CN101748194A

CN101748194A - Genetic detecting chip for hypertension and vasculopathy disease risks and application thereof

Info

Publication number: CN101748194A
Application number: CN200810204126A
Authority: CN
Inventors: 高平进; 张怡; 朱鼎良
Original assignee: Shanghai Research Institute Of Hypertension
Current assignee: Shanghai Research Institute Of Hypertension
Priority date: 2008-12-05
Filing date: 2008-12-05
Publication date: 2010-06-23

Abstract

The invention relates to a gene polymorphism detecting chip, in particular to a genetic detecting chip for hypertension and vasculopathy disease risks, which comprises a solid phase vector and a probe fixed on the solid phase vector, wherein the probe comprises a probe for detecting the related gene polymorphism of hypertension and vasculopathy, and the probe is hybridized with the nucleotide sequence and/or the complementary sequence of the related gene of the hypertension and vasculopathy disease risks to be detected. The invention also relates to a method for carrying out disease risk pharmacogenetic detection of hypertension and vasculopathy by the chip. The detecting chip of the invention can enable the medical personnel to master the genetic information of patients with cardiovascular diseases and cerebrovascular diseases such as hypertension and the like in time and instruct patients to take medicines reasonably and realize individuation medical treatment.

Description

Genetics detection chip of hypertension and vascular lesion disease risks and uses thereof

Technical field

The present invention relates to gene polymorphism detecting chip, relate in particular to genetics detection chip of a kind of hypertension and vascular lesion disease risks and uses thereof.

Background technology

Hypertension is a kind of common disease, and the countries in the world morbidity is up to 10%～20%.In recent years, China's hypertension incidence rate has obvious ascendant trend.Show that according to national resident's nutrition in 2002 and investigation of health conditions data China adult hypertension morbidity is 18.8%, there is hyperpietic about 1.6 hundred million in the whole nation.Hypertension causes the serious pathology of important organs such as the heart, brain, kidney, is the most important Hazard Factor of cardiovascular and cerebrovascular disease.In China, cardiovascular and cerebrovascular disease have become the first cause of the death, account for 44.4% of total death.In causing dead various Hazard Factor, hypertension ranks the first.Hypertension and relative disease bring great burden for society and family.According to statistics in 2003, the direct medical fee of China's hypertension was 30,000,000,000 yuans, caused directly and indirectly expending up to annual 3000 hundred million yuans of cardiovascular and cerebrovascular disease thus.

In disease of multifactorial inheritance such as hypertension, genovariation is given the patient susceptibility to disease, and it can be used as the hereditary biological marker of forecast disease risks.Compare with conventional risk factors, hereditary biological marker is deep into gene level, more accurately, reliably, is not subjected to medicine, food and various Effect of Environmental; And can be teenager even the early screening easy patient that goes out hypertension or cardiovascular and cerebrovascular disease more.Thereby hereditary biological marker research enjoys attention.If the SNP that selection is relevant with hypertension and vascular lesion formulates gene detecting chip, in the crowd, carry out examination, might find the high risk population of easy generation hypertension and vascular lesion in early days, and accomplish to prevent and treat early.

The pathologic basis that hypertension takes place is that blood vessel is reinvented blood vessel structure and the changing function that causes, and therefore hypertensive essence is a kind of vascular lesions.The target organ damage that hypertension causes also is the organ dysfunction attenuation process based on blood vessel injury.Blood vessel injury is the pathologic basis that hypertension takes place, and also is the architecture basics that hypertension is kept, developed.Therefore how taking easy, quick, accurate means is the high-risk patient that examination goes out hypertension and blood vessel injury in hypertension in early days; take to treat preventive measures early; all significant for the inverse amplification factor that improves cardiovascular disorder, the mortality ratio that reduces cardiovascular complication, the quality of life of improving patient, the national health care expenditures of minimizing etc., also be the important topic of present medical research and clinical application.

