CN102465170A - Method for detecting single nucleotide polymorphism (SNP) of BANK1 (B-cell scaffold protein with ankyrin repeats 1) gene - Google Patents
Method for detecting single nucleotide polymorphism (SNP) of BANK1 (B-cell scaffold protein with ankyrin repeats 1) gene Download PDFInfo
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Abstract
The invention which belongs to the fields of gene engineering and gene diagnosis provides a method for detecting SNP of a BANK1 gene. The method comprises the following steps: 1, designing a primer and a probe for amplifying an rs10516487 site or an rs17266594 site; 2, carrying out PCR amplification by utilizing the primer and the probe with sample DNA as a template, wherein concentrations of the upstream primer and the downstream primer in the primer are different, and the concentration of one is not less than five times the concentration of other one; and 3, adding a saturated dye, analyzing a melting degree curve, and determining the SNP. The method which can detect the polymorphism of the rs10516487 site and the rs17266594 site of the BANK1 gene in peripheral blood or a body fluid can be used to determine the attack possibility of autoimmtme diseases of systemic lupus erythematosus, rheumatoid arthritis, systemic chorionitis and the like. The method has the advantages of rapidness, simplicity, no pollution, and high detection result resolution. The invention also provides a corresponding detection kit.
Description
Technical field
The invention belongs to genetically engineered and gene diagnosis field, relate to the detection and the purposes of BANK 1 gene rs10516487 and rs17266594 site SNP (SNP).
Background technology
Systemic lupus erythematous (SLE) is the autoimmune disorder that a kind of multisystem is got involved, and has multiple autoantibody in patient's body.This disease is mainly in the women, all can damage skin, lung, blood vessel and the neural system of human body.Data shows, if the SLE family history is arranged, then the sickness rate of SLE is tens of times of common people.SLE also with ethnic group, national relevant.Far below crowd the America and Europe, and in China, the sickness rate of SLE is but high than American-European countries like Black American women's sickness rate.It is thus clear that this disease is relevant with heredity and environment.Think at present and possibly cause that the disorder of immunity of organism regulatory function, antigen-antibody and complement mixture deposition cause part or body tissue or organ injury by combined factors effects such as heredity, hormone and environment.SLE patient is often with significant T, bone-marrow-derived lymphocyte abnormal activation.Bone-marrow-derived lymphocyte has been brought into play important effect as the immune effector cell of classics in the SLE self-immunprocess.
Kozyrev in 2008 etc.
[1]Delivered the report with the SLE susceptibility first about BANK 1.They discover, BANK 1 is the tumor susceptibility gene of SLE, and two SNP sites on the BANK 1, and the onset relation of rs10516487 and rs17266594 and SLE is close.Chang etc.
[2]Investigation to area, Hong-Kong crowd has also confirmed this discovery.
Rs10516487 is positioned at the critical area that links IP3R
[1], be a non-synonym alternate SNP site.Synonym substitutes the aminoacid sequence that only changes codon but do not change institute's translated protein, it is generally acknowledged that it does not influence protein structure and function.But not synonym alternate codon mutation can cause coded amino acid whose change, thereby influences proteinic physiological function.This variation usually is the immediate cause that causes biological character to change.The 61st amino acids of rs10516487 becomes Histidine by l-arginine exactly.
Discover
[1], two kinds of hypotypes that BANK1 exists that one is long and the other is short.Short hypotype △ 2 hypotypes by name mean No. 2 exons of BANK1 gene and all lack, and the protein that causes its translation to generate has lacked the IP3R calmodulin binding domain CaM.Rs17266594 is positioned on this sequence tapping point (Fig. 1) just, and the allelic homozygote of its T has very typical branch point sequence (YNYTGAYYN) (Y: pyrimidine, N are represented any Nucleotide).Branch point sequence is to be positioned at nuclear mRNA intron and II class introne 3 ' near the conserved sequence of end.5 ' of the sequence tapping point of intron, intron-end splice junction (supplying the position) and 3 '-end splice junction (receiving the position) are that the correct montage of mRNA precursor is necessary.Therefore, the polymorphum of rs17266594 is directly connected to the montage efficient of BANK 1 gene △ 2 hypotypes.
