CN1880480A - PCR method for detection of allele specific primer and use in detection of KLOTHO gene polymorphism - Google Patents
PCR method for detection of allele specific primer and use in detection of KLOTHO gene polymorphism Download PDFInfo
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- CN1880480A CN1880480A CN 200610054212 CN200610054212A CN1880480A CN 1880480 A CN1880480 A CN 1880480A CN 200610054212 CN200610054212 CN 200610054212 CN 200610054212 A CN200610054212 A CN 200610054212A CN 1880480 A CN1880480 A CN 1880480A
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Abstract
The invention discloses an astopic primer-PCR method to detect allelomorph and appliance in the detecting course of KLOTHO gene polymorphism, which is characterized by the following: designing allelomorph astopic primer couple according to detected gene sequence and SNP information of corresponding positional point; designing PCR reacting system, PCR condition and product; analyzing detected result for reacting product after electrophoresis; fitting for detecting allelomorph of KLOTHO gene SNP.
Description
Technical field
The present invention relates to a kind of detection method of biological gene, particularly detection allele specific primer-PCR method and the application in detecting the old plain gene polymorphism of gram thereof
Background technology
The method of current widely used detection of biological gene SNP (comprising the detection that restrains old plain gene) has two big classes: classical enzyme blanking method and direct sequence measurement.Classical enzyme blanking method is owing to some SNP site does not find that as yet restriction endonuclease is restricted, and the product length that enzyme cutting method produces is uncontrollable, sometimes produce very short product (several bp), be difficult to observe and analyze (Xie Zhengxiang waits the genotypic combination distribution of 5 site SNP of 3 kinds of subtype gene of .Beta adrenergic receptor. the biomedical engineering magazine.2005; 22 (1): 99-103.), thereby in the recent period direct sequence measurement (Arking DE, et al.Association of Human aging with a functional variant of klotho.PNAS, 2002,99 (2): 856-861) of adopting more.Directly sequence measurement not but still need round pcr, and instrument complexity, costliness, operation sequence is loaded down with trivial details, is difficult to generally adopt and be not suitable for extensive screening.
Summary of the invention
The purpose of this invention is to provide a kind of without restriction endonuclease, need not check order, the saving funds are saved time, easy and simple to handle, and the bp number of the product that produces can be by the detection allele specific primer-PCR method and the application of this method in the allelotrope that detects the old plain gene SNP of gram of planner's control.
Concrete technical scheme is as follows:
A kind of detection allele specific primer-PCR method (ASP-PCR method) is characterized in that carrying out according to the following steps:
(1), right according to the SNP information design allele specific primer in detection gene order and corresponding site thereof;
(2), design PCR reaction system;
(3), the pre-sex change of design PCR condition, circulation, prolongation and PCR product;
(4), the interpretation of result of PCR reaction product prepares 2% sepharose, get sample on the PCR product 10 μ l, at voltage is 100V, electrophoresis is 1 hour under the power 5W condition, after electrophoresis finishes, gel places 0.5 μ g/L ethidium bromide solution dyeing 15 minutes, and the gel that will dye places under the ultraviolet imagery system takes pictures, and carries out interpretation of result then.
The core of the ASP-PCR method of detection of biological gene SNP is the right design of allele specific primer in the step (1).Can be divided into two big class: SASP-PCR (single formula ASP-PCR:Single Allele-Specific Primer-Polymerase ChainReaction) and NASP-PCR (nested type ASP-PCR:Nested Allele-SpecificPrimer-Polymerase Chain Reaction) according to the right method of design difference of step (1) allele specific primer.
