CN101343658A - Gene chip for detection of hyperpiesis individual medicine correlated gene mutation and uses thereof - Google Patents
Gene chip for detection of hyperpiesis individual medicine correlated gene mutation and uses thereof Download PDFInfo
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- CN101343658A CN101343658A CNA2007100358609A CN200710035860A CN101343658A CN 101343658 A CN101343658 A CN 101343658A CN A2007100358609 A CNA2007100358609 A CN A2007100358609A CN 200710035860 A CN200710035860 A CN 200710035860A CN 101343658 A CN101343658 A CN 101343658A
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Abstract
The invention relates to a gene chip used for detecting gene mutation relating to a high blood pressure personalized medicine. The gene chip for detecting the gene mutation relating to high blood pressure personalized medicine comprises a solid phase support, a gene probe (an oligonueleotide probe) fixed in the solid phase support sequentially and a PCR primer used for amplifying the mutated gene fragment in the sample; the gene probe (the oligonueleotide probe) and the PCR primer are designed aiming at one or two points of the gene mutation in ACE (I/D) and CYP3A5*3 and / or two or more points as follows: CYP2C9*3, CYP2C9*13, AGTR1(A1166C), CYP2D6*10, ADRB1(C1165G), TSC(C1784T), ADRB3(T727C), SCNN1G_rs5729, SCNN1G_rs5723, ENOSA_rs1799983 and GNB3(C825T). The invention provides the gene chip and applications thereof for conveniently, quickly and systematically detecting the gene mutation relating to high blood pressure personalized medicine so as to determine drug reactions.
Description
Technical field
The present invention relates to be used for the gene chip that hyperpiesis individual medicine detects because of sudden change.
Background technology
Hypertension has become global public health problem as puzzlement human physical and mental health's major disease: conservative estimation, and China's hyperpietic's number has surpassed 1.6 hundred million, and sickness rate rises year by year; Treatment to hypertension and complication thereof has become the whole world the 3rd big disease financial burden.Medicine be at present and in the future considerable time internal therapy hypertension and the main means of complication thereof.Because the individual difference of drug reaction (curative effect and untoward reaction), accepting has 20%～50% blood pressure not to be well controlled among the patient of pharmacological agent approximately.
The individual difference of drug reaction is a general phenomenon extremely clinically, the reason that produces this species diversity has many, comprise sex, age, body weight, disease condition can cause different patients to reaction appearance amount and qualitative difference with a kind of medicine in interior multiple factor, it is wherein most important that yet the pharmacogenetics result of study over more than 20 year shows, the most basic factor is an inherited genetic factors, it is drug metabolism enzyme, the gene pleiomorphism of transporter and acceptor (drug target) has caused the changing function of its proteins encoded, further make Plasma Concentration significantly different with drug susceptibility, in one word, heritable variation has caused the drug reaction individual difference.Present clinical antihypertensive drug commonly used (table 1) as follows:
Table 1
| Classification | Represent medicine | The market share |
| Calcium antagonist | Amlodipine, nifedipine, felodipine, nimodipine, levamlodipine | 42.06％ |
| Angiotensin converting enzyme inhibitor | Benazepril, fosinopril, perindopril, Yipingshu, enalapril, captopril | 19.14％ |
| Beta-blocker | Metoprolol, carvedilol, bisoprolol, esmolol | 14.97％ |
| Angiotensin II receptor antagonists | Losartan, valsartan, irbesartan, telmisartan | 13.98％ |
| The diuretic antihypertensive medicine | Hydrochlorothiazide, Furosemide, bumetanide | 2.55％ |
| Other | Terazosin, Doxazosin, Hypotensor No 0 (Triamterene+Reserpine+Hydrochlorothiazide+Dihydralazine+Chlordiazepoxide) | 7.31％ |