The polymorphum of rs10516487 and rs17266594 may have influence on the function of BANK 1 gene, thereby impels B-cell receptor to continue releasor through series reaction, causes the activation of B cell transition.
At present, the known SNP of examination detection method commonly used has restriction fragment length polymorphism (RFLP), single strand conformation polymorphism (SSCP), allele specific oligonucleotide fragment to analyze multiple technologies such as (ASO), denaturing gradient gel electrophoresis (DGGE), Taqman probe method, gene chip, order-checking.But shortcoming is respectively arranged, only can detect the SNP of restriction enzyme site like RFLP, no restriction enzyme site can not detect; RFLP, SSCP, ASO, DGGE will use electrophoresis method, and sensitivity is limited, and environment is had pollution; The Taqman probe method costs an arm and a leg; The detection in the unsuitable multiple sample of gene chip, minority SNP site; Though order-checking is the gold standard that Nucleotide detects, cost is higher, and the cycle is long, is not suitable for the disease association analysis of large sample.
(High-resolution melting, HRM) detecting SNP is new in recent years a kind of method of releasing to adopt high resolving power fusion method.It judges the character of product according to melting temperature (Tm) (Tm).Melting temperature (Tm) is by GC content, length, sequence and the decision of heterozygosis situation of PCR product.In conjunction with novel saturable dye (like SYTO 9) with have high-resolution PCR appearance (like Rotor-Gene 6000),, also can embody from melting temperature (Tm) even have only the change of a base
[3]Only need a kind of dyestuff just can satisfy the detection of any goal gene.If but in a sample, there are a plurality of sudden changes simultaneously, only depend on above method not judge.It is not high perhaps to work as dna sample purity, when primer dimer appears in pcr amplification, can change the detected melting temperature (Tm) of HRM, thereby influence result's judgement.Therefore, though Taqman probe method price is more expensive, and receives each fluorescent probe and only can detect multiple restriction such as target position to one, still extensively by clinical employing
[4]For the detection of extensive sample,, press for the analytical procedure of the ubiquity of a kind of specificity that has combined probe simultaneously and dyestuff in order to reduce cost.Unmarked probe high resolving power fusion method (unlabeled probe high resolution melting, unlabeled probe HRM) has met these requirements fully.
Unmarked probe method has been done further improvement on the basis of PCR: unmarked probe, dyestuff and the inconsistent primer of a pair of concentration of 3 ' end sealing.SYBR Green I has continued to use the more than ten years in PCR as a kind of dyestuff of classics.But because this is a kind of unsaturated dyestuff, can not fill up the ditch of dna double chain fully, the SYBR Green I meeting recombine under in the process that DNA unwinds, dissociating causes distortion as a result in the two strands of not unwinding.Excessive SYBR Green I also can suppress pcr amplification.In unmarked probe method, the SYBR Green I concentration that is fit to pcr amplification only can detect the higher product of melting temperature (Tm), can't judge the existence of heteroduplex
[5]
Reference:
1.?Kozyrev?SV,?Abelson?AK,Wojcik?J,?et?al.?Functional?variants?in?the?B-cell?gene?BANK1?are?associated?with?systemic?lupus?erythematosus.?Nat?genet,?2008,?40:?211-216.
2.?Chang?YK,?Yang?W,?Zhao?M,?et?al.?Association?of?BANK1?and?TNFSF4?with?systemic?lupus?erythematosus?in?Hong?Kong?Chinese.?Genes?Immun,?2009,?10:?414-420.
3.?Reed?GH,?Kent?JO,?Wittwer?CT.?High-resolution?DNA?melting?analysis?for?simple?and?efficient?molecular?diagnostics.?Pharmacogenomics,2007,8:?597-608.