SASP-PCR (single formula ASP-PCR:Single Allele-Specific Primer-Polymerase ChainReaction) method of design is: from 5 ' to 3 ' direction is the direction of template DNA, P-C (Primer-Common) is a consensus primer, this consensus primer is a downstream primer, and the antisense strand of template DNA is pressed the design of Nucleotide coupling rule; P-W (Primer-Wild) is the wild-type allele primer, and this design of primers is a upstream primer, and the positive-sense strand of template DNA is pressed the design of Nucleotide coupling rule; P-M (Primer-Mutant) is an anomaly allelotrope primer, this primer also be designed to upstream primer to the positive-sense strand of template by the design of Nucleotide coupling rule; P-W, P-M constitute two pairs of allele specific primers with downstream primer P-C respectively.When detecting Klotho gene G-395A SNP with SASP-PCR (single formula ASP-PCR), its PCR reaction conditions and product are:
G allelotrope
(i) pre-sex change: 94 ℃ of pre-sex change of 3min;
(ii) circulation: carry out 94 ℃ of 30s sex change of 35 round-robin altogether, 63 ℃ of 30s annealing, 72 ℃ of 30s extend;
(iii) stable: 72 ℃ of 10min;
(iv) product: 147bp
A allelotrope
(i) pre-sex change: 94 ℃ of pre-sex change of min;
(ii) circulation: carry out 94 ℃ of 30s sex change of 35 round-robin altogether, 65 ℃ of 30s annealing, 72 ℃ of 30s extend;
(iii) stable: 72 ℃ of 10min;
(iv) product: 148bp.
The method of design of NASP-PCR (nested type ASP-PCR:Nested Allele-Specific Primer-PolymeraseChain Reaction) is: from 5 ' to 3 ' direction is the direction of template DNA, and SSDNA is just sequence DNA; Outside upstream primer OSP (Outer Sense Primer) and downstream antisense allele specific primer DASP (Downstream Allele Specific Primer) form first inner primer to IPP-1 (Inner Primer Pair-1), and promptly the OSP-DASP primer is right; Outside downstream antisense primer OAP (Outer Anti-sense Primer) and upstream justice allele specific primer UASP (UPstream Allele Specific Primer) form second inner primer to IPP-2 (InnerPrimer Pair-2), and promptly the UASP-OAP primer is right; Outside downstream primer OAP and outside upstream primer OSP form the 3rd couple of primer OPP, and (outside primer is right: Outer Primer Pair); The starting point of wherein said UASP is in the left side of SNP; The starting point of DASP is on the right side of SNP, and all the special base with SNP is 3 ' end.When selecting the method for design detection NOS2 C-1173T SNP of NASP-PCR for use, PCR reaction conditions and product are:
(i) pre-sex change: 95 ℃ of pre-sex change of 5min;
(ii) circulation: carry out 94 ℃ of 30s sex change of 40 round-robin altogether, 57 ℃ of 30s annealing, 72 ℃ of 30s extend;
(iii) stable: 72 ℃ of 10min;
(iv) product: the product length of outer primer to producing: 331bp; Two inner primers to the product that produces are: 268bp and 102bp;
Two class ASP-PCR methods may be used to the detection of biological gene, and its main difference is: for the SASP-PCR method, and be with different allele specific primers to making twice PCR; NASP-PCR has two kinds of different allele specific primers and outside primer simultaneously to making PCR one time in same reaction system.The PCR reaction conditions and the product of two kinds of methods will be done corresponding adjustment.
