CN1616675A - Individual administration gene type diagnostic chip and its producing method and using method - Google Patents

Abstract

The present invention relates to gene technology field, and is gene diagnosis chip for individual administration and its making process and usage. The gene diagnosis chip includes bearing substrate and gene probes distributed on the substrate, and the gene probes contain oligonucleotide sequences complementary with the medicine metabolism and effect related gene mutant sites and have reference probes set. Each mutant site has two types of probes, wild type and mutant type, designed. The chip making process includes designing two types of probes of length 17-22 bp for each mutant site, synthesizing probes with DNA sequence synthesizing instrument, applying probes onto the substrate with chip sample applying instrument, curing the chip and storing at 4 deg.c. The usage of the gene diagnosis chip includes sampling, sample processing, PCR amplification, crossbreeding, washing, reading, result analysis and other steps.

Description

Individual administration gene type diagnostic chip and manufacture method and methods for using them thereof

Technical field

The invention belongs to the gene engineering field, relate to a kind of individual administration gene type diagnostic chip and manufacture method and methods for using them thereof.

Background technology

Gene chip (Gene chip) claims DNA chip (DNA CHIP), DNA array (DNA arrays), oligonucleotide microchip (oligonucleotide micro-chip) again, be meant many specific oligonucleotide fragments or gene fragment as probe, arrange regularly and be fixed on the upholder, hybridize by the base pairing principle with the gene of mark sample to be measured then, by detection system etc. chip is scanned again, and be equipped with computer system intensity of hybridization signal is analyzed and handled, thereby draw desired information rapidly.At present, biochip technology is applied in the life science every field just gradually.

The gene chip technology of preparing: the essence of gene chip is highly integrated oligonucleotide arrays, and the technology that the manufacturing gene chip at first will solve is how to locate synthetic highdensity nucleic acid probe on the chip slapper base.At present, the technology of preparing of gene chip mainly contains two kinds of the synthetic and synthetic back of original position point samples.

The application of gene chip in clinical disease diagnosis: from normal people's genome, isolate DNA and the DNA chip hybridization just can draw standard diagram.From patient's genome, isolate DNA and the DNA chip hybridization just can draw the pathology collection of illustrative plates.Characteristics such as this gene chip diagnosis technology is quick, efficient with it, sensitivity, economy, parallelization, automatization will become a modernization diagnosis new technology.For example, the p53 gene chip of Affymetrix company and Oncormed company cooperation research and development can be monitored the p53 transgenation of 50% above tumour patient, can play a role in early diagnosis of cancer.Gene chip has special advantages aspect the clinical diagnosis of diseases such as infectious diseases, heredopathia, severe transmissible disease and malignant tumour.Compare with traditional detection method, it can carry out the detection of multiple disease simultaneously at a chip to patient, do not need body to be in the immune response phase, can diagnose early, and the testing sample consumption is little; The hypotype and the variation of energy specific detection pathogenic micro-organism; Can help doctor and patient to be converted to generation, the evolution that " DNA, RNA, protein and interaction level thereof (phase III medical science) " understands disease from " system, blood vessel, tissue and cell level (subordinate phase medical science) ".These characteristics make the medical worker at short notice, can grasp a large amount of medical diagnosis on disease information, help the doctor to find correct treatment measure at short notice.

The diagnosis of heredopathia: along with finishing of the Human Genome Project, the genes involved of many heredopathias is by successive position fixes, for example obesity, senile dementia, psychosis, essential hypertension, thalassemia etc.The methods such as PCR-SSCP, craft or automatic sequencing, heteroduple analysis, albumen brachymemma detection that adopt when former studies sudden change and polymorphism, all these need pass through the electrophoresis link, can not satisfy on a large scale, the requirement of low consumption and automatization more.The applying gene chip method can overcome above-mentioned deficiency when detecting, and combines when detecting with archaeal dna polymerase or ligase enzyme and can obtain higher resolving power.The thalassemia gene diagnosis chip of at present domestic existing state plan fertility scientific research institution's exploitation is used to diagnose patient whether to carry thalassemic Disease-causing gene.