4.?Erali?M,?Palais?R,?Wittwer?C.?SNP?genotyping?by?unlabeled?probe?melting?Analysis.?Methods?Mol?Biol,?2008,?429:199-206.
5.?Zhou?L,?Myers?AN,?Vandersteen?JG,et?al.?Closed-tube?genotyping?with?unlabeled?oligonucleotide?probes?and?a?saturating?DNA?dye.?Clin?Chem,?2004,?50,?1328-1335.
Summary of the invention
The method that the purpose of this invention is to provide a kind of easy detection BANK1 gene pleiomorphism.This method has quick, simple, free of contamination characteristics, can detect all kinds of samples, comprises serum (slurry) and various body fluid.
Another object of the present invention provides a kind of test kit of easy detection BANK1 gene pleiomorphism.
Among the unmarked probe HRM, the unmarked probe in the asymmetric PCR reaction can not increase under the effect of polysaccharase because 3 ' end seals.Rely on the upstream and downstream primer can amplify a large amount of mutual paired purpose segments, after a primer exhausted, another excessive primer can continue the DNA amplification strand, and the free probe combines with these strands.Probe combines with the single stranded product that asymmetric PCR amplifies, and saturable dye is attached to probe/product and product/product combines in the two strands of formation.Saturable dye (like SYTO 9, LC Green) has stronger DNA binding ability and very low restraining effect, is applicable to the detection to single sequence change fully.
If product contains different allelotrope, then the melting temperature (Tm) of the two strands that forms of itself and probe can demonstrate evident difference.Mutual pairing between the product is like this equally.Behind the software analysis that carries through high resolution instrument, can clearly demonstrate different genotype.
In the negative derivative figure of fluorescence (dF/ dT), 2 fusion peaks appear in each sample to I haven't seen you for ages, and one is the fusion peak of the lower probe/product of melting temperature (Tm), and another then is the fusion peak of the higher product/product of melting temperature (Tm).If also contain other unknown base mutations in the amplified fragments, then except that above-mentioned 2, the 3rd fusion peak can appear also.
In the fusion peak of the lower probe/product of melting temperature (Tm), if the single stranded product and the probe that are amplified by template DNA mate fully, owing to need higher energy that two strands is unwind, melting temperature (Tm) can be higher; If SNP site and probe in the template DNA to be measured not exclusively mate, only need lower energy can open duplex structure, so melting temperature (Tm) is lower; If template DNA is a heterozygote, then its melting curve can have the characteristic at above-mentioned 2 kinds of fusion peaks simultaneously.
In the fusion peak of the higher product/product of melting temperature (Tm), when except that site to be measured, not having other sequence changes in the amplified fragments, the fusion peak of product/product is consistent with the fusion peak result of probe/product; When also containing the sequence change in other sites in the amplified fragments, then the fusion peak shape attitude of product/product can change, and there are other sequence changes in prompting.Because the difference at product between different bases/product fusion peak general < therefore 0.5 ℃ become general view to carry out more being prone to observe this base analog after amplify the part data-switching through software and change.In the double-spiral structure of DNA, the G-C base pair has 3 pairs of hydrogen bonds, and the A-T base pair has 2 pairs of hydrogen bonds; Therefore destroy that hydrogen bond needs pay more energy than A-T hydrogen bond between G-C; So the genotypic melting temperature (Tm) of CC/>GG is higher than TT/AA genotype, and heterozygote is insecure owing to combining, and melting temperature (Tm) is lower.
Through the result of combined with fluorescent derivative figure and general view, can judge the genotype of BANK1 gene polymorphism sites accurately.
The invention provides a kind of method of the BANK of detection 1 gene mononucleotide polymorphism, this method comprises successively: the primer and the probe of design amplification mononucleotide polymorphism site sequence; With the sample DNA is template, utilizes above-mentioned primer and probe to carry out pcr amplification; Add saturable dye; Analyze the melting degree curve, confirm SNP.Probe is according to the mononucleotide polymorphism site sequences Design.Primer is confirmed through follow-up PCR result according to the PCR principle design.