Klotho (KL) gene is the new gene relevant with aging that Kuro-o etc. finds when research essential hypertension in 1997.Discover: the KL gene is genes a kind of and life-span and various geriatric disease phenotypic correlations.The KL assignment of genes gene mapping of people and mouse is in karyomit(e) 13q12 zone.The about 50kb of KL full length gene, in its exons 1 and the side 2000bp sequence, the average content of G+C is 66.75%, be up to 89%, prompting has the formation on CPG island in this zone, and apart from about 6kb place, KL genetic transcription starting point upstream, a segmental disappearance of 8kb is arranged, this may be exactly reason (Matsumura Y, the et al.Identification of the human klothogene and its two transcripts encoding membrane and secreted klothoprotein.Biochem Biophys Res Commun.1998 Jan 26 of KL genetic expression disappearance; 242 (3): 626-30.).Have the F352V of Exon2 of report KL and two SNPs of C370S and coronary heart disease that relation is arranged, the KL-VS mutant is independent hazard factor (the Arking et al.Association of humanaging with a functional variant of klotho.Proc Natl Acad Sci U SA.2002 Jan 22 of coronary heart disease; 99 (2): 856-61; Arking et al.Association between afunctional variant of the KLOTHO gene and high-density lipoproteincholesterol, blood pressure, stroke, and longevity.Circ Res.2005Mar 4; 96 (4): 412-8.).The G-395A SNP of promoter region influences DNA and combination of proteins, thereby influence the expression of KL, and then influence particularly bone metabolism (Saito Y of metabolism, Kuroo M, Nabeshima Y, et al.The protective role of Klotho gene on vascularendothelium.Nippon Rinsho, 1999,57 (7); 1514-1518.).Therefore think that KL albumen may be a kind of instrument for circulation of body fluid factor or membrane-bound receptor composition, old and feeble and aging-related disease are being carried out corresponding the adjusting.Further investigation KL gene function will be for disclosing the mankind aging, provide important new thread with diseases associated with senescence and antidotal molecule mechanism, but study less at present.Use the method for the invention, not only go for the detection of any biological gene, can detect the SNP:G-395A in 3 sites of KLOTHO gene especially, F352V, C370S.Find G-395A relevant with diabetes (P＜0.002), wherein the morbidity of GG wild-type homozygote carrier's diabetes is minimum, is 0.277 times of AG heterozygote, homozygous 0.277 times of AA anomaly.F352V relevant with hypertension (P=0.0120), wherein heterozygote FV carrier has minimum hypertension morbidity, is 0.438 times of wild-type homozygote FF, is 0.429 times of anomaly homozygote VV; Also relevant with coronary heart disease (P＜0.0001), wherein heterozygote FV carrier is 0.024 times of wild-type homozygote FF, is 0.023 times of anomaly homozygote VV.C370S also relevant with coronary heart disease (P＜0.0001), wherein heterozygote CS carrier is 0.111 times of wild-type homozygote SS, is 0.110 times of anomaly homozygote CC.
The method of the invention can be used for studying gene (single nucleotide polymorphism) and with the getting in touch of disease, formulate (disease) individualized treatment scheme of gene level.
The beneficial effect of the method for the invention is:
1. universality is strong: do not need restriction endonuclease, can overcome the obstacle that restriction endonuclease is not found in some SNP site as yet.
2. economic implications is big: do not need expensive order-checking, do not need expensive restriction endonuclease.
3. easy and simple to handle: the enzyme that saves the PCR product is cut step, thereby has simplified operation, is easier to grasp.
4. be fit to national conditions: do not need restriction endonuclease, not under one's control, also needn't use sequencing technologies, thereby very suitable current China's actual conditions.
5. be easy to promote: this Technological Economy, practicality, easy and simple to handle are easy to grasp.
6. the bp number of the product of Chan Shenging can be controlled by the planner, is easy to observe and analyze.
Description of drawings
Fig. 1 is the ASP principle of design figure of SASP-PCR method
Fig. 2 is the ASP principle of design figure of NASP-PCR method
Fig. 3 is the G-395A site SNP electrophorogram of KLOTHO
Fig. 4 is F352V (T1062G) the site SNP electrophorogram of KLOTHO
Fig. 5 is the NOS2C-1173T site SNP electrophorogram of NOS
Specific embodiment (totally 3 examples)
Embodiment 1 usefulness SASP-PCR method detect the used primer of the G-395A site SNP of KLOTHO to and the PCR reaction conditions as follows:
(1) primer is right: public upstream primer method of design
Upstream primer: 5 '-AAT
GGCTCC AGCAATGTCC AG-3 ', Tm 60.0, GC%54.5
Downstream primer
G allelotrope: 5 '-AGAAAAG
CG CCGACCAACT TTC-3 ', Tm 58.4, GC%47.8;
A allelotrope: 5 '-G AGAAAAGGCG ACGACCA
CT TT
-3 ', Tm 58.7, GC%45.8.
