleotide sequence of the probe in the sites rs7412 is as follows：
P2-C：5'-FAM-CAGGCGCTTCTGCA-MGB-3'
P2-T：5'-VIC-CCAGGCACTTCTGCA-MGB-3.
6. the method according to claim 1 for carrying out APOE genetic tests using Taqman sonde methods, which is characterized in that institute
The PCR reaction systems for stating step (3) are 10 μ l：
7. the method according to claim 6 for carrying out APOE genetic tests using Taqman sonde methods, which is characterized in that institute
It is as follows to state PCR conditions：
95 DEG C, 15min；
95 DEG C, 30s, 60 DEG C 1min, 35 cycles.
8. carrying out the primer and probe of APOE genetic tests for Taqman sonde methods, which is characterized in that the primer includes
The primer of the primer and the sites rs7412 in the sites rs429358；The probe include the sites rs429358 probe and rs7412
The probe of point.
9. primer and probe according to claim 8, which is characterized in that the nucleotide of the primer in the sites rs429358
Sequence is as follows：
Forward primer：5'-CCGCGGTACTGCACCAG-3'
Reverse primer：5'-GGACGAGACCATGAAGGAGTT-3'
The nucleotide sequence of the probe in the sites rs429358 is as follows：
P1-C：5'-FAM-CCGCGCACGTCCT-MGB-3'
P1-T：5'-VIC-CGGCCGCACACGT-MGB-3'.
10. primer and probe according to claim 8, which is characterized in that it is characterized in that, the sites rs7412 are drawn
The nucleotide sequence of object is as follows：
Forward primer：5'-CCCCGGCCTGGTACACT-3'
Reverse primer：5'-CTGCGCAAGCTGCGTAA-3'
The nucleotide sequence of the probe in the sites rs7412 is as follows：
P2-C：5'-FAM-CAGGCGCTTCTGCA-MGB-3'
P2-T：5'-VIC-CCAGGCACTTCTGCA-MGB-3.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104862402A (en) * | 2015-05-29 | 2015-08-26 | 沈阳优吉诺生物科技有限公司 | Primers for detecting ApoE gene polymorphism, kit and PCR (polymerase chain reaction) method for primers or kit |
| CN105803099A (en) * | 2016-05-16 | 2016-07-27 | 钟诗龙 | Kit for simultaneously detecting SLCO1B1, APOE and LDLR gene multisite mutation |
| CN107099602A (en) * | 2017-05-27 | 2017-08-29 | 宁波美晶医疗技术有限公司 | It is a kind of at the same detect statins metabolic gene multisite mutation kit |
| CN107447030A (en) * | 2017-09-20 | 2017-12-08 | 苏州康吉诊断试剂有限公司 | The kit of Alzheimer's disease APOE genetic tests |
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2017
- 2017-12-19 CN CN201711371146.7A patent/CN108546747A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104862402A (en) * | 2015-05-29 | 2015-08-26 | 沈阳优吉诺生物科技有限公司 | Primers for detecting ApoE gene polymorphism, kit and PCR (polymerase chain reaction) method for primers or kit |
| CN105803099A (en) * | 2016-05-16 | 2016-07-27 | 钟诗龙 | Kit for simultaneously detecting SLCO1B1, APOE and LDLR gene multisite mutation |
| CN107099602A (en) * | 2017-05-27 | 2017-08-29 | 宁波美晶医疗技术有限公司 | It is a kind of at the same detect statins metabolic gene multisite mutation kit |
| CN107447030A (en) * | 2017-09-20 | 2017-12-08 | 苏州康吉诊断试剂有限公司 | The kit of Alzheimer's disease APOE genetic tests |
Non-Patent Citations (1)
| Title |
|---|
| ZAHID AHMAD, MD ET AL.: ""Low Prevalence of Mutations in Known Loci for Autosomal Dominant Hypercholesterolemia in a Multi-Ethnic Patient Cohort"", 《CIRC CARDIOVASC GENET.》 * |
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Application publication date: 20180918 |