CN101525618A - Susceptibility gene region closely related to psoriasis and method for detecting psoriasis susceptible population - Google Patents
Susceptibility gene region closely related to psoriasis and method for detecting psoriasis susceptible population Download PDFInfo
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Abstract
The invention relates to a susceptibility gene region closely related to psoriasis and a method for detecting psoriasis susceptible population. The susceptibility gene region contains 7 genes, that is, LCE3A, LCE3B, LCE3C, LCE3D, LCE3E, LCE2C and LCE2D. The method for detecting psoriasis susceptible population by the region is realized by the following steps: extracting genome DNA; amplifying PCR; purifying by SAP alkaline phosphatase; allowing single base extension reaction of SNP sites according to a UEP primer designed by the SNP sites in the region; scanning by a mass spectrometer, judging difference of the SNP sites in the susceptibility gene region according to different values of molecular weights of various single base extension products; and judging results. The invention discloses the susceptibility gene region closely related to psoriasis, and provides the method for accurately and effectively determining the psoriasis susceptible population according to hereditary properties of the susceptibility gene region.
Description
Technical field
The present invention relates to molecular genetics and dermatological field.Specifically, the present invention relates to one has the susceptibility gene region of close association to be used to detect the method for psoriasis susceptible population with psoriatic.
Background technology
In the tetter field, psoriatic (psoriasis) is a kind of common with erythema, the scales of skin that peel off is main performance, the most common chronic recurrent complex disease, classified as one of the sick field of 20th century human skin two big chronic diseases by the World Health Organization, this sick sickness rate up to 2%, and is 0.123% in Chinese population in white people, domestic have 5,000,000 individuality to suffer from approximately, and to have a net increase of the speed increase of 100,000 cases every year.This disease of psoriatic is commonly called as " psoriasis ", easily difficult radical cure of recurrence and protracted course of disease, and also joint damage appears in patient easily, causes dyskinesia.Along with socioeconomic growing, dermopathic harm more and more receives the concern of various circles of society, and extensive patients presses for and obtains the effective treatment.Think that at present psoriatic is the interactional polygenic disease of nature-nurture, wherein inherited genetic factors plays an important role in psoriatic morbidity.Therefore, the psoriatic Studies on Susceptibility Genes is to illustrate the most important clue of psoriatic pathogenesis.
Up to now, still not obvious in the achievement that obtains aspect the identification of complex disease and complex physiologic process genes involved thereof and the location.Reason is that mainly complex disease has stronger genetic heterogeneity, the uncertainty of hereditary pattern, and the interactive complicacy of environment and gene is aided with at present for the limitation of complex disease understanding etc.Progress along with science and technology, people recognize that more and more genetic flaw is the major reason that many complex diseases produce, the people is when running into psychology and social environment pressure, and those people that carry diseases predisposing gene more may suffer from psoriatic than the people who does not carry diseases predisposing gene.Inherited genetic factors also obtains family, twins basically and entrusts one's child to the care of sb. the epidemiology survey result's of son support.In science and technology growing today, how to detect tumor susceptibility gene and individual disease susceptibility from hereditary angle, to such an extent as to carry out further risk profile and diagnosis, become the severe problem that vast skin scientific and technical personnel and health care personnel face.Although diseases predisposing gene research is both at home and abroad carried out for many years, do not make a breakthrough as yet, rarely have the Study on Value result.For the genetic predisposition of how identifying inheritance susceptible gene and qualification test person, this area lacks comprehensively always, system, effective recognition method.
Summary of the invention
One of purpose of the present invention is to disclose and the closely related susceptibility gene region of psoriatic; Two of purpose is a kind of methods of judging psoriatic Susceptible population of hereditary property proposition by this susceptibility gene region.
The present invention is achieved by the following technical solutions:
A kind of and the closely related susceptibility gene region of psoriatic, this zone contains LCE3A, LCE3B, LCE3C, LCE3D, LCE3E, LCE2C, seven genes of LCE2D, the rs1886734 pleomorphism site that is positioned in this zone on the LCE3A gene is G, the rs4845454 pleomorphism site is T, the rs1011297 pleomorphism site is C, and the rs4845457 pleomorphism site is T, and the rs6687682 pleomorphism site is T, the rs1325508 pleomorphism site is G, and by the SNP site rs1886734 that is positioned on this gene, rs4845454, rs1011297, rs4845457, rs6687682, rs11205050, the haplotype GTCTTAG that rs1325508 forms, GTCTTAG, the individuality of GTTCCGA; The rs4845445 pleomorphism site that is positioned on the LCE3D gene is T, and the rs4085613 pleomorphism site is C, and the rs4112788 pleomorphism site is C, the rs11810844 pleomorphism site is T, and the rs4112789 pleomorphism site is T, and by the SNP site rs12046030 that is positioned on this gene, rs4845445, rs16834214, rs4085613, rs4112788, rs11810844, the haplotype ATCCCTT that rs4112789 forms, CCTCCTT, the individuality of CTTCCCC; The rs1925661 pleomorphism site that is positioned on the LCE2C gene is A, and by the SNP site rs1925661 that is positioned on this gene, the haplotype AA that rs6701538 forms, the individuality of AG; The rs942826 pleomorphism site that is positioned on the LCE2D gene is T, the rs1325507 pleomorphism site is T, the rs908929 pleomorphism site is G, the rs1332497 pleomorphism site is C, and the rs11205062 pleomorphism site is A, and by the SNP site rs942826 that is positioned on this gene, rs1325507, rs908929, rs1332497, the individuality of the haplotype TTGCA that rs11205062 forms; The rs7516108 pleomorphism site that is positioned on the LCE3E gene is C, and by the SNP site rs533917 that is positioned on this gene, rs7530609, rs4240887, rs4845790, rs4845791, rs4845792, rs17659389, the individuality of the haplotype ACACGGAC that rs7516108 forms.