(2) template and primer matching Design
1561 tAATTGGCTC CAGCAATGTC CAGccggagc ttctttgggc ctccgagtgg gagaaaagtg (AAT
GGCTC CAGCAATGTC CAG, Tm 68.0, GC%54.5) upstream primer and antisense template complementation 5 ' → 3 ' 22nt (upstream primer length, nt is a nucleotide unit, down together)
To G allelotrope
1681 tcggg
AAAG TTGGTCGGCG CCTTTTCTcc ccgacgaagc cgctccaggg ctgctctcag (
TTTC AACCAGCCGC
GAAAAGA Tm 66, GC%47.8) downstream primer and just template complementation 3 ' ← 5 ' 23nt
To A allelotrope
1681 tcggg
AAAG TTGGTCGGCG CCTTTTCTCc ccgacgaagc cgctccaggg (
TTTC
ACCAGCCGC GGAAAAGAG Tm 66, GC%47.8) downstream primer and just template complementation 3 ' ← 5 ' 24nt
Annotate: in the primer to add frame ash background color Nucleotide be artificial mispairing, to improve the primer performance; Ash background color Nucleotide is the special allelotrope of SNP.
(3) PCR reaction system
| The PCR reaction system | ||||||||
| Allele | Buffer (μl) | MgCl 2 (mM/L) | dNTP (μM/L) | P 1 (μM/L) | P 2 (μM/L) | Taq (U) | Template DNA (ng) | ddH 2O (μl) |
| G A | 2 2 | 1.5 1.5 | 125 125 | 0.41 0.41 | 0.415 0.41 | 0.5 0.5 | 100 100 | * * |
Annotate: *: add to 20 microlitres
(4) PCR condition and product
G allelotrope
(i) pre-sex change: 94 ℃ of pre-sex change of 3min;
(ii) circulation: carry out 94 ℃ of 30s sex change of 35 round-robin altogether, 63 ℃ of 30s annealing, 72 ℃ of 30s extend;
(iii) insulation: 72 ℃ of 10min;
(iv) product: 147bp
A allelotrope
(i) pre-sex change: 94 ℃ of pre-sex change of min;
(ii) circulation: carry out 94 ℃ of 30s sex change of 35 round-robin altogether, 65 ℃ of 30s annealing, 72 ℃ of 30s extend;
(iii) insulation: 72 ℃ of 10min;
(iv) product: 148bp
(5) the reaction product interpretation of result prepares 2% sepharose, gets sample on the PCR product 10 μ l,
At voltage is 100V, and electrophoresis is 1 hour under the power 5W condition, and after electrophoresis finished, gel placed 0.5 μ g/L ethidium bromide solution dyeing 15 minutes, and the gel that will dye places under the ultraviolet imagery system takes pictures.Its electrophorogram is as shown in Figure 3:
The standard substance of M representation DNA electrophoresis product bp value, G/G is the wild-type homozygote, and G/A is a heterozygote, and A/A is the mutant homozygote.
Embodiment 2 SASP-PCR methods detect F352V (T1062G) the site SNP of KLOTHO
1. primer is right: public upstream primer method of design
Upstream primer: 5 '-TACAATACT TCTTTCCGTC CC-3 ' Tm 53.9GC%42.9
Downstream primer:
T allelotrope: 5 '-
CTTTTTCGC AGATTCAGTA AA-3 ' Tm50.7, GC%32;
G allelotrope: 5 '-
TTTTTCGCA GATTCAGTAA C-3 ' Tm51.9, GC%38
Annotate: the grey background color Nucleotide in the primer is the special allelotrope of SNP.
2.PCR reaction system
| PCR Reaction System | ||||||||
| Allele | Buffer (μl) | MgCl 2 (mM/L) | dNTP (μM/L) | P 1 (μM/L) | P 2 (μM/L) | Taq (U) | Template DNA (ng) | ddH 2O (μl) |
| F V | 2 2 | 1.5 1.5 | 125 125 | 0.475 0.475 | 0.41 0.41 | 0.5 0.5 | 100 100 | * * |
Annotate: * means and adds to 20 microlitres
3.PCR condition and product
(1) pre-sex change: 94 ℃ of pre-sex change of 3min;
(2) circulation: carry out 94 ℃ of 30s sex change of 35 round-robin altogether, 54 ℃ of 30s annealing, 72 ℃ of 30s extend;
(3) insulation: 72 ℃ of 10min prolong;
(4) product: G allelotrope is 230bp, and T allelotrope is 231bp.