Through secular observation and research, discovery has many transgenations may be relevant with the reactivity of antihypertensive drug, and these transgenations comprise CYP2C9*3, CYP2C9*13, CYP2D6*10, CYP3A5*3, CYP3A4*17, CYP11B2 (C-344T), CYP11B2 (A3323G), ADD1 (Gly460Trp), GNB3 (C825T), GNAS (T131C), SCNN1B (T594M), SCNN1G_rs5729, SCNN1G_rs5723, ENOSA_rs1799983, TSC (C1784T), ACE (I/D), ACE2_rs2106809, ACE (C10514T), ACE (T10527C), ACE (A10578G), ACE (G12257A), ACE (G14480C), ACE (C14488A), ACE (A14521G), AGT (G-6A), AGT (M235T), AGT (T174M), AGT (A1237G), AGT (A-20C), AGTR1 (A1166C), AGTR1 (C573T), AGTR1 (A49954G), AGTR1 (T4955A), AGTR1 (T5052G), AGTR1 (C5245T), AGTR2 (C3123A), AGTR2 (G1675A), AGTR2 (G4297T), AGTR2 (A4303G), NR3C2 (C1825T), REN (A7174G), REN (T1456G), REN (A144G), REN (G134A), REN (T164G), REN (A204C), EDN1 (G5665T), NOS1 (Glu298Asp), ADRB1 (A145G), ADRB1 (G1165C), ADRB2 (Arg16Gly), ADRB3 (T727C), MTR (D919G), APOAIV (A1449G), APOAV (C31455T), APOB (100 G10108A), APOB (100C711T), LDLR (C16730T), LDLR (C20001T), LIPC (A110G), LPL (C9040G), LPL (G7315C), LPL (A7360G), LPL (Ser447Stop), SLC14A2_rs1123617, SLC14A2_rs3745009, MDR1 (G2677T), tens of kinds of MDR1 (C3435T) etc.
Through further clinical study, have in the said gene mutational site clinical evidence show its influence drug responsiveness mainly contain 30 surplus kind, shown in following table (table 2):
Table 2
| Drug categories | Relevant mutational site |
| Calcium antagonist | CYP3A5*3、SLC14A2_rs1123617、SLC14A2_rs3745009、 CYP3A4*17、MDR1(G2677T)、MDR1(C3435T) |
| Angiotensin converting enzyme inhibitor | ACE(I/D)、AGT(M235T)、AGTR1(A1166C)、MTR(D919G)、 ACE2_rs2106809 |
| Beta-blocker | GNB3(C825T)、GNAS(T131C)、AGT(G-6A)、AGT(M235T)、EDN1 (G5665T)、ADRB1(C1165G)、LDLR(C16730T)、CYP2D6*10 |
| Angiotensin II receptor antagonists | ACE(I/D)、AGTR1(A1166C)、CYP11B2(C-344T)、EDN1(G5665T)、 APOB(100C711T)、CYP2C9*3、CYP2C9*13 |
| Diuretic antihypertensive medicine and other | ADD1(Gly460Trp)、GNB3(C825T)、ACE(I/D)、AGT(G-6A)、 AGTR1(A1166C)、NOS1(Glu298Asp)、TSC(C1784T)、ADRB3 (T727C)、SCNN1G_rs5729、SCNN1G_rs5723、ENOSA_rs1799983 |
Above-described transgenation all has more greatly may be closely related in the intravital reactivity of people with antihypertensive drug, here said reactivity has comprised curative effect and two aspects of security of medicine, drug effect with dosage of the same race is in the patient who has different genotype, the somebody is effective just, the somebody is absolutely void, also the toxic side effects of medicine can appear in the somebody, therefore utilize the range gene detection technique that hyperpietic's above-mentioned genetic information is understood, adjust patient's types of medicines and dosage according to the result who detects, can make the best curative effect of medicine performance, prevent that to greatest extent toxic side effects from taking place, the personalized medicine new model of Here it is gene targeting.This kind medication pattern is accepted by increasing scientific research personnel, clinician and patient at present, 2006 in the 15th " international pharmacogenetics conference " (IUPHAR meeting) that China holds, inquired into the current situation of gene targeting personalized medicine jointly and, predicted the arrival in " personalized medicine " epoch comprehensively from hundreds of representatives of 27 countries in the popularization situation of various countries.
The method that at present genotype is detected has a lot, the most frequently used have methods such as polymerase chain reaction-restriction fragment length polymorphism analysis (PCR-RFLP method), sequence-specific PCR, craft or automatic sequencing, these methods not only complex operation, sense cycle are grown, can't be accomplished high-throughput, and the factor that influences detected result is numerous, wayward, be difficult to satisfy the requirement that clinical practice detects, only carry out at present at scientific research field.