The closely related susceptibility gene region of a kind of and psoriatic is used to detect the method for psoriasis susceptible population, it is characterized in that realizing by following steps:
(1) extracting genome DNA is extracted test kit with minim DNA and is extracted genomic dna;
(2) this zone of pcr amplification includes the purpose fragment in SNP site;
(3) use the SAP alkaline phosphatase to carry out purifying, remove the intact materials such as dNTP of unreacted;
(4) use the single base extension that carries out the SNP site according to the UEP primer of SNP site design in this zone;
(5) use mass spectrograph to scan, judge the difference in SNP site in this susceptibility gene region according to the difference of the molecular weight of each single-basic extension product;
(6) result judges, the difference of the haplotype of forming according to SNP site in this susceptibility gene region and by series of SN-striking P site is determined psoriatic Susceptible population.
The method that the closely related susceptibility gene region of a kind of and psoriatic is used to detect psoriasis susceptible population, realize by following steps:
(1) extracting genome DNA is extracted test kit with minim DNA and is extracted genomic dna;
(2) this zone of pcr amplification includes the purpose fragment in SNP site;
(3) after the PCR product carries out purifying, the PCR product is checked order;
(4) result judges, the difference of the haplotype of forming according to SNP site in this susceptibility gene region and by series of SN-striking P site is determined psoriatic Susceptible population.
Content of the present invention shows, a fragment gene zone that is positioned on the karyomit(e) 1q21 is the susceptibility gene region closely related with psoriatic, comprise LCE3A in this zone, LCE3B, LCE3C, LCE3D, LCE3E, LCE2C, seven genes of LCE2D (referring to Fig. 1), be positioned at the SNP site on these genes of this zone and there are differences, thereby be associated with psoriatic susceptibility by haplotype and normal people that these sites are formed.As the crowd who belongs to one of following situation is psoriatic Susceptible population: the rs1886734 pleomorphism site that is positioned in this zone on the LCE3A gene is G, the rs4845454 pleomorphism site is T, the rs1011297 pleomorphism site is C, the rs4845457 pleomorphism site is T, the rs6687682 pleomorphism site is T, the rs1325508 pleomorphism site is G, and by the SNP site (rs1886734, rs4845454, the rs1011297 that are positioned on this gene, rs4845457, rs6687682, rs11205050, rs1325508) the haplotype GTCTTAG of Zu Chenging, GTCTTAG, the individuality of GTTCCGA; The rs4845445 pleomorphism site that is positioned on the LCE3D gene is T, and the rs4085613 pleomorphism site is C, and the rs4112788 pleomorphism site is C, the rs11810844 pleomorphism site is T, and the rs4112789 pleomorphism site is T, and by the SNP site (rs12046030 that is positioned on this gene, rs4845445, rs16834214, rs4085613, rs4112788, rs11810844, rs4112789) the haplotype ATCCCTT of Zu Chenging, CCTCCTT, the individuality of CTTCCCC; The rs1925661 pleomorphism site that is positioned on the LCE2C gene is A, and by the SNP site (rs1925661, rs6701538) the haplotype AA of Zu Chenging, the individuality of AG that are positioned on this gene; The rs942826 pleomorphism site that is positioned on the LCE2D gene is T, the rs1325507 pleomorphism site is T, the rs908929 pleomorphism site is G, the rs1332497 pleomorphism site is C, and the rs11205062 pleomorphism site is A, and by the SNP site (rs942826 that is positioned on this gene, rs1325507, rs908929, rs1332497, rs11205062) individuality of the haplotype TTGCA of Zu Chenging; The rs7516108 pleomorphism site that is positioned on the LCE3E gene is C, and by the SNP site (rs533917, the rs7530609 that are positioned on this gene, rs4240887, rs4845790, rs4845791, rs4845792, rs17659389, rs7516108) individuality of the haplotype ACACGGAC of Zu Chenging.
This susceptibility gene region is to be positioned at karyomit(e) 1q21 upper epidermis differentiation mixture (epidermaldifferentiation complex, EDC) part in the LCE gene family zone in the zone, comprise above-mentioned seven genes, the hornification coating albumen of the class differentiation in late period of encoding.A lot of dermatosis all exists epidermis propagation and barrier function damage, such as ordinary type ichthyosis, and atopic dermatitis, psoriasis vulgaris etc.Epidermis mainly is made of keratinocyte, quantity accounts for more than 80% of epidermic cell, according to differential period and characteristics, by being deep to shallow stratum basale (basal layer), spinous layer (spinous layer), granular layer (granular layer) and the stratum corneum (stratum corneum) of being divided into.Psoriatic important pathophysiological characteristics are epidermal keratinocytes normal obviously acceleration of multiple fission at stratum basale, the mitoschisis cycle foreshortened to 37.5 hours from normal 311 hours, thereby also cause epidermal transit time (cell travels to the cuticular time by stratum basale) to shorten to 3～4 days by normal 28 days, Parakeratotic phenomenon has appearred in histopathology.Thereby people infer it may is because the interior genetic variation of keratinocyte has caused this kind phenomenon, people infer that also the T lymphocyte discharges cytokine (IL-1 in the stimulation of the environment that comes from the outside and the activatory immunity system in addition, IL-6, IL-8, IL-10, IL-12, IFN-γ etc.) also may inspire and participate in psoriatic course of disease development by stimulating the keratinocyte multiple fission too fast.By the research to the existing member of LCE gene family, discovery can be according to its position and proteic homology in karyomit(e), can be divided into again three subgroups (LCE1X, LCE2X, LCE3X).Wherein, LCE first and second subgroups mainly are high expression levels in skin, are not even identified and express extremely to hang down in inner epithelium (as tongue, esophagus); LCE the 3rd subgroup is a low expression level on skin, and at inner epithelium expression level height.First and second subgroup of LCE is expressed under ultraviolet irradiation obviously and is raised, and the 3rd subgroup is all insensitive to uviolizing except LCE3E.