4.PCR reaction product analytical procedure
Preparing 2% sepharose, get sample on the PCR product 10 μ l, is 100V at voltage, and electrophoresis is 1 hour under the power 5W condition, and after electrophoresis finished, gel placed 0.5 μ g/L ethidium bromide solution dyeing 15 minutes, and the gel that will dye places under the ultraviolet imagery system takes pictures.Its electrophorogram such as accompanying drawing 4:
The standard substance of M representation DNA electrophoresis product bp value, T/T is the wild-type homozygote,
T/G is a heterozygote, and G/G is the mutant homozygote
Embodiment 3 NASP-PCR methods detect the NOS2C-1173T site SNP of NOS
(1) the design primer is right
The outside primer to (OSP-OAP), allele specific primer to (UASP-DASP), primer length (LP), GC value (GC) and AT value (AT) as following table.The nt of unit is the Nucleotide number.
* Nei Nucleotide is the special allelotrope of SNP.
(2) the PCR reaction system of detection NOS2C-1173T site SNP
Detect the PCR reaction system of NOS2C-1173T site SNP, reaction system such as following table, the reaction system volume is 20 μ l.
The reaction system table of NOS2C-1173T site SNP
| SNP | ddH 2O (uL) | Buffer (μl) | MgCl 2 (mM) | dNTP (μM) | P1 (μmol/L) | P2 (μmol/L) | P-wild (μmol/L) | P-mutated (μmol/L) | Taq polymerase (U) | Template DNA (ng) |
| NOS2 C-1173T | * | 2 | 1.5 | 200 | 0.125 | 0.125 | 0.5 | 0.5 | 0.5 | 100 |
Annotate: * means and adds to 20 microlitres
(3) the PCR reaction conditions of detection NOS2C-1173T site SNP
PCR reaction conditions such as following table:
The PCR reaction conditions table of NOS2C-1173T site SNP
| Genotype | Predenaturation | Denaturation | Annealing | Extension | Cycle numbe |
| NOS2 C-1173T | 95℃5min | 94℃30s | 57℃30s | 72℃30s | 40 |
Annotate: Genotype: genotype, Predenaturation: pre-sex change, Denaturation: sex change, Annealing): annealing, Extension: extend Cycle number: cycle number.
(4) product length and electrophoresis result
Product length:
The product length of outer primer to producing: 331bp; Two inner primers to the product that produces are: 268bp and 102bp;
Electrophoresis result is seen accompanying drawing 5: Marker is the standard substance of marker DNA electrophoresis product bp value among the figure, and commodity are called DL2000; CT represents that NOS2 C-1173T is a heterozygote, is feature so that three electrophoresis bands to be arranged; CC represents that NOS2C-1173T is the wild-type homozygote, is feature with two electrophoresis bands of close together (63bp); TT represents the anomaly homozygote of NOS2C-1173T, is feature with two electrophoresis bands of distance (229bp) far away.
Claims (5)
1. one kind is detected allele specific primer-PCR method, it is characterized in that carrying out according to the following steps:
(1), right according to the SNP information design allele specific primer in detection gene order and corresponding site thereof;
(2), design PCR reaction system;
(3), the pre-sex change of design PCR condition, circulation, prolongation and PCR product;
(4), the interpretation of result of PCR reaction product prepares 2% sepharose, get sample on the PCR product 10 μ l, at voltage is 100V, electrophoresis is 1 hour under the power 5W condition, after electrophoresis finishes, gel places 0.5 μ g/L ethidium bromide solution dyeing 15 minutes, and the gel that will dye places under the ultraviolet imagery system takes pictures, and carries out interpretation of result then.