Gene chip (gene chip), claim the DNA chip again, it is one of great science and technology progress of the tool characteristics of the times that in biological high-tech area, occurred in recent years, it is with the gene probe (oligonucleotide probe of a large amount of gene informations in the energy reflected sample, the cDNA clone, PCR product etc.) be fixed on solid support in an orderly manner (as aldehyde radical, amino, sulfydryl, the slide glass or the silicon chip of carboxyl isoreactivity base group modification, nylon membrane, nitrocellulose filter) goes up the formation array, by carrying out hybridization with actual sample or amplified production and to the detection of hybridization signals such as fluorescence, only need single test, but obtain the information of all genes to be checked with regard to high-throughput ground.Compare with additive method, the detection method of gene chip have various product parallel processing capability, fast, the required sample size of analysis speed few, pollute less, advantage such as simple to operate, cheap.Therefore utilizing biochip technology to detect and the closely-related transgenation of hypertension drug reaction individual difference, is best choice instantly, has important practical significance.
U.S. Roche Holding Ag released a kind of gene test product " AmpliChip CYP450 " in 2005, be used to detect 26 kinds of transgenations (comprising CYP2D6*10) of CYP2D6 and 2 kinds of transgenations of CYP2C19, can be used to instruct clinical use via CYP2D6 and the metabolic multiclass medicine of CYP2C19 according to detected result.Although this product has detected CYP2D6*10, but when instructing high blood medicine individuation pharmacological agent, there is bigger weak point, mainly show as: (1) has only CYP2D6*10 that bigger occurrence frequency is arranged in Chinese in 26 kinds of transgenations of the CYP2D6 that detects, and the genotype of CYP2C19 and the use of antihypertensive drug do not have direct relation; (2) reactive relevant with β 1 adrenoceptor retarding agent except CYP2D6*10, ADRB1 (C1165G) in addition.With reactive closely-related CYP2C9*3 and the AGTR1 (A1166C) of comprising simultaneously of angiotensin receptor blocking agent, it is nonsensical to detect a kind of genotype merely; (2) in secular hypertension therapeutic process, can use multiple medicine, therefore need treat the detection that genes involved carries out system hypertension drug.
Chinese patent publication number CN1616675, open day on May 18th, 2005, the name of innovation and creation is called " individual administration gene type diagnostic chip and manufacture method and methods for using them thereof ", this application case discloses a kind of individual administration gene type diagnostic chip and manufacture method and methods for using them thereof, this diagnosing chip comprises carrying substrates and be the several genes probe that array distributes on substrate, gene probe is at CYP2C9*3, CYP2C19*2, CYP2D6*10, ADRB1 (A145G), ADRB1 (G1165C), these 6 gene mutation site designs relevant of AGTR1 (A1166C) with drug metabolism and effect, being provided with in the gene probe array on the chip with reference to probe, is ACCTTGGAATGTCCACAG with reference to the sequence of probe.Wild-type and two kinds of probes of mutant have been designed in each mutational site.The weak point of this invention is only can detect CYP2C9*3, CYP2C19*2, CYP2D6*10, ADRB1 (A145G), ADRB1 (G1165C), these 6 transgenations of AGTR1 (A1166C), wherein the reactivity of CYP2C19*2 and hypertension drug treatment does not have direct correlation, then occurrence frequency is lower for ADRB1 (A145G), both are all less than the necessity that detects, the detected result of remaining 4 transgenations then can not fully be used to instruct hypertensive individuation pharmacological agent, in addition, this invention only is provided with a positive with reference to probe, feminine gender is not set with reference to probe at each mutational site, this also will have influence on the accuracy of detected result.
Summary of the invention
The objective of the invention is to solve the deficiencies in the prior art, at ubiquitous drug reaction individual difference phenomenon in the present hypertension drug therapeutic process, provide a kind of and can convenient, fast, systematically detect the transgenation of hypertension personalized medicine, determine the gene chip and the application thereof of drug responsiveness.