At first in Chinese han population, choose 1160 psoriatics and 1166 normal artificial research objects (being complementary) at aspects such as age and sexes.All psoriatics' clinical diagnosis is confirmed by at least two experts of Dermatology Department, meets international tetter Case definition, and these patients have two place's skin lesions at least.Case and mate to impinging upon on age and the sex.
Psoriatic and normal controls extract anticoagulation 5ml by peripheral vein, extract genomic dna with the DNA extraction test kit.Step is as follows: 1. draws 1.0ml cell pyrolysis liquid (FG1) in the 2.0ml centrifuge tube of carrying out mark, gets the 400ul blood sample and add corresponding centrifuge tube, put upside down mixing 20-30 time, occur up to no blood clot, and 16, centrifugal 4 minutes of 000g.2. carefully topple over supernatant, thieving paper drains and adds sex change damping fluid/Proteinase K (FG2/PK) solution 200ul after 30 seconds, every adding portion need shake mixing immediately, occurs up to acellular agglomerate, and whether treat all to add the back scrutiny has the cracking fragmentary sample.If any, should thoroughly shake fully.3. of short duration centrifugal after, 65 ℃ of water-baths 20 minutes need be stayed the mouth of pipe about 1/3 more than the liquid level during water-bath, open in case heating causes the pipe lid, impurity such as moisture content enter in the pipe.4. of short duration centrifugal after, add the 200ul Virahol, turn upside down for several times, visible cotton-shaped DNA precipitation has the blackish green agglomerate as finding in the pipe, must firmly shake, and agglomerate is shaken into clear liquor.5. 16, centrifugal 4 minutes of 000g carefully topples over supernatant, and thieving paper drained 30 seconds, as seen manages end adularescent DNA precipitation.6. add 200ul 70% ethanol, the flaky precipitate of carefully upspringing, 16, centrifugal 5 minutes of 000g.7. carefully topple over supernatant, thieving paper drained 20 minutes, add 110ul hydration damping fluid (FG3), 65 ℃ of water-baths 20 minutes, shaking table spends the night, (Nanodrop Spectrophotometer ND-1000) detects the DNA quality, and all samples are diluted to 50ng/ml with standby with FG1 for electrophoresis and ultraviolet spectrophotometer.
(San Diego, USA) chip carries out the somatotype of SNP in the full genome range to utilize illumina Human 610-Quad BeadChips.Step is roughly as follows: at first the genomic dna that detects sample is carried out complete genomic pcr amplification, then amplified production is used the endonuclease section of cutting into pieces, hybridize with the oligonucleotide in previously selected 610,000 SNP sites on this chip, and carry out single-basic extension, according to different fluorescent signal on the not isolabeling of extending base on the SNP site, draw the difference of base on the different SNP site at last by the difference of fluorescent signal then.
According to above-mentioned primary dcreening operation result's data, use the Cochran-Armitage trend test to estimate the related of genotype and phenotype, thereby draw the series of SN-striking P site that between psoriatic and normal population, has significant difference.From these SNP sites that there were significant differences, preferentially choose 64 lower SNP sites of p value and carried out confirmatory experiment, (sequenom Inc) carries out genetic analysis once more to them, analyzes its difference to use MassARRAY system.This time confirmatory experiment independently carries out among case-check sample group at two, first sample cluster is made up of the 5182 routine psoriatics and the 6516 routine normal controls of China Han, and second sample cluster is made up of the 539 routine psoriatics and the 824 routine normal controls of Chinese Xinjiang Uygur.Roughly step is as follows: the genomic DNA that at first extracts above-mentioned crowd, this zone of pcr amplification includes the purpose fragment in SNP site, use the SAP alkaline phosphatase to carry out purifying thereafter, remove the intact materials such as dNTP of unreacted, use the single base extension that carries out the SNP site according to the UEP primer of SNP site design in this zone then, the utilization mass spectrograph scans after using gummy purifying, judge the genotype in SNP site according to the difference of each base molecular weight, carry out the result at last and judge.In data statistic analysis, use the Cochran-Armitage trend test to estimate the related of genotype and phenotype, thereby determine psoriatic Susceptible population according to the significance of difference of the SNP loci gene type between patient and the normal control; Simultaneously, according to the haplotype that the genotype data in the series of SN-striking P site that is obtained by aforesaid method is formed, use Haploview method calculates the haplotype difference between patient and the normal control, thereby determines psoriatic Susceptible population.