Osteopontin (osteopontin OPN) is a kind of important cell adhesion and chemokine, and it mainly brings into play the effect of cell signal molecules by two kinds of mechanism: the one, combine with the integral protein family molecule with intramolecularly RGD primitive; The 2nd, rely on mode with cell surface adhesion gC D44 with non-RGD and combine.All mediated cell sticks, moves and breeds two kinds of modes of action by activating cells internal specific signal transducting system.In recent years, existing researchist finds that OPN and blood vessel injury and blood vessel reinvent relevant (Li G, Chen SJ, Oparil S, et al.Direct in vivo evidence demonstrating neointimal migration of adventitialfibroblasts after balloon injury of rat carotid artery.Circulation, 2000,101:1362-1365).RhoA/ROK regulates the active signal of interest path transduction of cardiovascular function path.RhoA albumen (Ras homolog gene family, member A) is a member in the Rho subfamily of Rho family in the small G-protein superfamily, be acknowledged as the main setter of the Ca2+ sensibilized of contractile protein now, be responsible for the enhancing assembly that vascular smooth muscle cell is shunk.Rho albumen starts the activation that Rho connects kinases (Rho associated kinase) as a kind of molecular switch, the Rho kinases of ROCK α genes encoding is as the specific biological effect of effector molecule performance in Rho downstream, the contraction of mediation smooth muscle cell and non-smooth muscle cell, the change of the sticking of cell, migration, propagation, apoptosis and cytoskeleton, thus participate in a series of physiological and pathological process in the body.Upstream signals different in the hypertension vascular conditions can be assembled to the RhoA activation, the RhoA/Rho kinases has vital role (Masumoto A in hypertension, HirookaY, Shimokawa H, Hironaga K, SetoguchiS, Takeshita A.Possible involvement of Rho-kinase in the pathogenesis ofhypertension in humans.Hypertension.2001; 38:1307-1310.).These changes are important steps of hypertension pathology process.Therefore, select SNP above-mentioned and hypertension and vascular lesion genes involved, formulate gene detecting chip, in the crowd, carry out screening, might find the high risk population that hypertension and vascular lesion take place, and in time carry out preventing and controlling.

Summary of the invention

The technical problem to be solved in the present invention provides genetics detection chip of a kind of hypertension and vascular lesion disease risks and uses thereof, this chip can effectively detect the polymorphism of hypertension and vascular lesion genes involved, and auxiliary doctor in time finds the diagnostic method of treatment hypertension and vascular lesion disease risks.In addition, the present invention also provides the method for using this chip detection hypertension and vascular lesion related gene polymorphism.

In order to solve the problems of the technologies described above, the present invention is achieved through the following technical solutions:

In one aspect of the invention, the genetics detection chip of a kind of hypertension and vascular lesion disease risks is provided, comprise solid phase carrier and the probe that is fixed on the described solid phase carrier, described probe comprises the probe that detects hypertension and vascular lesion related gene polymorphism, and described probe and hypertension to be measured and nucleotide sequence and/or its complementary sequence of vascular lesion genes involved are hybridized.Solid phase carrier described in the present invention can be selected the known carrier in field for use, as long as described carrier is compatible with described reactant, it is just passable can not influence detected result.Preferably, solid phase carrier of the present invention can be a kind of in slide, silicon chip, nitrocellulose filter, nylon membrane and the macromolecular material or their arbitrary combination.

Described hypertension to be measured and vascular lesion disease risks genes involved comprise OPN, RhoA, ROK α gene.

Described probe can be DNA, RNA, DNA-RNA mosaic, PNA or derivatives thereof.The length of described probe combines as long as can finish with purpose nucleotide sequence specificity without limits.Because different probe length has different influences to hybridization efficiency, signal specificity, the length of described probe is 14 base pairs usually at least, the longlyest generally be no more than 30 base pairs, and purpose nucleotide sequence complementary length is between 15-25 the base pair being the best.Self complementary sequence of described probe is most preferably less than 6 base pairs, in order to avoid influence hybridization efficiency.

The probe of detection chip of the present invention is DNA, comprising:

(1) with sequence shown in (a) SEQ ID NO:1～SEQ ID NO:33 of OPN gene recombination to be measured, (b) complementary strand of every sequence in the sequence shown in SEQ ID NO:1～SEQ ID NO:33, and/or every sequence has the sequence of at least 70% homology (c) and in the sequence shown in SEQ ID NO:1～SEQ ID NO:33;

(2) with sequence shown in (a) SEQ ID NO:34～SEQ ID NO:48 of ROK α gene recombination to be measured, (b) complementary strand of every sequence in the sequence shown in SEQ ID NO:34～SEQ ID NO:48, and/or every sequence has the sequence of at least 70% homology (c) and in the sequence shown in SEQ ID NO:34～SEQ ID NO:48;

(3) with sequence shown in (a) SEQ ID NO:49～SEQ ID NO:54 of RhoA gene recombination to be measured, (b) complementary strand of every sequence in the sequence shown in SEQ ID NO:49～SEQ ID NO:54, and/or every sequence has the sequence of at least 70% homology (c) and in the sequence shown in SEQ ID NO:49～SEQ ID NO:54.