For addressing the above problem, the present invention by the following technical solutions:
A kind of hyperpiesis individual medicine that is used for is because of the gene chip that sudden change detects, and comprises solid support, is fixed on the gene probe (oligonucleotide probe) on the solid support and be used for the segmental PCR primer of amplified sample mutator gene in order; Wherein said gene probe (oligonucleotide probe) and PCR primer are to design at one or two gene mutation site design among ACE (I/D), the CYP3A5*3 and/or at two or more following sites: CYP2C9*3, CYP2C9*13, AGTR1 (A1166C), CYP2D6*10, ADRB1 (C1165G), TSC (C1784T), ADRB3 (T727C), SCNN1G_rs5729, SCNN1G_rs5723, ENOSA_rs1799983, GNB3 (C825T).
Gene mutation site involved in the present invention is may carrying out obtaining after the strict examination by the gene mutation site relevant with the reactivity of antihypertensive drug of his-and-hers watches 2 disclosures.Concrete steps are:
1) collects sample, gather clinical data, extracting DNA.
2) carry out gene type respectively according to table 2 pair each mutational site, determine its occurrence frequency in Chinese population simultaneously, remove the low excessively site of mutation frequency.
3) remaining each mutational site is carried out the experiment of clinical pharmacokinetics and the pharmacodynamics test of large sample respectively.
4) statistical study, the reactivity of removing antihypertensive drug does not have the obviously mutational site of influence.
5) mutational site to filtering out, select the hyperpietic and carry out genotype detection in " individuation pharmacological agent consulting and inspection center ", adjust therapeutic regimen according to detected result, further carry out clinical trail, observe the medication effect, verify the validity of each mutational site detection, remove the site of not having clear meaning in actual applications direction of medication usage.
6) determine target gene of the present invention mutational site.
Through the examination of his-and-hers watches 2 contents, the gene mutation site that the present invention finally determines is as shown in table 3:
Table 3
| The sudden change title | Functional meaning | Related drugs |
| CYP2C9*3 | The metabolic enzyme function significantly reduces | Angiotensin II receptor antagonists |
| CYP2C9*13 | The metabolic enzyme function significantly reduces | Angiotensin II receptor antagonists |
| AGTR1(A1166C) | Receptor sensitivity descends | Angiotensin II receptor antagonists |
| CYP2D6*10 | The metabolic enzyme function significantly reduces | Beta-blocker |
| ADRB1(C1165G) | Receptor sensitivity strengthens | Beta-blocker |
| ACE(I/D) | The susceptibility of action target spot changes | Angiotensin converting enzyme inhibitor |
| CYP3A5*3 | The metabolic enzyme function significantly reduces | Calcium antagonist |
| TSC(C1784T) | Drug susceptibility reduces | The diuretic antihypertensive medicine |
| ADRB3(T727C) | Drug susceptibility reduces | The diuretic antihypertensive medicine |
| SCNN1G_rs5729 | Drug susceptibility strengthens | The diuretic antihypertensive medicine |
| SCNN1G_rs5723 | Drug susceptibility strengthens | The diuretic antihypertensive medicine |
| ENOSA_rs1799983 | Drug susceptibility reduces | The diuretic antihypertensive medicine |
| GNB3(C825T) | Drug susceptibility strengthens | The diuretic antihypertensive medicine |
According to table 3, the present invention proposes the selection in the mutational site of preferred version, for example: the combination of CYP2C9*3, CYP2C9*13 and AGTR1 (A1166C) can be used for instructing the medication of angiotensin II receptor antagonists; The combination of CYP2D6*10 and ADRB1 (C1165G) can be used for instructing the medication of beta-blocker; The independent detection of ACE (I/D) can be instructed the medication of angiotensin converting enzyme inhibitor; The independent detection of CYP3A5*3 can be instructed the medication of calcium antagonist; The combination of TSC (C1784T), ADRB3 (T727C), SCNN1G_rs5729, SCNN1G_rs5723, ENOSA_rs1799983 and GNB3 (C825T) can be used for instructing the medication of diuretic antihypertensive medicine; The recombinant of above-mentioned Sites Combination can be used to instruct the use of corresponding multiple depressor.Most preferred scheme is that these 13 sites are all detected, and its detected result can instruct the reasonable use of clinical 5 hypotensor things commonly used.