The diagnosis of infectious diseases: follow the progress of pathogenic micro-organism genome plan, make the gene diagnosis cause pathogeny imcrobe infection become possibility.Biochip technology has not only avoided loaded down with trivial details and time-consuming pathogenic micro-organism to cultivate, and does not need the appearance of antibody by the time, for the pathogenic micro-organism diagnosis provides strong technique means.

The diagnosis of tumour and treatment: biochip technology has been widely used for tumor tissues and the difference of healthy tissues genetic expression on the mRNA level, for research tumour mechanism provides strong tool.Utilize this technology to distinguish at present to the tumour cell subgroup that comprises kinds of tumors diseases such as leukemia, lymphoma, cutaneous melanoma and mammary cancer, can also utilize this technology that treatment plan is assessed and the new drug evaluating drug effect, in addition can also be, develop and the prediction that lapses to provides the molecule foundation to the generation of tumour.

Other application: gene chip also is widely used in a plurality of Application Areass such as screening, Resistant strain and susceptibility detection of drug screening, mechanism of drug action research, toxicologic study, genescan, environmental chemistry poisonous substance.It is believed that in new century, gene chip will be brought into play enormous function in the gene diagnosis of human diseases, for All Around The World brings huge social benefit and economic interests.

The present domestic gene chip that is used for clinical diagnosis: the sharp hepatitis B virus mutation checking gene chip of section advanced in years of a. Shanghai associating genome company exploitation is mainly used in the early discovery hepatitis B virus rummy is held up fixed resistance tendency.B. the thalassemia gene diagnosis chip of state plan fertility scientific research institution exploitation is used to diagnose thalassemic Disease-causing gene.C. also have in addition: the tubercule bacillus resistance chip of the south of the River, Zhejiang biotech firm exploitation, the SP GC-200 type hereditary defect detection chip of the preeminent company in Shaanxi exploitation.

The individual difference of drug metabolism and effect: clinically, the dosage of same medicine effectively may be inoperative to patient's second to patient's first, and patient third is then had side effect.Aspect curative effect of medication and side effect, patient's response difference is very big.This mainly is owing to there are differences on patient's genetics that (single nucleotide polymorphism SNP), causes medicine is produced different reactions.For example to have be metabolic by cytochrome P 450 enzymes in human body more than 50% to the medicine of widespread use clinically at present, if undergoing mutation, the gene of patient CYP2D6 enzyme will cause weakening in the intravital metabolism of people through the metabolic tens of kinds of medicines of this kind of enzyme, disappearance or enhancing, these medicines comprise Dextromethorphane Hbr, morphine monomethyl ether, Desipramine, clomipramine, imipramine, fluoxetine, paroxetine, haloperidol, nortriptyline, trilafon, thioridazine, flecainide, encainide, Debrisoquine, metoprolol, bufuralol, timolol, carvedilol, sparteine, mexiletine, Propafenone etc.Nearly 51% CYP2D6 enzyme gene is active more weak genotype among the Chinese.If utilize biochip technology that the patient is diagnosed earlier, prescribe again, just can implement the individual treatment of optimizing to patient.

The defective of existing clinical application method: in a lot of treatment of diseases processes, often inappropriate medication takes place and the side effect that causes or can't reach therapeutic purpose because of underdosage.Therefore carry out the meaning that personalized medicine has particularly important at the genotype of these sufferers.In the past, the doctor adjusted patient's medication if desired, needed to observe for a long time, and patient also needs to do a large amount of detections.May need to extract sequence of operations such as DNA, pcr amplification, restriction nuclease enzyme inscribe for genetic reasons to certain susceptibility sense if suspect patient, be unfavorable for detecting simultaneously the variation of several genes.The process procedures complexity of this adjustment medication, time-consuming, incur loss through delay and cure the disease opportunity,

Summary of the invention

Technical problem to be solved by this invention is at present in the treatment of diseases process, the situation that the kind of medication and dosage are difficult to determine provides a kind of assist clinicians easily and efficiently to formulate the individual administration gene type diagnostic chip and the manufacture method and methods for using them thereof of personalized medicine scheme.