Preferably, the probe of detection chip of the present invention is selected from sequence shown in SEQ ID NO:1～SEQ IDNO:54.

Sequence involved in the present invention is as follows:

Described probe sequence can comprise 1～10 base mismatch, preferably, can comprise 1～5 base mismatch, more preferably, can comprise 1～2 base mismatch.

Detection chip of the present invention also comprises at least a contrast probe, and described contrast probe is selected from: negative control probe, positive control probe, hybridization contrast probe and immobilization contrast probe.

Described probe can be fixed on the solid phase carrier by connecting arm.Connecting arm can provide one the space is sterically hindered to reduce freely for probe forms double-stranded part, carrying out [the Afanassiev V that helps hybridization, HanemannV, Wolfl S.Preparation of DNA andprotein micro arrays on glass slides coated with an agarose film.NucleicAcids Res.2000,28:e66; USA Patent No.5556752].Connecting arm is long more, and hybridization efficiency is high more.Typical connecting arm comprises 15～30 functional group length.Connecting arm can be selected the functional group of appropriate form for use, as the mosaic of Poly T (A, C or G), C atom or polyethylene glycol and Poly T (A, C or G), poly-ethanol, polyester, poly-ammonia, poly-sulfuric ester and its composition.

Described probe or connecting arm are fixed on the solid phase carrier by link molecule.Probe stationary can be passed through the realization of C-C key to carrier, for example, and the voltalef surface; Or better use siloxane bond (glass or silicon-dioxide use when making upholder).The siloxane bond bonding can be by upholder and link molecule Trichloromonosilane base or radical reaction such as trialkoxysilyl finish.Aminoalkyl group silane, hydroxyalkyl silane, 2-hydroxyethyl one aminopropyl triethoxysilane, hydroxyethyl one aminopropyl triethoxysilane or hydroxypropyl triethoxyl silane all are surface adsorption groups of great use.

Described probe can be modified, and modifying method can be 5 '-NH2 modification, 5 '-SH modification, 5 '-Poly T (A, C or G) modification, 5 '-biotin modification, 3 '-NH2 modification, 3 '-SH modification, 3 '-Poly T (A, C or G) modification and 3 '-biotin modification etc.

Described probe can have one or several, even is all through mark, and that described mark comprises is fluorescein-labelled, biotin labeling, radioelement mark, enzyme labelling and FRET (fluorescence resonance energy transfer) mark.

In another aspect of this invention, provide a kind of using method of said chip, comprised the steps:

(1) carries and the nucleotide sequence of hypertension to be measured and vascular lesion disease risks genes involved and/or the probe that its complementary sequence is hybridized at surface of solid phase carriers point;

(2) nucleic acid of extracting testing sample;

(3) the purpose nucleotide sequence of preparation hypertension to be measured and vascular lesion genes involved;

(4) the purpose nucleotide sequence of markers step (3);

(5) be loaded under the condition that the probe on the solid phase carrier hybridizes with the described point of step (1) being suitable for, add purpose nucleotide sequence, and make it react the enough time through mark;

(6) result of detection hybridization.

In another aspect of this invention, also provide a kind of another kind of using method of said chip, comprised the steps:

(1) nucleotide sequence of mark and hypertension to be measured and vascular lesion genes involved and/or its complementary sequence probe of hybridizing;

(2) nucleic acid of extracting testing sample;

(4) carry the described purpose nucleotide sequence of step (3) at surface of solid phase carriers point;

(5) be loaded under the condition that the purpose nucleotide sequence on the solid phase carrier hybridizes with the described point of step (4) being suitable for, add probe, and make it react the enough time through mark;

(6) result of detection hybridization.

The preparation of described purpose nucleotide sequence can comprise the step of amplification, directly increases with the cell that contains nucleic acid in the isolating target sample, and also available extractive target nucleic acid directly increases.As from whole blood, separating white corpuscle, directly make template, amplification purpose nucleotide sequence with isolating white corpuscle or with the extractive nucleic acid of whole blood with magnetic bead.Strand that amplification obtains or double-stranded DNA or RNA can contain fluorescence or biotin labeling, and the DNA of mark or RNA can not purifiedly be directly used in hybridization.With the preferred target nucleotide molecule of chip hybridization described in the present invention be single stranded nucleic acid molecule, after double chain acid molecule is handled through sex change etc. also in chip hybridization of the present invention.