According to the present invention, the described gene probe (oligonucleotide probe) that is fixed on the solid support comprise can with the detection probes of hypertension drug therapeutic response genes involved fragments specific hybridization and corresponding feminine gender with reference to probe and the positive with reference to probe; Detection probes comprises forward wild-type probe, forward mutation type probe, reverse wild-type probe, inverse transition type probe, and feminine gender comprises that with reference to probe the forward feminine gender is with reference to probe and oppositely negative with reference to probe; Forward probe and corresponding reverse probe satisfy the base complementrity pair principle, it is identical that both detect effect, optional one gets final product, therefore each site all can select forward probe groups (comprising that forward wild-type probe, forward mutation type probe and forward feminine gender are with reference to probe) or reverse probe groups (comprising reverse wild-type probe, inverse transition type probe and oppositely negative with reference to probe) to be fixed on the solid support, each the bar probe both forward and reverse directions that is same site is identical, and the probe both forward and reverse directions of different loci can be different; Each the bar probe that belongs to the same probe groups in same site is consistent on sequence length, removes in the based composition of sequence outside the base in mutational site, and other bases all are consistent.
The detection probes sequence in described each site (comprise forward wild-type probe, forward mutation type probe, oppositely wild-type probe, inverse transition type probe) shown in the sequence of SEQ ID NO.1-52 in the appended sequence table, described feminine gender with reference to probe (comprising that the forward feminine gender is with reference to probe and oppositely negative with reference to probe) and the positive with reference to probe sequence shown in the sequence of appended SEQ ID NO.53-81.
Described gene probe 3 ' end or 5 ' end can increase spacerarm or carry out chemical group and modify; The spacerarm of described increase can be poly-thymus gland thuja acid or poly-TEG, and described chemical group is modified and be can be the amination modification.
The described segmental PCR primer sequence of amplified sample mutator gene that is used for is shown in the sequence of SEQ ID NO.82-107 in the appended sequence table.
The present invention is directed to each mutational site (mutant) two kinds of situations of not undergoing mutation (wild-type) and undergo mutation and respectively designed positive and negative two reference sequences that are used for synthesising probing needle, can synthesize the probe stationary in several or whole mutational sites on solid support at different antihypertensive drugs in use, wherein the detection probes in each site comprises a pair of forward probe (forward wild-type probe and forward mutation type probe) or a pair of reverse probe of selecting at random (oppositely wild-type probe and inverse transition type probe), this arranges be consistent on the formation (except the mutational site) to probe at sequence length and base, the sequence of each bar probe is for example synthesized the forward wild-type and the forward mutation type probe of following different lengths by any continuous 14～25 based compositions on the corresponding canonical sequence (sequence SEQ ID NO.1-52) at CYP2C9*3:
Forward wild-type probe I (SEQ ID NO.1 the 14th～27 bit base sequence, totally 14 bases)
5’-GAGATAC
TTGACC-3’
Forward mutation type probe I (SEQ IDNO.3 the 14th～27 bit base sequence, totally 14 bases)
5’-GAGATAC
TTGACC-3’
Forward wild-type probe II (SEQ ID NO.1 the 11st～29 bit base sequence, totally 19 bases)
5’-CCAGAGATAC
TTGACCTT-3’
Forward mutation type probe I I (SEQ ID NO.3 the 11st～29 bit base sequence, totally 19 bases)
5’-CCAGAGATAC
TTGACCTT-3’
Forward wild-type probe III (SEQ ID NO.1 the 9th～31 bit base sequence, totally 23 bases)
5’-GTCCAGAGATAC
TTGACCTTCT-3’
Forward mutation type probe I II (SEQ ID NO.3 the 9th～31 bit base sequence, totally 23 bases)
5’-GTCCAGAGATAC
TTGACCTTCT-3’
Prepare chip A, B and C with above-mentioned sequence synthetic probe according to the method for embodiment 1, method amplification CYP2C9*3 wild-type and mutant plasmid according to embodiment 1, respectively with chip A, B and C hybridization, result such as Fig. 3, Fig. 4 and shown in Figure 5: the probe of different lengths has all been obtained results of hybridization preferably, proves that thus the probe of Different Alkali radix all has good sensitivity and specificity.