The technical scheme that solution the technology of the present invention problem is adopted is that this individual administration gene type diagnostic chip comprises carrying substrates and be the several genes probe that array distributes on substrate, gene probe contains the complementary oligonucleotide sequence in drug metabolism and effect associated gene mutation site, result of study according to pharmacogenomics, the incidence of these mutational sites in the crowd is higher, and pharmacokinetics or drug effect to the clinical medicine commonly used of some disease have a significant effect, and (determining according to clinically the medicine commonly used of some disease being searched with the individual difference of its drug effect of gene mutation site is in close relations, and occurrence frequency is than higher, the mutational site that using value is arranged).In addition, for the control chip quality, also can be provided with positive on the chip in the gene probe array with reference to probe.Each mutational site design wild type gene probe array and two kinds of probes of mutated genes probe array, its probe length is 17-22bp.It is a plurality of point samples zones that a plurality of gene probe arrays can be set on each carrying substrates.Carrying substrates can adopt solid-phase medias such as silicon chip, sheet glass, plastic sheet, nylon membrane, and chemically modified can be passed through in its surface.

Individual administration gene type diagnostic chip manufacture method of the present invention is: according near the base sequence the gene locus of required detection, each mutational site design wild-type and two kinds of probes of mutant, design length is the probe of 7-22bp, use dna sequence dna synthesizer synthesising probing needle, with spot sample device probe is selected on substrate by certain spacing, the point sample area can be adjusted according to the factors such as size of number of probes, probe spacing, point, behind the point sample chip pressed the different of probe and substrate combination, take appropriate means to be cured.Use immediately then or be stored in 4 ℃ standby.

Carrying substrates can adopt solid-phase medias such as silicon chip, sheet glass, plastic sheet, nylon membrane.For improving probe and substrate bonded efficient and detection sensitivity, probe and substrate can be done further to modify and the bag quilt.Can be poly-lysine bag quilt, agar glycolyx, amino or aldehyde radical modification as its substrate surface treatment mode.

The application method of individual administration gene type diagnostic chip of the present invention is:

(1) sampling: from the patient 1-2ml that takes a blood sample on one's body;

(2) sample process

Use the genome DNA extraction test kit to extract DNA;

The operation steps that below relates to fluorescent dye primer and product thereof all (has in the darkroom of red light) under the lucifuge condition carries out.

(3) PCR reaction amplification

Use the gene mutation site Auele Specific Primer by the amplification of PCR reaction pair DNA sample, use fluorescent substance or isotropic substance that primer or dNTP are carried out mark.

(4) hybridization

Get PCR product mixed solution, add with reference to solution and through the hybridization solution of preheating, mixing, 3 minutes postposition of sex change are on ice, all transfer to the point sample zone (contrast point sample matrix positioning chip) of individual administration gene type diagnostic chip, add cover glass (noting not having bubble); Hybridization adds several dripping in the cabin, to keep humidity; Chip is put into the hybridization cabin, and thermostat container or water bath heat preservation one hour is put in sealing hybridization cabin then into.

(5) develop a film

Open the hybridization cabin, take out chip, wash out cover glass with elutriant, then chip is put into elutriant, room temperature (25 ℃ ± 10 ℃) was placed 5 minutes, with sterilization distilled water flushing two times.Dry under the room temperature.

(6) read sheet

Recommend to use the ScanArray4000 scanner under 60-85 scanning intensity, to scan.

(7) interpretation of result

Adopt artificial interpretation or use the software kit analysis.Provide the medication proposal on adjustments according to gene type assay result and related data.

The present invention utilizes this chip only to need patient 1-2 milliliter blood, just can obtain the result rapidly in 3-4 hour, and cooperates special software, provides clear and definite suggestion to the clinician.Adopt this new diagnostic method, the doctor can adjust prescription before patient's medication, has significantly reduced unnecessary misery and the economical load of patient.And, adopt biochip technology, can detect a plurality of variant sites simultaneously, once judging patient may be to which medicaments insensitive or tolerance.Detected result can instruct its medication throughout one's life.