The purpose nucleotide sequence can use any suitable amplification method to carry out enrichment, as: polymerase chain reaction (polymerase chain reaction, PCR), multiplex PCR, ligase chain reaction (ligase chain reaction, LCR), rolling circle amplification (rolling cycle amplification, RCA), based on the amplification of nucleotide sequence (nucleic acid sequence-basedamplification, NASBA), strand displacement amplification (strand displacementamplification, SDA) and the amplification of transcriptive intermediate (transcription medicatedamplification, TMA) etc.

In one embodiment of the invention, the pcr amplification method is adopted in the preparation of purpose nucleotide sequence, and this method the primer contains nucleotide chain or its complementary strand of sequence shown in SEQ ID NO:55～SEQ ID NO:90.

Described probe or purpose nucleotide sequence all are suitable for mark.Probe is at the synthetic mark of introducing, and the purpose nucleotide sequence can be introduced mark in amplification, and perhaps mark is introduced with suitable method in the amplification back.

Suitable mark comprises fluorescent mark, labelled with radioisotope, chromophoric group, twinkler, FRET, enzyme, vitamin H or the aglucon of special combination part is arranged.

The hybridization of the inventive method can be carried out under any suitable temperature, and as 25 ℃～65 ℃, described hybridization time is 2 minutes～18 hours.Can change rigorous degree, the hybridization specificity of hybridization conditions to improve or to reduce hybridization.

The disease risks detection chip and the application thereof of hypertension of the present invention and vascular lesion, make the medical worker in time grasp a large amount of genetic information of cardiovascular and cerebrovascular diseases patient such as hypertension, and guide its rational use of drug, realize the individuation medical treatment, improve the validity of treatment and the toxic side effect of minimizing medicine.For pharmaceutical manufacturer, detection chip of the present invention can make it select suitable clinical trial crowd, thereby improves curative effect of medication, reduces the inefficiency and the toxic side effect of medicine, shortens the R﹠D cycle of medicine.

Description of drawings

Fig. 1 is the figure as a result of the electrophoresis detection after the multiplex PCR amplification in the embodiments of the invention 1;

Fig. 2 is the figure as a result of the electrophoresis detection behind the PCR product fragmentation in the embodiments of the invention 1;

Fig. 3 is the results of hybridization figure of the disease risks detection chip of the hypertension of the embodiment of the invention 1 and vascular lesion.

Embodiment

The present invention is further detailed explanation below in conjunction with drawings and Examples.

Embodiment 1 is reverse hybridized

1. the preparation of gene chip

(1) probe dissolving

With every probe TE solution dilution of sequence probe shown in the SEQ ID NO:1～SEQ ID NO:54 of synthetic, final concentration is 10mM.With concentration be the probe of 10mM and PBS solution that concentration is 200mM in the medium volume mixture of 384 orifice plates, seal 384 orifice plates with adhesive sheet, vibration is 2 minutes under the room temperature, and is centrifugal ,-20 ℃ of preservations are used in order to point sample.

(2) point sample

The probe that designs and synthesizes in advance is downloaded on the solid phase carrier sheet base of materials such as slide, silicon chip by contact point sample or ink jet type point of sample.The sheet base adopts Cell AssociatesCSS-100 aldehyde radical sheet base, the point sample instrument of Ominigrid 100 models of GeneMachine company, humidity: 65-75% (being as the criterion) with FullMoon sheet base, temperature is a point sample under 25 ℃ the condition, after point sample finishes, after placing half an hour, chip is taken out, drying at room temperature is preserved.

2. the processing of testing sample and mark

(1) amplification of human gene group DNA's extracting and goal gene

Adopt FlexiGene DNA Kit (250) (QIAGEN, Cat.No.51206) genomic dna in the test kit extracting human peripheral.Get 1ul and carry out electrophoresis (1% sepharose, 0.5 * TBE, EB, 80MV, 1.5 hours electrophoresis), at FR-200 ultraviolet and visible analytical equipment photographs photo, and contrast marker (Lambda DNA/EcoRI+HindIII) carries out quantitatively.