On the basis of the detection probes of having synthesized each site,, can design corresponding feminine gender respectively with reference to probe and positive at each site synthetic detection probes with reference to probe in order to obtain each site hybridization signal accurately; Negative with reference to probe and corresponding detection probes (comprising wild-type and mutant probe) basically identical on sequence length and base arrangement formation, just different with detection probes on the mutational site, both forward and reverse directions according to detection probes, it is oppositely negative with reference to probe with reference to probe or one that a corresponding forward feminine gender all can be synthesized in each site, both also satisfy base complementrity pair principle and identical with reference to effect, only need to select to get final product according to the direction of detection probes; Positive then is to select one section to arrange in base with detection probes and to have highly conforming oligonucleotide sequence on constituting with reference to probe, each site can a shared positive with reference to probe.The feminine gender of the hypertension drug that the present invention relates to treatment genes involved with reference to probe (comprising that the forward feminine gender is with reference to probe and oppositely negative with reference to probe) and the positive with reference to probe sequence shown in the sequence of SEQ ID NO.53-81 in the appended sequence table.
The present invention is directed to each site and designed positive and negative two sequences that are used for synthetic feminine gender with reference to probe, each feminine gender with reference to the sequence of probe by on the corresponding canonical sequence (sequence SEQ ID NO.53-81) any continuous 14～25 based compositions, the feminine gender in each site with reference to probe sequence direction, sequence length and base arrange constitute on corresponding wild-type probe and mutant probe basically identical, just the base on the mutational site is different with detection probes.The wild-type probe TCCAGAGATAC that has synthesized a forward such as CYP2C9*3 with reference to SEQ IDNO.1
TTGACCTTCTCCACCAGCCT has synthesized the mutant probe TCCAGAGATAC of a forward with reference to SEQ IDNO.3
TTGACCTTCTCCACCAGCCT then should arrange the forward feminine gender that is consistent with above-mentioned wild-type probe and mutant probe on constituting with reference to probe TCCAGAGATAC in length, direction and base according to synthetic one of the sequence of SEQIDNO.53
TTGACCTTCTC CACCAGCCT; CYP2D6*10 has synthesized a reverse wild-type probe GCACGCTAC with reference to SEQ ID NO.13 for another example
CACCAGGCCCCCTG has synthesized an inverse transition type probe GCACGCTAC with reference to SEQ IDNO.15
CACCAGGCCCCCTG then should arrange the reverse feminine gender that is consistent with above-mentioned wild-type probe and mutant probe on constituting with reference to probe GCACGCTAC in length, direction and base according to synthetic one of the sequence of SEQ ID NO.60
CACCAGGCCCCCTG.The forward feminine gender in same site with reference to probe with oppositely negative when the chip detection be identical with reference to probe with reference to effect, prerequisite is that negative the arrangement in length, direction and base with reference to probe and corresponding wild-type and mutant probe is consistent on constituting.
It is negative that application in gene chip is made and detected belongs to this area scientific research personnel's technique known with reference to probe with the positive with reference to probe, negative by being provided with reference to probe and positive with reference to probe, can control in the crossover process non-specific hybridization to the interference of signal value and the assessment of crossover process.For example, at CYP2C9*3 design wild-type probe, mutant probe and negative with reference to probe, sequence is as follows:
With above-mentioned 4 sequence of points on same chip; the CYP2C9*3 that increases respectively wild and the sudden change plasmid; hybridize with reference to positive reference of probe complementary and chip with product and with the positive; the hybridization scintigram as shown in Figure 6; we are as can be seen from Fig. 6; no matter use wild plasmid or mutant plasmid amplified production; negative all do not have hybridization signal with reference to probe; and the positive is stablized and specific signals with reference to probe signals, illustrates that the feminine gender among the present invention can play the effect of control crossover process really with reference to probe with reference to the probe and the positive.
The sequence characteristic design that is used for the segmental PCR primer of amplified sample mutator gene according to hypertension drug treatment associated gene mutation of the present invention is reacted one or more mutator genes that can increase by PCR.The present invention preferably utilizes asymmetric PCR that mutator gene is increased, asymmetric PCR is a large amount of single stranded DNA of a pair of primer amplification generation with inequality, this is called unrestricted primer (high density primer) and restricted primer (lower concentration primer) to primer, its ratio is generally 50～100: 1, and the asymmetric PCR technology belongs to the known general technology of this area scientific research personnel.The sequence of SEQ IDNO.82-107 is the canonical sequence that can be used for synthesizing the PCR primer among the present invention in the appended sequence table.