2. detection allele specific primer-PCR method as claimed in claim 1, it is right to it is characterized in that step (1) designs allele specific primer by the following method: from 5 ' to 3 ' direction is the direction of template DNA, P-C is a consensus primer, this consensus primer is a downstream primer, and the antisense strand of template DNA is pressed the design of Nucleotide coupling rule; P-W is the wild-type allele primer, and this design of primers is a upstream primer, and the positive-sense strand of template DNA is pressed the design of Nucleotide coupling rule; P-M is an anomaly allelotrope primer, this primer also be designed to upstream primer to the positive-sense strand of template by the design of Nucleotide coupling rule; P-W, P-M constitute two pairs of allele specific primers with downstream primer P-C respectively.
3. detection allele specific primer-PCR method as claimed in claim 1, it is right to it is characterized in that step (1) designs allele specific primer by the following method: from 5 ' to 3 ' direction is the direction of template DNA, SSDNA is just sequence DNA; Outside upstream primer OSP and downstream antisense allele specific primer DASP form first inner primer to IPP-1, and promptly the OSP-DASP primer is right; Outside downstream antisense primer OAP and upstream justice allele specific primer UASP form second inner primer to IPP-2, and promptly the UASP-OAP primer is right; Outside downstream primer OAP and outside upstream primer OSP form the 3rd couple of primer OPP; The starting point of wherein said UASP is in the left side of SNP; The starting point of DASP is on the right side of SNP, and all the special base with SNP is 3 ' end.
4. detect the application of allele specific primer-PCR method in the allelotrope of the old plain gene G-395A SNP of gram detects.
5. detect the application of allele specific primer-PCR method in the allelotrope of the NOS2 C-1173T site SNP that detects NOS.
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| CN102861343A (en) * | 2012-10-17 | 2013-01-09 | 南方医科大学 | Application of secretory type Klotho in preparing medicine for treating chronic renal failure |
| CN103443274A (en) * | 2011-03-31 | 2013-12-11 | 积水医疗株式会社 | Sample nucleic acid for single nucleotide polymorphism detection purposes, pcr primer for preparing sample for single nucleotide polymorphism detection purposes, and method for preparing sample for single nucleotide polymorphism detection purposes wh |
| CN103937880A (en) * | 2014-03-20 | 2014-07-23 | 苏州大学附属第二医院 | Detection of SLC2A9 gene single nucleotide polymorphism (SNP) |
| US9447460B2 (en) | 2011-01-12 | 2016-09-20 | Sekisui Medical Co., Ltd. | Method for detecting single nucleotide polymorphisms |
| US9481881B2 (en) | 2011-01-12 | 2016-11-01 | Sekisui Medical Co., Ltd. | Eluent for ion-exchange chromatography, and method of analyzing nucleic acid chains |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102465182A (en) * | 2010-10-29 | 2012-05-23 | 株式会社爱茉莉太平洋 | Detection kit for detecting skin active substance and method for detecting skin active substance |
| US9447460B2 (en) | 2011-01-12 | 2016-09-20 | Sekisui Medical Co., Ltd. | Method for detecting single nucleotide polymorphisms |
| US9481881B2 (en) | 2011-01-12 | 2016-11-01 | Sekisui Medical Co., Ltd. | Eluent for ion-exchange chromatography, and method of analyzing nucleic acid chains |
| CN103443274A (en) * | 2011-03-31 | 2013-12-11 | 积水医疗株式会社 | Sample nucleic acid for single nucleotide polymorphism detection purposes, pcr primer for preparing sample for single nucleotide polymorphism detection purposes, and method for preparing sample for single nucleotide polymorphism detection purposes wh |
| CN102861343A (en) * | 2012-10-17 | 2013-01-09 | 南方医科大学 | Application of secretory type Klotho in preparing medicine for treating chronic renal failure |
| CN103937880A (en) * | 2014-03-20 | 2014-07-23 | 苏州大学附属第二医院 | Detection of SLC2A9 gene single nucleotide polymorphism (SNP) |
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