Carry out the amplification of purpose nucleotide sequence with the primer (SEQ ID NO:55～76) of artificial synthetic OPN, the primer (SEQ ID NO:77～86) of ROK α, the primer (SEQ ID NO:87～90) of RhoA.Pcr amplification carries out with 30 μ l reaction systems, reaction system is concentration 0.16 μ M, the genomic dna 10ng of 0.3mM dNTP, 10mM Tris-HCl, 50mM KCl, 2mM MgCl2,20%Q solution (Qiagen), upstream and downstream primer, Taq enzyme 0.6U (Takara).Use Touch-down PCR response procedures [Don RH, CoxPT, Wainwright BJ, Baker K, Mattick JS. ' Touchdown ' PCR tocircumvent spurious priming during gene amplification.Nucleic AcidsRes.1991,19:4008]: 94 ℃ of sex change 5min; 94 ℃ of sex change 40s, 64 ℃ of annealing 1min, each circulation reduces by 0.5 ℃, and 72 ℃ are extended 50s, totally 10 circulations; 94 ℃ of sex change 40s then, 59 ℃ of annealing 40s, 72 ℃ are extended 50s, totally 30 circulations; Last 72 ℃ are extended 5min.PCR finishes the back and detects amplification with 1.5% sepharose.

When carrying out multi-PRC reaction, the primer of sequence shown in SEQ ID NO:55～SEQ ID NO:90 to be put into a reaction system increase, system is 50 μ l.The reaction system of multiplex PCR is: every kind of dNTP 0.3 μ mol/L, Tricine-KOH (PH8.7) 40mmol/L, KCl 16mmol/L, MgCl23.5mmol/L, BSA 3.75 μ g/ml, every primer 2 μ mol/L, and DNA 80ng and 2.2 * Titanium Taq archaeal dna polymerase (Clontech, USA).Multi-PRC reaction condition: 95 ℃ of sex change 3min; 95 ℃ of sex change 30s, 66 ℃ of annealing 2min, 68 ℃ are extended 4min, totally 40 circulations; Last 68 ℃ prolong 10min.Behind the pcr amplification, get 3 μ 1PCR reaction product and do agarose gel electrophoresis, electrophoresis result is seen Fig. 1.These PCR products can be used for following hybridization step after treatment.

(2) PCR product purification and fragmentation

All PCR products of each sample mix, with QIAquick PCR Purification Kit (Qiagen, Cat.No.28106) purifying.The PCR product of purifying after the concentration, carries out fragmentation with DNase I after measured.The reaction system of fragmentation comprises: 30 μ l purified pcr products (10 μ g), 10 * DNase I damping fluid of 4 μ l, the DNase I of 0.12 μ l, the ddH2O of 5.88 μ l.Reaction conditions is that 37 ℃ of temperature are bathed 5min, 95 ℃ of 15min then.Product behind the fragmentation carries out 4% agarose gel electrophoresis, guarantees that most fragments is in 30-200 base pair, and electrophoresis detection result as shown in Figure 2.

(3) fluorescein-labelled

Utilize deoxynucleotidyl transferase to carry out fluorescein-labelled at 3 ' end, 40 μ l reaction systems of mark comprise: 25 μ l fragmentation PCR products, 5 * deoxynucleotidyl transferase damping fluid of 8 μ l, the CY3-N6-ddCTP of 1 μ l (1mM), the deoxynucleotidyl transferase of 3 μ l (20U/ μ l), the ddH2O of 3 μ l.Reaction conditions is that 37 ℃ of temperature are bathed 120min, then 95 ℃ of heating 15min.

3. hybridization, washing and result detect

95 ℃ of sex change 10min of fluorescently-labeled PCR product, place immediately on ice, be used for hybridization, hybridization 20 μ l systems comprise: fluorescein-labeled PCR product 15 μ l, 20 * SSPE, 1.2 μ l, 1%Triton 0.2 μ l, 10 * Denhandts, 0.9 μ l, methane amide 0.5 μ l, ddH2O 2.2 μ l.Reaction conditions is that 48 ℃ of temperature are bathed 120min, use 1 * lavation buffer solution I (5 * SSC then in succession, 0.1%SDS), 1 * lavation buffer solution II (2 * SSC, 0.1%SDS) and 1 * lavation buffer solution III (1 * SSC) respectively washs 10min at 42 ℃, washs 0.5min with ddH2O at last.