Description of drawings

Fig. 1 is an individual administration gene type diagnostic chip structure iron of the present invention

Fig. 2 is used for essential hypertension individual administration gene type diagnostic chip orthographic plan

Among the figure: the gene probe 4-that 1-carrying substrates 2-point sample zone 3-array distributes labels the 5-of place with reference to probe 6-wild type gene probe array 7-mutated genes probe array

Embodiment

Below the hypertension individual administration gene type diagnostic chip that is used for constructed in accordance be that the present invention is described in further detail for example.

According to clinically hypertensive patient's medicine commonly used being searched the gene mutation site in close relations with the individual difference of its metabolism and drug effect, and be chosen among the Chinese occurrence frequency than 7 gene mutation sites higher, that using value is arranged, manufacture the genotype detecting chip that detects these several mutational sites.The individual administration gene type diagnostic chip that is used for the hypertensive patient contains the probe that detects 7 gene mutation sites, these 7 gene mutation sites are: CYP2C9*3, CYP2C19*2, CYP2C19*3, CYP2D6*10, ADRB1 (A145G) (β 1 adrenergic receptor gene), ADRB1 (G1165C) (β 1 adrenergic receptor gene), AGTR1 (A1166G) (1 receptor gene of hypertensin 2).

Substrate is handled: substrate adopts glass medium, and its surface treatment mode adopts amination to modify.Concrete steps are:

(1) in 2mol/L NaOH-70% ethanolic soln, behind the cleaning slide 2h, uses distilled water flushing 5 times, dry and be cooled to room temperature for 110 ℃;

(2) slide is placed 1% 3-aminopropyl trimethoxysilane-95% acetone soln soak 2min;

(3) take out back acetone rinsing 10 times, each 5min;

(4) 110 ℃ of dry 45min;

(5), handle 2h in the 4-benzene diisothiocyanic acid salts solution at 1 of 1g/L;

(6) use washed with methanol 5min, acetone rinsing 5min, drying at room temperature.

The probe preparation:

(1) oligonucleotide of synthetic required sequence (polyT that comprises 10～15 bases of 5 ' end) on dna synthesizer;

(2) on the dna sequence dna synthesizer, oligonucleotide poly (T) molecular arm is introduced active aliphatic amino arm.

(3) with the amido modified oligonucleotide of HPLC purifying 5 ' end, behind the centrifugal drying, be dissolved in 100mmol/LNa ₂CO ₃/ NaHCO ₃Solution (PH9.0), concentration are 2mmol/L.

Manufacturing of gene chip: by the essential hypertension individual administration gene type diagnostic chip plane structure chart that is used for shown in Figure 2, on the substrate 1 of a glass medium, be divided into two gene probe point sample zones 2, the area in each point sample zone is 10mm*8mm, the probe of gene probe 3 the 1st row that the array in each point sample zone distributes and the probe of the 1st row are all positive with reference to probe 5, the 2nd behavior mutational site CYP2C9*3 gene probe, the 3rd behavior mutational site CYP2C19*2 gene probe, the 4th behavior mutational site CYP2C19*3 gene probe, the 5th behavior mutational site CYP2D6*10 gene probe, the 6th behavior mutational site ADRB1 (A145G) (β 1 adrenergic receptor gene) gene probe, the 7th behavior mutational site ADRB1 (G1165C) (β 1 adrenergic receptor gene) gene probe, eighth row are mutational site AGTR1 (A1166G) (1 receptor gene of hypertensin 2) gene probe.The two separated into two parts of array that these 7 mutational site gene probes form: wild type gene probe array 6 and mutated genes probe array 7.

Print and the printing aftertreatment:

(1) probe solution is respectively got 2 μ l, presses above-mentioned format print to the substrate of handling with point sample instrument;

(2) 37 ℃ of sealing treatment 1h;

(3) with 1% ammonia soln washing 5min, again with distilled water washing 3 times;

(4) use immediately after the drying at room temperature or be stored in 4 ℃ standby.

Application method:

(1) test kit: see Table 1

Listed reagent composition in the table 1:

Primer mixture 1: contain be useful on the amplification CYP2C9*3, CYP2C19*2, CYP2C19*3, CYP2D6*10, the fluorescent dye primer of the multiplex PCR of 5 gene mutation sites of AGTR1 (A1166G);

Primer mixture 2: contain be useful on the amplification ADRB1 (A145G), the fluorescent dye primer of the multiplex PCR of 2 gene mutation sites of ADRB1 (G1165C);

Positive in solution: the synthetic positive is with reference to the probe complementary sequence, through fluorescent mark.