By primary dcreening operation experiment and confirmatory experiment, can find to be arranged in one section closely linked Block zone that karyomit(e) 1q21 goes up this zone of LCE gene cluster, in this zone series of SN-striking P site and existed between psoriatic and normal controls by the haplotype that series of SN-striking P site is formed and to have the significance difference opposite sex.Comprise LCE3A in this Block zone, LCE3B, LCE3C, LCE3D, LCE3E, LCE2C, seven genes of LCE2D, the position thereon the SNP site and the difference of the haplotype formed by series of SN-striking P site be associated with psoriatic susceptibility.By the analysis of haplotype frequency difference between patient and normal people of forming to these SNP sites and by series of SN-striking P site, we can draw the variation of each concrete base in site and by the relation of its haplotype difference formed and psoriatic susceptibility.As the crowd who belongs to one of following situation is psoriatic Susceptible population: the rs1886734 pleomorphism site that is positioned in this zone on the LCE3A gene is G, the rs4845454 pleomorphism site is T, the rs1011297 pleomorphism site is C, the rs4845457 pleomorphism site is T, the rs6687682 pleomorphism site is T, the rs1325508 pleomorphism site is G, and by the SNP site (rs1886734, rs4845454, the rs1011297 that are positioned on this gene, rs4845457, rs6687682, rs11205050, rs1325508) the haplotype GTCTTAG of Zu Chenging, GTCTTAG, the individuality of GTTCCGA; The rs4845445 pleomorphism site that is positioned on the LCE3D gene is T, and the rs4085613 pleomorphism site is C, and the rs4112788 pleomorphism site is C, the rs11810844 pleomorphism site is T, and the rs4112789 pleomorphism site is T, and by the SNP site (rs12046030 that is positioned on this gene, rs4845445, rs16834214, rs4085613, rs4112788, rs11810844, rs4112789) the haplotype ATCCCTT of Zu Chenging, CCTCCTT, the individuality of CTTCCCC; The rs1925661 pleomorphism site that is positioned on the LCE2C gene is A, and by the SNP site (rs1925661, rs6701538) the haplotype AA of Zu Chenging, the individuality of AG that are positioned on this gene; The rs942826 pleomorphism site that is positioned on the LCE2D gene is T, the rs1325507 pleomorphism site is T, the rs908929 pleomorphism site is G, the rs1332497 pleomorphism site is C, and the rs11205062 pleomorphism site is A, and by the SNP site (rs942826 that is positioned on this gene, rs1325507, rs908929, rs1332497, rs11205062) individuality of the haplotype TTGCA of Zu Chenging; The rs7516108 pleomorphism site that is positioned on the LCE3E gene is C, and by the SNP site (rs533917, the rs7530609 that are positioned on this gene, rs4240887, rs4845790, rs4845791, rs4845792, rs17659389, rs7516108) individuality of the haplotype ACACGGAC of Zu Chenging.
Comprise LCE3A according to the present invention by one section, LCE3B, LCE3C, LCE3D, LCE3E, LCE2C, the method for the variation property judgement psoriasis susceptible population of SNP site in seven gene regions of LCE2D and the haplotype is made up of series of SN-striking P site can be carried out the examination of method as follows to the crowd who the psoriatic clinical symptom do not occur.Risk factor should be reduced in daily life to psoriasis susceptible population as far as possible,, psoriatic sickness rate can be reduced as stimulation of environmental factors etc.This is an important use of the present invention.
According to the present invention, can adopt gene engineering method to carry out gene therapy targetedly to psoriatic patient.In addition, the existence of disease gene can cause the damaged of protein structure and function or change, and hinders or disturb the interaction of biomacromolecule in the specific biochemical pathway.Just because of the difference of base and the haplotype be made up of series of SN-striking P site on the SNP site in this susceptibility gene region can cause protein expression and dysfunction.The present invention further detects protein function, furthers investigate psoriatic mechanism of causing a disease, and the development of new medicine is carried out based on the individuation pharmacological agent of genetic background and established crucial basis, and will bring very considerable society and economy benefit.
Embodiment
By extracting experimenter's genomic dna, contain LCE3A, LCE3B to one section, LCE3C, LCE3D, LCE3E, LCE2C, the SNP site is carried out and the analysis of the haplotype be made up of series of SN-striking P site in the Block zone of seven kinds of genes of LCE2D, judges psoriatic Susceptible population.Specific practice is:
Embodiment 1
(sequenom, Inc) the haplotype difference that detects LCE gene SNP site difference in this Block zone and draw is thus judged psoriatic Susceptible population below by MassARRAY system.