Chip after the washing after drying, scans (also can with other laser scanner) with GenePix 4000B confocal laser scanner.The results of hybridization that chip after the scanning hybridization obtains as shown in Figure 3, handle image with GenePix Pro again and obtain data file, then the data file being analyzed the disease risks result that just can obtain hypertension and vascular lesion, is that the patient selects suitable medicine and rational dosage thereby instruct the clinician.

As Fig. 3, each SNP genotype of this sample is respectively rs2428127A/GGG, rs11730582T/C CC, rs6839524C/G CG, rs6840362T/C CC, rs7695531A/G AA, rs6811536T/C CC, rs2853750A/G AG, rs11728697T/CTC, rs10516799C/G CC, rs1126772A/G AA, 8975T/CTT, rs11693061T/CCC, rs2271621G/T TT, rs978906A/G AA, rs13018466T/CTT, rs9808232G/T GT, rs7621003T/C TT, rs6784820A/G AA.Two bases of each SNP are represented hypertension and vascular disease step-down danger/high-risk allelotrope respectively.Point out its danger that hypertension and vascular lesion take place higher as high-risk allelotrope after the pattern detection.

The hybridization of embodiment 2 forwards

1. the amplification of the disease risks genes involved of hypertension and vascular lesion and chip preparation

Primer (the SEQ ID NO:87～90) amplification gene of the primer (SEQ ID NO:77～86) of the primer of OPN (SEQ ID NO:55～76), ROK α, RhoA.Pcr amplification carries out with 100 μ l reaction systems, and reaction system is 0.3mM dNTP, 10mM Tris-HCl, 50mM KCl, 2mM MgCl2,20%Q solution (Qiagen), 0.01 μ M SHV-F, 0.2 μ M SHV-R, 100ng genomic dna, 3U Ex Taq enzyme (Takara).Loop parameter: 94 ℃ of sex change 5min; 94 ℃ of sex change 40s, 65 ℃ of annealing 1min, 72 ℃ are extended 1min, totally 40 circulations; Last 72 ℃ are extended 5min.PCR product QIAquick PCRPurification Kit (Qiagen, Cat.No.28106) purifying.The PCR production concentration of purifying is adjusted to 400ng/ μ l.

With concentration be the PCR product of 400ng/ μ l and 100% DMSO in the medium volume mixture of 384 orifice plates, seal 384 orifice plates with adhesive sheet, vibration is 2 minutes under the room temperature, and is centrifugal.The PCR product of 200ng/ μ l is downloaded on the solid phase carrier sheet base of materials such as slide, silicon chip by contact point sample or ink jet type point of sample.Adopt the point sample instrument of the Ominigrid100 model of GeneMachine company, humidity: 65-75% (being as the criterion) with FullMoon sheet base, temperature is a point sample under 25 ℃ the condition, point sample finish place half an hour after, chip is put in the saturated aqueous common salt box, and 37 ℃ of aquations are spent the night, and take out next day, 600mJ is crosslinked, and the crosslinked chip that finishes can use.

2. chip hybridization

The hybridization system comprises: the fluorescently-labeled oligonucleotide probe of 2nM (SEQ IDNO:1～SEQ ID NO:54), 1.2 μ l, 20 * SSPE, 0.2 μ l 1%Triton, 0.9 μ l, 10 * Denhandts, 0.5 μ l methane amide, 2.2 μ l ddH2O.Reaction conditions is that 50 ℃ of temperature were bathed 2 hours, use 1 * lavation buffer solution I (5 * SSC then in succession, 0.1%SDS), 1 * lavation buffer solution II (2 * SSC, 0.1%SDS) and 1 * lavation buffer solution III (1 * SSC) respectively washs 10min at 42 ℃, at last with ddH2O washing 10 seconds.Chip after the washing after drying, scans (also can with other laser scanner) with GenePix 4000B confocal laser scanner.