Hybridization solution: 0.2%SDS

50 * elutriant: 10%SDS

(2) experimental implementation step

1. sampling: from the patient 1ml that takes a blood sample on one's body;

2. sample process

Use Takara genome DNA extraction test kit to extract DNA;

The operation steps that below relates to primer and product thereof must (have in the darkroom of red light) under the lucifuge condition carries out.

3. PCR reaction

All primers that test kit provided that contain the listed reagent of table 1 are primer mixture, please abundant earlier dissolving mixing before using.

(A) in the reaction system of 25 μ l, contain primer mixture 1 (reagent 2) 2.5 μ l, template 50-100ng, 1 * PCR damping fluid, MgCl ₂2.0mM, dNTPs 0.2mM and 2U Taq enzyme.

PCR program: 94 ℃ of pre-sex change 3 minutes; 94 ℃ of sex change are 30 seconds then, 56 ℃ of renaturation 30 seconds, and 72 ℃ were extended 35 circulations like this 40 seconds; 72 ℃ were extended 5 minutes again.

(B) in the reaction system of 25 μ l, contain primer mixture 2 (reagent 3) 2.5 μ l, template 50-100ng, 1 * PCR GC Buffer II, MgCl ₂1.5mM, dNTPs 0.2mM and 2U LA Taq enzyme.

PCR program: 94 ℃ of pre-sex change 3 minutes; 94 ℃ of sex change are 30 seconds then, 60 ℃ of renaturation 1 minute, and 72 ℃ were extended 30 circulations like this 1 minute; 72 ℃ were extended 7 minutes again.

Attention: the abundant mixing of mentioned component, add Taq enzyme and mixing at last, but can not thermal agitation; Place the PCR pipe on the PCR instrument, with PCR conversion zone on the aluminium-foil paper parcel PCR instrument.

4. hybridization

With (A) and (B) PCR product mixing, mixing, get 7 μ l PCR product mixed solutions, add positive with reference to solution (reagent 4) 1 μ l and through hybridization solution (reagent 5) the 6 μ l of 48 ℃ of preheatings, mixing, 3 minutes postposition of 94 ℃ of sex change are all transferred to the point sample zone (please contrast point sample matrix positioning chip) of chip on ice, add cover glass (noting not having bubble); Hybridization adds several dripping in the cabin, to keep humidity; Chip is put into the hybridization cabin, and 48 ℃ of (± 1 ℃) thermostat containers or water bath heat preservation one hour is put in sealing hybridization cabin then into.

5. develop a film

Open the hybridization cabin, take out chip, wash out cover glass with 1X elutriant (reagent 6), then chip is put into the staining jar that fills 1X elutriant (reagent 6), room temperature (25 ℃ ± 10 ℃) was placed 5 minutes, with sterilization distilled water flushing two times.Dry under the room temperature.

6. read sheet

Use the ScanArray4000 scanner under 60-85 scanning intensity, to scan.

7. interpretation of result

Adopt artificial interpretation or use the software kit analysis.Provide the medication proposal on adjustments according to gene type assay result and related data:

Genotype is the individuality of CYP2C9*3, and losartan (Losartan) should use heavy dose of.

Genotype is the individuality of CYP2C19*2 and CYP2C19*3, and Proprasylyte should use smaller dose.

Genotype is the individuality of CYP2D6*10, and metoprolol, Proprasylyte, bisoprolol carvedilol and Odizem all should use smaller dose.

Genotype is the individuality of ADRB1 (G1165C), should use smaller dose when using metoprolol or atenolol USP 23.

Genotype is the individuality of AGTR1 (A1166G), should use low dose when using losartan (Losartan).