The upstream primer that carries out in the primer of pcr amplification is different with downstream primer concentration, a kind of 5 times of being not less than alternative concentration.For example, downstream primer concentration is 10 times of upstream primers, 15 times, and 20 times, 25 times, 30 times, etc.
Described sample is blood ingredient or body fluid.
Described saturable dye is SYTO 9 or LC Green (commercially available, Invitrogen company for example, the U.S.).
Concentration and probe concentration is not less than the concentration difference of two kinds of primers.Concentration and probe concentration also can be slightly less than the concentration difference of upstream and downstream primer, and perhaps concentration and probe concentration is equal to that high primer of concentration.
In one embodiment of the invention, the site of described SNP is rs10516487.With shown in the SEQ ID NO 2, described probe sequence is shown in SEQ ID NO 3 like SEQ ID NO 1 for described primer sequence.
In another embodiment of the present invention, the site of described SNP is rs17266594.With shown in the SEQ ID NO 5, described probe sequence is shown in SEQ ID NO 6 like SEQ ID NO 4 for described primer sequence.
Particularly, present method behind the extracting DNA, adopts the dna segment that the Asymmetric Polymerization PCR (PCR) that has added unmarked probe amplifies to be needed from sample, add the saturated fluorescence dyestuff, along with decrease of temperature detects its change in fluorescence.Each sample has 2 fusion peaks, and the fusion peak that temperature is lower is used to judge that the base in the probe coverage changes, and the higher fusion peak of temperature is used to judge that base changes in the whole purpose segment.
1. extracting DNA from blood plasma.
2. design primer.
3. design is to the probe in site to be measured, mark fluorescent not, and 3 ' end seals with C3.
4. the adding optical dye carries out the asymmetric PCR amplification like SYTO 9.After the end, on high resolving power PCR appearance, detect melting temperature (Tm), the variation of sample fluorescent signal when analysis temperature descends.
5. analyze sample to be tested BANK1 gene rs10516487 and rs17266594 polymorphum.
Accordingly, the invention provides a kind of test kit of the BANK of detection 1 gene mononucleotide polymorphism, this test kit comprises the primer and the probe of amplification mononucleotide polymorphism site sequence.Can also comprise saturable dye, damping fluid etc.
The detection BANK 1 gene mononucleotide polymorphism method of invention can be used to predict the susceptibility of suffering from systemic lupus erythematous, rheumatoid arthritis or systemic sclerosis.
Two sites of BANK1 gene rs10516487 and rs17266594 are the susceptibility loci of SLE, and the probability that the crowd who carries the CT haplotype suffers from SLE can be used for assessing the risk that individuality is suffered from this disease for 1.6 times of the normal people.
△ 2 hypotypes of BANK1 mean No. 2 exons of BANK1 gene and all lack, the protein delation IP3R land that its translation obtains.Rs17266594 is positioned on this sequence tapping point of BANK1 just.Therefore, the polymorphum of rs17266594 is directly connected to the montage efficient and the proteic function of translation acquisition of BANK 1 gene.Rs10516487 is positioned at the critical area that links IP3R, is a non-synonym alternate SNP site.The polymorphum of rs10516487 and rs17266594 can have influence on the function of BANK 1 gene, thereby impels B-cell receptor to continue releasor through series reaction, causes the activation of B cell transition.
The invention provides a kind of method of the BANK of detection 1 gene mononucleotide polymorphism, can adopt this method to detect BANK1 gene rs10516487 and rs17266594 polymorphum in peripheral blood or the body fluid.Suffer from the possibility of autoimmune disorders such as systemic lupus erythematous, rheumatoid arthritis, systemic sclerosis with judgement.This method operation has quick, simple, free of contamination characteristics, and detected result resolving power is high, can detect all kinds of samples, comprises serum (slurry) and various body fluid.The present invention also provides the relevant detection test kit.