Sequence table

＜110〉Shanghai Research Institute of Hypertension

＜120〉genetics detection chip of hypertension and vascular lesion disease risks and uses thereof

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ttttcaggag?ggaagagaac?taaac 25

<210>37

<211>25

<212>DNA

＜213〉artificial sequence

<400>37

ttttctataa?aaaagtttga?ataaa 25

<210>38

<211>25

<212>DNA

＜213〉artificial sequence

<400>38

ttttctataa?aaaattttga?ataaa 25

<210>39

<211>25

<212>DNA

＜213〉artificial sequence

<400>39

ttttctataa?aaaactttga?ataaa 25

<210>40

<211>25

<212>DNA

＜213〉artificial sequence

<400>40

tttttcacac?tacaatgcac?acaag 25

<210>41

<211>25

<212>DNA

＜213〉artificial sequence

<400>41

tttttcacac?tacagtgcac?acaag 25

<210>42

<211>25

<212>DNA

＜213〉artificial sequence

<400>42

tttttcacac?tacactgcac?acaag 25

<210>43

<211>25

<212>DNA

＜213〉artificial sequence

<400>43

ttttccttgc?ttaatatgga?acgtt 25

<210>44

<211>25

<212>DNA

＜213〉artificial sequence

<400>44

ttttccttgc?ttaacatgga?acgtt 25

<210>45

<211>25

<212>DNA

＜213〉artificial sequence

<400>45

ttttccttgc?ttaagatgga?acgtt 25

<210>46

<211>25

<212>DNA

＜213〉artificial sequence

<400>46

tttttatgga?atcagtttct?ctaca 25

<210>47

<211>25

<212>DNA

＜213〉artificial sequence

<400>47

tttttatgga?atcattttct?ctaca 25

<210>48

<211>25

<212>DNA

＜213〉artificial sequence

<400>48

tttttatgga?atcactttct?ctaca 25

<210>49

<211>25

<212>DNA

＜213〉artificial sequence

<400>49

ttttagaggg?cttttaggac?agaaa 25

<210>50

<211>25

<212>DNA

＜213〉artificial sequence

<400>50

ttttagaggg?ctttcaggac?agaaa 25

<210>51

<211>25

<212>DNA

＜213〉artificial sequence

<400>51

ttttagaggg?ctttgaggac?agaaa 25

<210>52

<211>25

<212>DNA

＜213〉artificial sequence

<400>52

tttttcctga?gcacattgac?ctcat 25

<210>53

<211>25

<212>DNA

＜213〉artificial sequence

<400>53

tttttcctga?gcacgttgac?ctcat 25

<210>54

<211>25

<212>DNA

＜213〉artificial sequence

<400>54

tttttcctga?gcaccttgac?ctcat 25

<210>55

<211>10

<212>DNA

＜213〉artificial sequence

<400>55

gagtaaacta 10

<210>56

<211>10

<212>DNA

＜213〉artificial sequence

<400>56

aaacataccc 10

<210>57

<211>10

<212>DNA

＜213〉artificial sequence

<400>57

tactcgaaat 10

<210>58

<211>10

<212>DNA

＜213〉artificial sequence

<400>58

acttattgaa 10

<210>59

<211>10

<212>DNA

＜213〉artificial sequence

<400>59

atgaaaaagt 10

<210>60

<211>10

<212>DNA

＜213〉artificial sequence

<400>60

ggagcagaac 10

<210>61

<211>10

<212>DNA

＜213〉artificial sequence

<400>61

cagcaaaac 10

<210>62

<211>10

<212>DNA

＜213〉artificial sequence

<400>62

atgagtagag 10

<210>63

<211>10

<212>DNA

＜213〉artificial sequence

<400>63

tttaaggtat 10

<210>64

<211>10

<212>DNA

＜213〉artificial sequence

<400>64

attttattag 10

<210>65

<211>10

<212>DNA

＜213〉artificial sequence

<400>65

tgaagattat 10

<210>66

<211>10

<212>DNA

＜213〉artificial sequence

<400>66

ttaacacagg 10

<210>67

<211>10

<212>DNA

＜213〉artificial sequence

<400>67

tagtgaaaga 10

<210>68

<211>10

<212>DNA

＜213〉artificial sequence

<400>68

tctatagaat 10

<210>69

<211>10

<212>DNA

＜213〉artificial sequence

<400>69

cttggacaaa 10

<210>70

<211>10

<212>DNA

＜213〉artificial sequence

<400>70

tcttcattaa 10

<210>71

<211>10

<212>DNA

＜213〉artificial sequence

<400>71

tatttttttt 10

<210>72

<211>10

<212>DNA

＜213〉artificial sequence

<400>72

ctctagattt 10

<210>73

<211>10

<212>DNA

＜213〉artificial sequence

<400>73

tttctcagtt 10

<210>74

<211>10

<212>DNA

＜213〉artificial sequence

<400>74

ttacagggag 10

<210>75

<211>10

<212>DNA

＜213〉artificial sequence

<400>75

caggcacaaa 10

<210>76

<211>10

<212>DNA

＜213〉artificial sequence

<400>76

tctgcccctt 10

<210>77

<211>10

<212>DNA

＜213〉artificial sequence

<400>77

ggaggaggca 10

<210>78

<211>10

<212>DNA

＜213〉artificial sequence

<400>78

gtccttagat 10

<210>79

<211>10

<212>DNA

＜213〉artificial sequence

<400>79

cacatctgtc 10

<210>80

<211>10

<212>DNA

＜213〉artificial sequence