Individual administration gene type diagnostic chip index of the present invention:

Recall rate (number of samples that has detected/sample sum)＞95%

Accuracy (number of samples that correctly detects/detect sample sum)＞99%

Repeatability (number of times that same sample correctly detects/always detect number of times)＞99%

Table 1 hypertension individual administration gene type diagnostic chip test kit content

The reagent numbering	Reagent name	Quantity	Storage and remarks
				??1	Gene chip	5 (2 person-portions/sheet)	4 ℃ of preservations
??2	Primer mixture 1	1 pipe	Add ddH before the use 2O is dissolved to 50 μ l ,-20 ℃ of preservations
				??3	Primer mixture 2	1 pipe	Add ddH before the use 2O is dissolved to 50 μ l, and-20 ℃ keep in Dark Place
??4	Sun ginseng solution	???15μl	-20 ℃ keep in Dark Place
				??5	Hybridization solution	1 pipe (100 μ l)	4 ℃ of preservations
??6	The 50X elutriant	1 bottle (20ml)	Normal temperature is preserved, and adds ddH before the use 2O is diluted to 1X
				??7	Cover glass	15	Frangible
??8	The hybridization cabin	1	Room temperature
				??9	The location slide	1	For having the slide of point sample matrix frame trace, be used for the location during hybridization
Other composition	Working instructions	1 part

Table 2 hypertension individual administration gene type diagnostic chip reagent composition

The reagent numbering	Reagent name	Composition
			????5	Hybridization solution	????0.2％SDS
????6	The 50X elutriant	????10％SDS

Table 3 hypertension individual administration gene type diagnostic chip probe sequence

The probe title	Sequence (5 '-3 ')
		Positive in probe	????ACCTTGGAATGTCCACAG
The CYP2C19*2 wild-type probe	????CCCAGGAACCCACCGGA
		CYP2C19*2 mutant probe	????GAAGCCTATTTCCCAGT
The CYP2C9*3 wild-type probe	????CCAGCATCCCTGGATCCG
		CYP2C9*3 mutant probe	????TCCAGGTTTTACGCGGC

The CYP2D6*10 wild-type probe	????ATCCCGCATGAGTAGCG
		CYP2D6*10 mutant probe	????TGAGTAGCGTGATTGCC
?ADRB1(145A)	????CTTAAAGCCCCGATTGGA
		?ADRB1(145G)	????GTTAAGCGAAGGCCGGCC
?ADRB1(1165G)	????GGACCGAGCAGTCCCGCA
		?ADRB1(1165C)	????ATTGCCGAGCAGTCCGGT
?AGTR1(1166A)	????AAGGGAAAATGAGCATTA
		?AGTR1(1166G)	????CTAAACAAATGAGCCTTA

Table 4 hypertension individual administration gene type diagnostic chip primer sequence

Primer or complementary sequence title	Sequence (5 '-3 ')
		Positive in the probe complementary sequence	????CTGTGGACATTCCAAGGT
The CYP2C19*2 forward primer	????TTGCGGCTAACGAGAGGATTTG
		The CYP2C19*2 reverse primer	????TTCGGGATATGAAGCAACAGTG
The CYP2C9*3 forward primer	????CTTTTCGTCCATCAGTTTTTCGA
		The CYP2C9*3 reverse primer	????TCCTTTTCCCGGCATCAGTTTTT
The CYP2D6*10 forward primer	????TCAAAAGCAGGTCGAACTATAA
		The CYP2D6*10 reverse primer	????TTTCGCAGAGCTGGTGGCATAT
The ADRB1 forward primer	????CCGACACTTTCATGGCGCCTGG
		The ADRB1 reverse primer	????GGAGCAAATTGTTGTGTTCCAATC
The ADRB1 forward primer	????TTTCGGAGCCCATTTGGAGCGGTA
		The ADRB1 reverse primer	????GAACCCATTTTGGCGGTAGTGAGG
The AGTR1 forward primer	????CCGGAAGCCATCACGCGTAGCAGA
		The AGTR1 reverse primer	????GCTTAAGGCGAGGGTTCGTGATGG

Sequence table

＜110〉week, grand Hao

＜120〉individual administration gene type diagnostic chip and manufacture method and methods for using them thereof

<150>CN03124852.7

<151>2003-09-22

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<221>prim_bind

<222>(1)..(24)

<400>24

gaacccattt?tggcggtagt?gagg?????????????????????????????24

<210>25

<211>24

<212>DNA

＜213〉human (Homo sapiens)