Description of drawings
Fig. 1: rs10516487 and rs17266594 site synoptic diagram.Rs10516487 is a non-synonym alternate SNP site, causes the 61st amino acids to become Histidine by l-arginine; Rs17266594 is positioned on the sequence tapping point, has typical branch point sequence.
Fig. 2: the fluorescence in rs10516487 site is born derivative figure (dF/ dT).The left side is the fusion peak of the lower probe/product of melting temperature (Tm), and the right side is the fusion peak of the higher product/product of melting temperature (Tm).Red line is represented the CC genotype, and blue line is represented the CT genotype, and yellow line is represented the TT genotype.
Fig. 3: the general view of rs10516487 site product/product melting curve.Red line is represented the CC genotype, and blue line is represented the CT genotype, and yellow line is represented the TT genotype.
Fig. 4: the fluorescence in rs17266594 site is born derivative figure (dF/ dT).The left side is the fusion peak of the lower probe/product of melting temperature (Tm), and the right side is the fusion peak of the higher product/product of melting temperature (Tm).Red line is represented the TT genotype, and blue line is represented the TC genotype, and yellow line is represented the CC genotype.
Fig. 5: the general view of rs17266594 site product/product melting curve.Red line is represented the TT genotype, and blue line is represented the TC genotype, and yellow line is represented the CC genotype.
Embodiment
Embodiment 1:rs10516487 loci polymorphism detects
1. DNA extraction:
Use the vacuum test tube of YD 30 (EDTA) anti-freezing to gather venous blood 2ml, use QIAamp DNA Extract Kit (German Qiagen company) test kit to extract DNA.
2. design of primers:
Use Primer 3 softwares (http://frodo.wi.mit.edu/) design primer,, obtain primer through artificial screening and PCR experimental verification.Upstream primer F:5 '-ACATTTGTAAGACGTTAAGTTCAGCA-3 ' of rs10516487 (SEQ ID NO 1), downstream primer R:5 '-ATGATATATGAAGAAGATGCTGAGGA-3 ' (SEQ ID NO 2), expanding fragment length 150 bp.Primer is synthetic by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd.
3. probe design:
Use Primer Premier 5.0 software design probes; The probe of rs10516487 is 5 '-GAAAAGAGAAATTCTCCAAGCGATATAACAGGATG-3 ' (SEQ ID NO 3); Probe is synthetic by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd; 3 ' end is done the C3 sealing, prevents that probe from extending.
4. pattern detection:
Use TaKaRa PCR test kit (precious biological, Japan) to carry out pcr amplification.Reaction system comprises: 0.1
L TaKaRa Taq HS (5 U/ μ l), 2
L 10 * PCR Buffer (-) (Mg
2+Free), 1.2
L MgCl
2(25 mM), 1.6
L dNTP Mixture (each 2.5 mM), upstream primer F 1
L (1
M), downstream primer R 1
L (10
M), probe 1
L (10
M), add water to TV 19.3
L adds 0.7 then
The l sample DNA.The pcr amplification condition: 95 ℃ of 2 min, 94 ℃ of 30 s, 58 ℃ of 30 s, 72 ℃ of 30 s, after 50 circulations, 72 ℃ of 5 min extends.Use Eppendorf personal gene-amplificative instrament (Eppendorf company; German) amplification back adding 0.7
l SYTO 9 dyestuffs (50
M) (Invitrogen company; The U.S.); Again sample hose is transferred to Rotor-Gene 6000 (Mortlake Corbett Research company; Australia), carry out the high resolving power liquation.Condition is: 95 ° of C 2 min, and after 25 ° of C 2 min pre-treatment, melting temperature (Tm) rises to 88 ° of C from 55 ° of C, and 0.2 ° of C of every rising gathers a secondary data, obtains high resolving power melting curve figure.