<400>80

aattaatata 10

<210>81

<211>10

<212>DNA

＜213〉artificial sequence

<400>81

actcttccag 10

<210>82

<211>10

<212>DNA

＜213〉artificial sequence

<400>82

atcttttcta 10

<210>83

<211>10

<212>DNA

＜213〉artificial sequence

<400>83

cagtttttca 10

<210>84

<211>10

<212>DNA

＜213〉artificial sequence

<400>84

cctttactca 10

<210>85

<211>10

<212>DNA

＜213〉artificial sequence

<400>85

agcagtaaaa 10

<210>86

<211>8

<212>DNA

＜213〉artificial sequence

<400>86

ctcccccc 8

<210>87

<211>10

<212>DNA

＜213〉artificial sequence

<400>87

aaaccctcca 10

<210>88

<211>10

<212>DNA

＜213〉artificial sequence

<400>88

ttattttatt 10

<210>89

<211>10

<212>DNA

＜213〉artificial sequence

<400>89

ctccgcacaa 10

<210>90

<211>10

<212>DNA

＜213〉artificial sequence

<400>90

ttagtgagcg 10

Claims

1. the genetics detection chip of hypertension and vascular lesion disease risks, it comprises solid phase carrier and the probe that is fixed on the described solid phase carrier, described probe comprises the probe that detects hypertension and vascular lesion related gene polymorphism, nucleotide sequence and/or its complementary sequence of described probe and hypertension to be measured and vascular lesion disease risks genes involved are hybridized, it is characterized in that, described hypertension to be measured and vascular lesion disease risks genes involved are OPN, ROK α and RhoA gene, and described probe comprises:

2. the genetics detection chip of hypertension as claimed in claim 1 and vascular lesion disease risks is characterized in that, described probe is DNA, RNA, DNA-RNA mosaic, PNA or derivatives thereof.

3. the genetics detection chip of hypertension as claimed in claim 1 and vascular lesion disease risks is characterized in that described probe can be modified.

4. the genetics detection chip of hypertension as claimed in claim 1 and vascular lesion disease risks is characterized in that, described solid phase carrier is a kind of in slide, silicon chip, nitrocellulose filter, nylon membrane and the macromolecular material or their arbitrary combination.

5. the using method of the genetics detection chip of hypertension as claimed in claim 1 and vascular lesion disease risks is characterized in that, comprises the steps:

(1) carries the described probe of claim 1 at surface of solid phase carriers point;

(2) nucleic acid of extracting testing sample;

(3) prepare the hypertension to be measured and the purpose nucleotide sequence of vascular lesion genes involved;

(4) the purpose nucleotide sequence of markers step (3);

(6) result of detection hybridization.

6. method as claimed in claim 5 is characterized in that, that the mark of the described purpose nucleotide sequence of step (4) comprises is fluorescein-labelled, biotin labeling, electrochemiluminescence mark, radioelement mark or enzyme labelling.

7. the using method of the genetics detection chip of hypertension as claimed in claim 1 and vascular lesion disease risks is characterized in that, comprises the steps:

(1) the described probe of mark claim 1;

(2) nucleic acid of extracting testing sample;

(6) result of detection hybridization.

8. method as claimed in claim 7 is characterized in that, that the described probe mark of step (1) comprises is fluorescein-labelled, biotin labeling, FRET (fluorescence resonance energy transfer) mark, electrochemiluminescence mark, radioelement mark or enzyme labelling.

9. as claim 5 or 7 described methods, it is characterized in that, the preparation of the purpose nucleotide sequence of described step (3) comprises pcr amplification reaction, and this reaction the primer contains nucleotide chain or its complementary strand of sequence shown in SEQID NO:55～SEQ ID NO:90.

10. as claim 5 or 7 described methods, it is characterized in that the hybridization temperature in the described step (5) is 25 ℃～65 ℃, hybridization time is 2 minutes～18 hours.