<220>

<221>prim_bind

<222>(1)..(24)

<400>25

ccggaagcca?tcacgcgtag?caga?????????????????????????????24

<210>26

<211>24

<212>DNA

＜213〉human (Homo sapiens)

<220>

<221>prim_bind

<222>(1)..(24)

<400>26

gcttaaggcg?agggttcgtg?atgg?????????????????????????????24

Claims (10)

1, a kind of individual administration gene type diagnostic chip, comprise carrying substrates (1) and on substrate, be the several genes probe (3) that array distributes, it is characterized in that gene probe (3) contains the complementary oligonucleotide sequence in drug metabolism and effect associated gene mutation site.

2, individual administration gene type diagnostic chip according to claim 1, it is characterized in that these mutational sites are that occurrence frequency in the crowd is higher, with the individual difference at the medicine of some disease is in close relations clinically, and the pharmacokinetics or the drug effect of these medicines had a significant effect, has the mutational site of using value.

3, individual administration gene type diagnostic chip according to claim 1 is characterized in that being provided with reference to probe (5) in gene probe on the chip (3) array.

4, individual administration gene type diagnostic chip according to claim 1 is characterized in that each mutational site design wild type gene probe array (6) and (7) two kinds of probes of mutated genes probe array, and its probe length is 17-22bp.

5, according to claim 1 or 2 or 3 or 4 described individual administration gene type diagnostic chips, it is characterized in that the individual administration gene type diagnostic chip that is used for the hypertensive patient contains the probe that detects 7 gene mutation sites, these 7 gene mutation sites are: CYP2C9*3, CYP2C19*2, CYP2C19*3, CYP2D6*10, ADRB1 (A145G), ADRB1 (G1165C), AGTR1 (A1166G).

6, individual administration gene type diagnostic chip according to claim 1, it is characterized in that can being provided with on each carrying substrates (1) a plurality of gene probe arrays is a plurality of point samples zone (2).

7, individual administration gene type diagnostic chip according to claim 1 is characterized in that carrying substrates can adopt solid-phase medias such as silicon chip, sheet glass, plastic sheet, nylon membrane, and probe and substrate can be done to modify and the bag quilt.

8, individual administration gene type diagnostic chip according to claim 7 is characterized in that the substrate surface treatment mode can be poly-lysine bag quilt, agar glycolyx, amino or aldehyde radical modification.

9, a kind of individual administration gene type diagnostic chip manufacture method, it is characterized in that according near the base sequence the gene locus of required detection, each mutational site design wild-type and two kinds of probes of mutant, design length is the probe of 17-22bp, use dna sequence dna synthesizer synthesising probing needle, with spot sample device with probe points on substrate, behind the point sample with the chip solidification treatment, be stored in 4 ℃ standby.

10, a kind of application method of individual administration gene type diagnostic chip may further comprise the steps:

(1) sampling: from the patient 1-2ml that takes a blood sample on one's body;

(2) sample process

Use the genome DNA extraction test kit to extract DNA;

(3) PCR reaction amplification

Use Auele Specific Primer by the amplification of PCR reaction pair DNA sample, use fluorescent substance or isotropic substance that primer or dNTP are carried out mark;

(4) hybridization

Get PCR product mixed solution, add positive with reference to solution and through the hybridization solution of preheating, mixing, 3 minutes postposition of sex change are all transferred to the point sample zone of individual administration gene type diagnostic chip on ice, add cover glass; Hybridization adds several dripping in the cabin, to keep humidity; Chip is put into the hybridization cabin, and thermostat container or water bath heat preservation one hour is put in sealing hybridization cabin then into;

(5) develop a film

Open the hybridization cabin, take out chip, wash out cover glass with elutriant, then chip is put into elutriant, room temperature (25 ℃ ± 10 ℃) was placed 5 minutes, and is with sterilization distilled water flushing two times, dry under the room temperature;

(6) read sheet

Recommend to use the ScanArray4000 scanner under 60-85 scanning intensity, to scan;

(7) interpretation of result

Adopt artificial interpretation or use the software kit analysis.Provide the medication proposal on adjustments according to gene type assay result and related data.

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