5. polymorphism analysis
In the negative derivative figure of fluorescence (dF/ dT), what be positioned at the left side is the fusion peak of the lower probe/product of melting temperature (Tm).If the rs10516487 site base of template DNA is cytosine(Cyt) (C), the single stranded product and the probe that then amplify mate fully, and melting temperature (Tm) is higher, and the fusion peak takes over; If this site base of template DNA is thymus pyrimidine (T), then strand can't mate with probe fully, and melting temperature (Tm) is lower, takes back in the fusion peak; If template DNA is a heterozygote, this site base portion is C, and part is T, and then its melting curve can have the characteristic (Fig. 2) at above-mentioned 2 kinds of fusion peaks simultaneously.
What be positioned at the right side is the fusion peak of the higher product/product of melting temperature (Tm).Through software data-switching is become general view, sequence change is observed in the local back of amplifying of the melting curve of product/product.The CC genotype Tm in rs10516487 site is the highest, is positioned at the right side, the TT genotype secondly, and the CT genotype since combine insecure, Tm minimum (Fig. 3).
Through the result of combined with fluorescent derivative figure and general view, can accurately judge the genotype in BANK1 gene rs10516487 site.
1. DNA extraction:
Use the vacuum test tube of YD 30 (EDTA) anti-freezing to gather venous blood 2ml, use QIAamp DNA Extract Kit (German Qiagen company) test kit to extract DNA.
2. design of primers:
Use Primer 3 softwares (http://frodo.wi.mit.edu/) design primer,, obtain primer through artificial screening and PCR experimental verification.Upstream primer F:5 '-AGGACTTTCATAGAGTTTTTCTCTGG-3 ' of rs17266594 (SEQ ID NO 4), downstream primer R:5 '-CATTCCTCAGCATCTTCTTCA-3 ' (SEQ ID NO 5), expanding fragment length 185 bp.Primer is synthetic by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd.
3. probe design:
Use Primer Premier 5.0 software design probes, the probe of rs17266594 is 5 '-TAATAATTTAACCTGCTGATAGCATTGCAAATAT-3 (SEQ ID NO 6).Probe is synthetic by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd, and 3 ' end is done the C3 sealing, prevents that probe from extending.
4. pattern detection:
Use TaKaRa PCR test kit (precious biological, Japan) to carry out pcr amplification.Reaction system comprises: 0.1
L TaKaRa Taq HS (5 U/ μ l), 2
L 10 * PCR Buffer (-) (Mg
2+Free), 1.2
L MgCl
2(25 mM), 1.6
L dNTP Mixture (each 2.5 mM), upstream primer F 1
L (0.67
M), downstream primer R 1
L (10
M), probe 1.5
L (10
M), add water to TV 19.3
L adds 0.7 then
The l sample DNA.The pcr amplification condition: 95 ℃ of 2 min, 94 ℃ of 30 s, 58 ℃ of 30 s, 72 ℃ of 30 s, after 50 circulations, 72 ℃ of 5 min extends.Use Eppendorf personal gene-amplificative instrament (Eppendorf company; German) amplification back adding 0.7
l SYTO 9 dyestuffs (50
M) (Invitrogen company; The U.S.); Again sample hose is transferred to Rotor-Gene 6000 (Mortlake Corbett Research company; Australia), carry out the high resolving power liquation.Condition is: 95 ° of C 2 min, and after 40 ° of C 2 min pre-treatment, melting temperature (Tm) rises to 83 ° of C from 57 ° of C, and 0.2 ° of C of every rising gathers a secondary data, obtains high resolving power melting curve figure.
5. polymorphism analysis
In the negative derivative figure of fluorescence (dF/ dT), what be positioned at the left side is the fusion peak of the lower probe/product of melting temperature (Tm).If the rs17266594 site base of template DNA is thymus pyrimidine (T), the single stranded product and the probe that then amplify mate fully, and melting temperature (Tm) is higher, and the fusion peak takes over; If this site base of template DNA is cytosine(Cyt) (C), then strand can't mate with probe fully, and melting temperature (Tm) is lower, takes back in the fusion peak; If template DNA is a heterozygote, this site base portion is T, and part is C, and then its melting curve can have the characteristic (Fig. 4) at above-mentioned 2 kinds of fusion peaks simultaneously.