1. the extraction of genomic dna
The experimenter extracts anticoagulation 5ml by peripheral vein, extracts genomic dna with the DNA extraction test kit.Step is as follows: 1. draws 1.0ml cell pyrolysis liquid (FG1) in the 2.0ml centrifuge tube of carrying out mark, gets the 400ul blood sample and add corresponding centrifuge tube, put upside down mixing 20-30 time, occur up to no blood clot, and 16, centrifugal 4 minutes of 000g.2. carefully topple over supernatant, thieving paper drains and adds sex change damping fluid/Proteinase K (FG2/PK) solution 200ul after 30 seconds, every adding portion need shake mixing immediately, occurs up to acellular agglomerate, and whether treat all to add the back scrutiny has the cracking fragmentary sample.If any, should thoroughly shake fully.3. of short duration centrifugal after, 65 ℃ of water-baths 20 minutes need be stayed the mouth of pipe about 1/3 more than the liquid level during water-bath, open in case heating causes the pipe lid, impurity such as moisture enter in the pipe.4. of short duration centrifugal after, add the 200ul Virahol, turn upside down for several times, visible cotton-shaped DNA precipitation has the blackish green agglomerate as finding in the pipe, must firmly shake, and agglomerate is shaken into clear liquor.5. 16, centrifugal 4 minutes of 000g carefully topples over supernatant, and thieving paper drained 30 seconds, as seen manages end adularescent DNA precipitation.6. add 200ul 70% ethanol, the flaky precipitate of carefully upspringing, 16, centrifugal 5 minutes of 000g.7. carefully topple over supernatant, thieving paper drained 20 minutes, the hydration damping fluid (FG3) that adds 110ul, 65 ℃ of water-baths 20 minutes, shaking table spends the night, (NanodropSpectrophotometer ND-1000) detects the DNA quality, and all samples are diluted to 50ng/ml with standby with FG1 for electrophoresis and ultraviolet spectrophotometer.
2.PCR amplification contains the purpose fragment in SNP site
Design can amplify the upstream and downstream primer that contains the SNP site in above-mentioned seven kinds of gene regions, disposes 0.5uMPCR primer pond, comprises the upstream and downstream primer of each array of corresponding tuple.By following system configurations PCRCocktail.
| Reagent | Concentration | Volume (μ L) |
| Other water of HPLC level | N/A | 1.750 |
| The 10X PCR damping fluid that contains 15mM MgCl2 | 1.25x(1.875mM MgCl2) | 0.625 |
| 25mM MgCl2 | 1.625mM | 0.325 |
| 25mM dNTP mixed solution | 500uM | 0.100 |
| 0.5uM SEQ ID No.5-12 primer | 0.1uM | 1.000 |
| 5U/ μ l HotStar archaeal dna polymerase | 1Unit | 0.200 |
| Cumulative volume [μ L] | n/a | 4.000 |
The PCR Cocktail that adds 4ul is in 384 hole PCR plates and mixing.If instrument and the corresponding operable words of method, this step can be used liquid processor.
Add in each holes of DNA to 384 new hole PCR plates of 1ul 10ng/ul (now being called sample plane).If instrument and the corresponding operable words of method, this step can be used liquid processor.When finishing, it is centrifugal then to seal plank.
384 hole PCR plates are put on the PCR instrument of a standard and use program:
95℃ 15min
72℃ 3min
4℃ forever
3.SAP purifying experiment
According to following table configuration SAP mixed solution.
| Reagent | Concentration | Volume (ul) |
| The autoclaving pure water | n/a | 1.53 |
| The SAP damping fluid | 0.24x | 0.17 |
| SAP alkaline phosphatase (1.7U/ul) | 0.5U | 0.30 |
| Cumulative volume [uL] | n/a | 2 |
Draw 2ul SAP, mixed solution is to 384 hole PCR plates.When finishing, shrouding is also centrifugal.
The according to the form below program is hatched:
37℃ 40min
85℃ 5min
4℃ ∞
4. single base extension
Extend the primer mixing pit according to following rule preparation 8/10/14uM iPLEX:
1) with multiple reaction divided by 3.
2) the oligonucleotide extension primer final concentration of calculating LOW Mass is 8uM
3) the oligonucleotide extension primer final concentration of calculating MEDIUM Mass is 10uM
4) the oligonucleotide extension primer final concentration of calculating HIGH Mass is 14uM
5) volume of calculating surplus water
IPLEX extends primer according to the following table preparation:
| Reagent | Concentration | Volume (ul) |
| The autoclaving pure water | N/A | 0.619 |
| IPLEX Buffer damping fluid | 0.222X | 0.200 |
| IPLEX stops mixed solution | 1X | 0.200 |
| IPLEX extends the primer mixed solution | 0.84/1.04/1.25 | 0.940 |
| IPLEX extension enzyme | 1X | 0.041 |
| The SAP+PCR mixed solution | n/a | 7.000 |
| Cumulative volume [uL] | n/a | 9.000 |
Draw 2ul SAP mixed solution to 384 hole PCR plates.When finishing, shrouding is also centrifugal.
The according to the form below program is hatched:
94℃ for 30sec
72℃ for 3min
4℃ forever
5. gummy purifying
Use liquid processor to add 16ul water in 384 hole PCR plates, add 6mg purifying natural gum in 384 hole PCR plates, rotated 1 hour.
6. put coremaking sheet and scanning of the mass spectrum
Divide at trace and to carry out the point sample speed that the point sample volume is optimized the optimum of seeking sample on the liquid system.Utilize then trace divide liquid system from 384 hole PCR plate application of samples to (San Diego is on the SpectroCHIP chip of MassArray system USA) from Sequenom.Use the MALDi TOF scanning of the mass spectrum of this system then, judge the genotype in this each SNP site, fragment gene zone.
7. the result judges
The result judges, the difference of the haplotype of forming according to SNP site in this susceptibility gene region and by this a series of SNP site is determined psoriatic Susceptible population.
Embodiment 2
Detect the difference of LCE gene SNP site in this Block zone below by gene sequencing, judge psoriatic Susceptible population.
1. the extraction of genomic dna
Extract genomic dna after experimenter's peripheral vein extracts anticoagulation, step is seen first part in the implementation method one.