What be positioned at the right side is the fusion peak of the higher product/product of melting temperature (Tm).Through software data-switching is become general view, sequence change is observed in the local back of amplifying of the melting curve of product/product.The CC genotype Tm in rs17266594 site is the highest, is positioned at the right side, the TT genotype secondly, and the TC genotype since combine insecure, Tm minimum (Fig. 5).
Through the result of combined with fluorescent derivative figure and general view, can accurately judge the genotype in BANK1 gene rs17266594 site.
Claims (10)
1. a method that detects BANK 1 gene mononucleotide polymorphism is characterized in that, this method comprises successively:
The primer and the probe of design amplification mononucleotide polymorphism site sequence; Described mononucleotide polymorphism site is rs10516487 or rs17266594;
With the sample DNA is template, utilizes above-mentioned primer and probe to carry out pcr amplification; Upstream primer in the primer is different with downstream primer concentration, a kind of 5 times of being not less than alternative concentration;
Add saturable dye;
Analyze the melting degree curve, confirm SNP.
2. the method for claim 1 is characterized in that, described sample is blood ingredient or body fluid.
3. the method for claim 1 is characterized in that, described saturable dye is SYTO 9 or LC Green.
4. the method for claim 1 is characterized in that, concentration and probe concentration is not less than the concentration difference of two kinds of primers.
5. the method for claim 1 is characterized in that, the site of described SNP is rs10516487.
6. method as claimed in claim 5 is characterized in that, with shown in the SEQ ID NO 2, described probe sequence is shown in SEQ ID NO 3 like SEQ ID NO 1 for described primer sequence.
7. the method for claim 1 is characterized in that, the site of described SNP is rs17266594.
8. method as claimed in claim 7 is characterized in that, with shown in the SEQ ID NO 5, described probe sequence is shown in SEQ ID NO 6 like SEQ ID NO 4 for described primer sequence.
9. a test kit that detects BANK 1 gene mononucleotide polymorphism is characterized in that, this test kit comprises the primer and the probe of amplification mononucleotide polymorphism site rs10516487 or rs17266594 sequence.
10. the application of the described method of claim 1 is characterized in that, utilizes the susceptibility of detected result prediction trouble systemic lupus erythematous, rheumatoid arthritis or the systemic sclerosis of BANK 1 gene mononucleotide polymorphism.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110452803A (en) * | 2019-08-27 | 2019-11-15 | 东南大学 | A kind of nucleic acid rapid amplifying detection method and device |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20100099101A1 (en) * | 2008-09-26 | 2010-04-22 | Genentech, Inc. | Methods for treating, diagnosing, and monitoring lupus |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20100099101A1 (en) * | 2008-09-26 | 2010-04-22 | Genentech, Inc. | Methods for treating, diagnosing, and monitoring lupus |
Non-Patent Citations (3)
| Title |
|---|
| KOZYREV 等: "Functional variants in the B-cell gene BANK1 are associated with systemic lupus erthematosus", 《NATURE GENETICS》, vol. 40, no. 2, 29 February 2008 (2008-02-29), pages 211 - 215, XP002478892 * |
| 张鑫: "PXK基因多态性与汉族人系统性红斑狼疮易感性关联分析", 《中国优秀硕士学位论文全文数据库》, no. 4, 15 April 2010 (2010-04-15), pages 065 - 89 * |
| 郜莉娜: "无标记探针检测单核苷酸多态性分子诊断技术的建立和应用", 《中国优秀硕士学位论文全文数据库 医药卫生科技辑》, no. 7, 15 July 2010 (2010-07-15), pages 060 - 35 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110452803A (en) * | 2019-08-27 | 2019-11-15 | 东南大学 | A kind of nucleic acid rapid amplifying detection method and device |
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Application publication date: 20120523 |