2.PCR amplification contains the purpose fragment in SNP site
Design can amplify the upstream and downstream primer of LCE gene SNP site in this Block zone, by following system configurations
| Reagent | Concentration | Volume (μ L) |
| Other water of HPLC level | N/A | 1.750 |
| The 10X PCR damping fluid that contains 15mM MgCl2 | 1.25x(1.875mM MgCl2) | 0.625 |
| 25mM MgCl2 | 1.625mM | 0.325 |
| 25mM dNTP mixed solution | 500uM | 0.100 |
| 0.5uM primer | 0.1uM | 1.000 |
| 5U/ μ l HotStar archaeal dna polymerase | 1Unit | 0.200 |
| Cumulative volume [μ L] | n/a | 4.000 |
The PCR Cocktail that adds 4ul is in 384 hole PCR plates and mixing.If instrument and the corresponding operable words of method, this step can be used liquid processor.
Add in each holes of DNA to 384 new hole PCR plates of 1ul 10ng/ul (now being called sample plane).If instrument and the corresponding operable words of method, this step can be used liquid processor.When finishing, it is centrifugal then to seal plank.
384 hole PCR plates are put on the PCR instrument of a standard and use program:
95℃ 15min
72℃ 3min
4℃ forever
3.PCR product directly checks order
At first carry out the purifying of PCR product, ABI 3730 sequenators are used in the censorship order-checking behind the purifying.
4. the result judges
The result judges, the difference of the haplotype of forming according to the SNP site of learning after the order-checking in this susceptibility gene region and by this a series of SNP site is determined psoriatic Susceptible population.
Sequence table
＜110〉institute of internal medicine of First Attached Hospital, Anhui Medical Univ.
＜120〉the closely related susceptibility gene region of a kind of and psoriatic and be used to detect the method for psoriasis susceptible population
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<170>Patent In Version 3.1
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TAAGGAGCTT GCCCATCCTT TTCCTT[G/T]TTT CTCCATTATG TGTTTTAATT CT 52
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ACAAATTCAG AAAGGACTCA GCAGGG[C/T]AAT TCTCATTTCA TGCATTTGTC GT 52
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TTTGTTAAGT TTTAGTGTCA GAGCTA[C/T]GCT GGAAGTAACT TTGCCCTTAT AT 52
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CACCACTGAC CACTGCAATA CCATGA[C/T]CAT AGGTAGTAAC ACTGGTGGGA TC 52
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CTCTGTCTGG ACGTCTCTTT ATTCTC[C/T]AAT CTCTTTCCTT TTTTTTTTCT TG 52
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ATGACTCAGG AAAATGTTGG GGAAAT[A/G]GCT CAAGTACCAA CAGGGAAATA AA 52
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<213>Homo sapiens
<400>7
GAGATTCTGT TTGTCTTGGG GAAAGT[A/G]AAA GAAAGAGAAC AAGCGTCTCT GC 52
<210>8
<211>52
<212>DNA
<213>Homo sapiens
<400>8
CATAGAGAGG ACAACAGACA CAGGGA[C/T]GTA CCAGAGGGTG GGGAATAGAA GG 52
<210>9
<211>52
<212>DNA
<213>Homo sapiens
<400>9
TCAAACTGCC TATTTATTTT TACTGC[A/C]AAG TCTAAGAACT CAGGATTTCT AA 52
<210>10
<211>52
<212>DNA
<213>Homo sapiens
<400>10
AAAACCTTTA GACTACAATT AAAAGA[C/T]GGG TAAAGAAAAA CATGGCCTCA GC 52
<210>11
<211>52
<212>DNA
<213>Homo sapiens
<400>11
CAACAAATAG AGATGAAATA ATTGGA[C/T]GTC CATATGTAAA AGAAATCATA CC 52
<210>12
<211>52
<212>DNA
<213>Homo sapiens
<400>12
TAATTTACTA TCAAAGTTAT CATTTT[C/T]GCT TGGATGGTGA ATTTTTGTGT GT 52
<210>13
<211>52
<212>DNA
<213>Homo sapiens
<400>13
ACAGTGGACC ACGGATGTTA GAGCTG[A/C]AAC CAGAGATGGA GATAAGAAAC TA 52
<210>14
<211>52
<212>DNA
<213>Homo sapiens
<400>14
CATCCCTTAT TCTTACTTGA TTGCTG[C/T]CTG TGGTATAAAA GTAAGGTGTT AC 52
<210>15
<211>52
<212>DNA
<213>Homo sapiens
<400>15
TATCAGTGGC TTCCAGGTGC TTTAGC[A/G]GGC TGGGAGATAT TGAGAAGTTA CT 52
<210>16
<211>52
<212>DNA
<213>Homo sapiens
<400>16
ACATGTGATT AAGTCAGAGG TCAGTA[A/G]TCC ATGGCTGGTA TGGTGGTTCC CT 52
<210>17
<211>52
<212>DNA
<213>Homo sapiens
<400>17
CAGCTCCAGC ACCTCATCTG CTCTGA[C/T]GCC CTAGGGACAT GTCTGTGCTC TT 52
<210>18
<211>52
<212>DNA
<213>Homo sapiens
<400>18
ATGGGCTTTT CTCATGCAAA GAAGTG[C/T]TTC AGAGATTCTA GTGGACTGTG AG 52
<210>19
<211>52
<212>DNA
<213>Homo sapiens
<400>19
AGGAAAACAT GTCCAAGATA GCCTAC[A/G]GGA CCCAAAAGGA GGAATGAAAG AC 52
<210>20
<211>52
<212>DNA
<213>Homo sapiens
<400>20
GAAAATTTAA AAGACTGATC CGAAAT[C/T]ACT TTTTGGCTTA ATTTTAAGTT CT 52
<210>21
<211>52
<212>DNA
<213>Homo sapiens
<400>21
TTATCATATG CAAGTTTCCT GATTCC[A/G]GCA ATGGATTCAA TACAATTGCT TC 52
<210>22
<211>52
<212>DNA
<213>Homo sapiens
<400>22
AGTCTCTCTT GACTACCTCC CTGGAC[C/T]TTG CCCCTTGTGT AGGTCATACT TT 52
<210>23
<211>52
<212>DNA
<213>Homo sapiens
<400>23
CAGACTCCAT CCCCTGCTGC TACTTA[A/G]CAG ATAAGGCCTG CATGCCTGGG CT 52
<210>24
<211>52
<212>DNA
<213>Homo sapiens
<400>24
GATTACTCAT GAGAAACCTT ACAAGC[A/C]AGA AGAAATTGGG GGCACATTTA CA 52
<210>25
<211>52
<212>DNA
<213>Homo sapiens
<400>25
ATTTCTCTCT TTGCACCTGT CACCTA[A/G]TTA GCTTTCAAGG ATCCCTTCCT AC 52
<210>26
<211>52
<212>DNA
<213>Homo sapiens
<400>26
CTGCTTCAAT TTCAACAAAT TTCTCT[C/T]TTT TTTTAAATAC CTATTGGTGC GT 52
<210>27
<211>52
<212>DNA
<213>Homo sapiens
<400>27
CTCTTTTTTT AAATACCTAT TGGTGC[A/G]TAT CTGAATATCT GATATTAAAC TT 52
<210>28
<211>52
<212>DNA
<213>Homo sapiens
<400>28
TGATCCCTAA ATTTTAAGAA TTAATT[G/T]ATA GGATTTGACA AAATCCTTTT TT 52
<210>29
<211>52
<212>DNA
<213>Homo sapiens
<400>29
AGACTATCCA TTCATCACCT TCCTGT[A/G]TAC TCACCAAGAT GCCTGACAAG AG 52
<210>30
<211>30
<212>DNA
＜213〉synthetic
<400>30
ACGTTGGATG CATGCTTGGG AAGTACTTTT 30
<210>31
<211>29
<212>DNA
＜213〉synthetic
<400>31
ACGTTGGATG CATAAGGAGC TTGCCCATC 29
<210>32
<211>17
<212>DNA
＜213〉synthetic
<400>32
TGCCCATCCT TTTCCTT 17
<210>33
<211>30
<212>DNA
＜213〉synthetic
<400>33
ACGTTGGATG GGGTCACAAA TTCAGAAAGG 30
<210>34
<211>30
<212>DNA
＜213〉synthetic
<400>34
ACGTTGGATG GCTCCAATCA ACATCTGACG 30
<210>35
<211>23
<212>DNA
＜213〉synthetic
<400>35
GACAAATGCA TGAAATGAGA ATT 23
<210>36
<211>30
<212>DNA
＜213〉synthetic
<400>36
ACGTTGGATG TTTGAAAACG TCAAACTGCC 30
<210>37
<211>30
<212>DNA
＜213〉synthetic
<400>37
ACGTTGGATG CAGAGATTTT AGAAATCCTG 30
<210>38
<211>19
<212>DNA
＜213〉synthetic
<400>38
ATCCTGAGTT CTTAGACTT 19
<210>39
<211>30
<212>DNA
＜213〉synthetic
<400>39
ACGTTGGATG GCAAGGCTGA AAACCTTTAG 30
<210>40
<211>30
<212>DNA
＜213〉synthetic
<400>40
ACGTTGGATG AGGATTACAG ACAGCTGAGG 30
<210>41
<211>21
<212>DNA
＜213〉synthetic
<400>41
ttGCCATGTT TTTCTTTACC C 21
<210>42
<211>30
<212>DNA
＜213〉synthetic
<400>42
ACGTTGGATG CAAGAAAGTA TGACCTACAC 30
<210>43
<211>30
<212>DNA
＜213〉synthetic
<400>43
ACGTTGGATG ACTTCTTCAG GGACAGTCTC 30
<210>44
<211>15
<212>DNA
＜213〉synthetic
<400>44
ACTACCTCCC TGGAC 15
Claims (3)
1, a kind of and the closely related susceptibility gene region of psoriatic, it is characterized in that this zone contains LCE3A, LCE3B, LCE3C, LCE3D, LCE3E, LCE2C, seven genes of LCE2D, the rs1886734 pleomorphism site that is positioned in this zone on the LCE3A gene is G, the rs4845454 pleomorphism site is T, the rs1011297 pleomorphism site is C, and the rs4845457 pleomorphism site is T, and the rs6687682 pleomorphism site is T, the rs1325508 pleomorphism site is G, and by the SNP site rs1886734 that is positioned on this gene, rs4845454, rs1011297, rs4845457, rs6687682, rs11205050, the haplotype GTCTTA6 that rs1325508 forms, GTCTTAG, the individuality of GTTCCGA; The rs4845445 pleomorphism site that is positioned on the LCE3D gene is T, and the rs4085613 pleomorphism site is C, and the rs4112788 pleomorphism site is C, the rs11810844 pleomorphism site is T, and the rs4112789 pleomorphism site is T, and by the SNP site rs12046030 that is positioned on this gene, rs4845445, rs16834214, rs4085613, rs4112788, rs11810844, the haplotype ATCCCTT that rs4112789 forms, CCTCCTT, the individuality of CTTCCCC; The rs1925661 pleomorphism site that is positioned on the LCE2C gene is A, and by the SNP site rs1925661 that is positioned on this gene, the haplotype AA that rs6701538 forms, the individuality of AG; The rs942826 pleomorphism site that is positioned on the LCE2D gene is T, the rs1325507 pleomorphism site is T, the rs908929 pleomorphism site is G, the rs1332497 pleomorphism site is C, and the rs11205062 pleomorphism site is A, and by the SNP site rs942826 that is positioned on this gene, rs1325507, rs908929, rs1332497, the individuality of the haplotype TTGCA that rs11205062 forms; The rs7516108 pleomorphism site that is positioned on the LCE3E gene is C, and by the SNP site rs533917 that is positioned on this gene, rs7530609, rs4240887, rs4845790, rs4845791, rs4845792, rs17659389, the individuality of the haplotype ACACGGAC that rs7516108 forms.
2. be used to detect the method for psoriasis susceptible population according to claims 1 described susceptibility gene region a kind of and that psoriatic is closely related, it is characterized in that realizing by following steps:
(1) extracting genome DNA is extracted test kit with minim DNA and is extracted genomic dna;
(2) this zone of pcr amplification includes the purpose fragment in SNP site;
(3) use the SAP alkaline phosphatase to carry out purifying, remove the intact materials such as dNTP of unreacted;
(4) use the single base extension that carries out the SNP site according to the UEP primer of SNP site design in this zone;
(5) use mass spectrograph to scan, judge the difference in SNP site in this susceptibility gene region according to the difference of the molecular weight of each single-basic extension product;
(6) result judges, the difference of the haplotype of forming according to SNP site in this susceptibility gene region and by series of SN-striking P site is determined psoriatic Susceptible population.
3. be used to detect the method for psoriasis susceptible population according to claims 1 described susceptibility gene region a kind of and that psoriatic is closely related, it is characterized in that realizing by following steps:
(1) extracting genome DNA is extracted test kit with minim DNA and is extracted genomic dna;
(2) this zone of pcr amplification includes the purpose fragment in SNP site;
(3) after the PCR product carries out purifying, the PCR product is checked order;
(4) result judges, the difference of the haplotype of forming according to SNP site in this susceptibility gene region and by series of SN-striking P site is determined psoriatic Susceptible population.
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| CN200910116500A CN101525618A (en) | 2009-04-08 | 2009-04-08 | Susceptibility gene region closely related to psoriasis and method for detecting psoriasis susceptible population |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105969859A (en) * | 2016-05-13 | 2016-09-28 | 深圳市核子基因科技有限公司 | Mammary-cancer-susceptible-gene detection chip and preparation method thereof |
| CN109207575A (en) * | 2017-07-02 | 2019-01-15 | 复旦大学附属华山医院 | A kind of gene marker and detection kit for predicting methotrexate treatment psoriasis clinical efficacy |
| CN112080559A (en) * | 2019-06-14 | 2020-12-15 | 复旦大学附属华山医院 | Application of PPP1CB gene SNP locus in preparation of product for detecting psoriasis vulgaris susceptibility |
| CN114196748A (en) * | 2021-11-09 | 2022-03-18 | 安徽医科大学 | Early prediction biomarker and prediction model for acute pancreatitis and construction method thereof |
| CN114371135A (en) * | 2021-10-25 | 2022-04-19 | 孙良丹 | Evaluation system for evaluating psoriasis and application |
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2009
- 2009-04-08 CN CN200910116500A patent/CN101525618A/en active Pending
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105969859A (en) * | 2016-05-13 | 2016-09-28 | 深圳市核子基因科技有限公司 | Mammary-cancer-susceptible-gene detection chip and preparation method thereof |
| CN109207575A (en) * | 2017-07-02 | 2019-01-15 | 复旦大学附属华山医院 | A kind of gene marker and detection kit for predicting methotrexate treatment psoriasis clinical efficacy |
| CN109207575B (en) * | 2017-07-02 | 2022-02-11 | 复旦大学附属华山医院 | Gene marker and detection kit for predicting clinical effect of methotrexate on treating psoriasis |
| CN112080559A (en) * | 2019-06-14 | 2020-12-15 | 复旦大学附属华山医院 | Application of PPP1CB gene SNP locus in preparation of product for detecting psoriasis vulgaris susceptibility |
| CN112080559B (en) * | 2019-06-14 | 2023-09-01 | 复旦大学附属华山医院 | Application of SNP locus of PPP1CB gene in preparation of product for detecting susceptibility to psoriasis vulgaris |
| CN114371135A (en) * | 2021-10-25 | 2022-04-19 | 孙良丹 | Evaluation system for evaluating psoriasis and application |
| CN114371135B (en) * | 2021-10-25 | 2024-01-30 | 孙良丹 | Evaluation system for evaluating psoriasis and application |
| CN114196748A (en) * | 2021-11-09 | 2022-03-18 | 安徽医科大学 | Early prediction biomarker and prediction model for acute pancreatitis and construction method thereof |
| CN114196748B (en) * | 2021-11-09 | 2024-01-30 | 安徽医科大学 | Early-stage acute pancreatitis prediction biomarker, prediction model and construction method thereof |
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