When using, reality can select continuous 17～25 the base synthetic primers on the corresponding sequence (sequence SEQ ID NO.82-107) of several or whole mutational sites (site during with synthesising probing needle is selected consistent), the primer of the invention described above can synthesize by method well known to those skilled in the art, the primer in each site can be placed apart after synthesizing, and also can use mix primer.Can when above-mentioned primer is synthetic, carry out suitable mark for ease of carrying out result's detection simultaneously, described labelling groups comprises: digoxin molecule (DIG), biotin molecule (Bio), fluorescein and derivative molecular (FITC etc.) thereof, other fluorescence molecules (as Cy3, Cy5 etc.), alkaline phosphatase (AP), horseradish peroxidase (HRP) etc., the detection method of these marks and marking method thereof and each marker all has been routine techniques well-known in the art.This unrestricted primer (high density primer) of testing preferred right title PCR carries out the Cy5 mark of 5 ' end, makes 5 of synthetic strand PCR product ' end have fluorescent mark, so that read the result in follow-up scanning process.If a certain site has been selected the forward probe for use when constituting probe array, then the preceding primer in this site (F primer) is the unrestricted primer of asymmetric PCR, should carry out mark to it; If a certain site has been selected reverse probe for use when constituting probe array, then the back primer in this site (R primer) is the unrestricted primer of asymmetric PCR, should carry out mark to it.
Probe of the present invention and primer can detect at a transgenation, also can be used in combination to detect a plurality of transgenations:
According to the synthetic detection probes sequence of the canonical sequence shown in the SEQ ID NO.1-12, synthetic negative according to the canonical sequence shown in the SEQ ID NO.53-58 with reference to probe, synthetic positive according to the canonical sequence shown in the SEQ ID NO.81 with reference to probe, primer according to the synthetic amplified sample of the canonical sequence shown in the SEQ ID NO.82-87, gene chip of Gou Chenging and detection reagent like this, can be used for detecting reactive closely-related CYP2C9*3 with angiotensin II receptor antagonists, the individual reaction of patient to angiotensin II receptor antagonists such as losartans determined in three kinds of transgenations of CYP2C9*13 and AGTR1 (A1166C).
According to the synthetic detection probes sequence of the canonical sequence shown in the SEQ ID NO.13-20, synthetic negative according to the canonical sequence shown in the SEQ ID NO.59-62 with reference to probe, synthetic positive according to the canonical sequence shown in the SEQ ID NO.81 with reference to probe, primer according to the synthetic amplified sample of the canonical sequence shown in the SEQ ID NO.88-91, gene chip of Gou Chenging and detection reagent like this, can be used for detecting reactive closely-related CYP2D6*10 with beta-blocker, the individual reaction of patient to beta-blockers such as carvedilols determined in two kinds of transgenations of ADRB1 (C1165G).
According to the synthetic detection probes sequence of the canonical sequence shown in the SEQ ID NO.21-24, synthetic negative according to the canonical sequence shown in the SEQ ID NO.63-66 with reference to probe, synthetic positive according to the canonical sequence shown in the SEQ ID NO.81 with reference to probe, primer according to the synthetic amplified sample of the canonical sequence shown in the SEQ ID NO.92-93, gene chip of Gou Chenging and detection reagent like this, can be used for detecting this transgenation of reactive closely-related ACE (I/D) with angiotensin converting enzyme inhibitor, determine that the patient is to enalapril, the individual reaction of angiotensin converting enzyme inhibitors such as captopril.
According to the synthetic detection probes sequence of the canonical sequence shown in the SEQ ID NO.25-28, synthetic negative according to the canonical sequence shown in the SEQ ID NO.67-68 with reference to probe, synthetic positive according to the canonical sequence shown in the SEQ ID NO.81 with reference to probe, primer according to the synthetic amplified sample of the canonical sequence shown in the SEQ ID NO.94-95, gene chip of Gou Chenging and detection reagent like this, can be used for detecting this transgenation of reactive closely-related CYP3A5*3 with calcium antagonist, determine the individual reaction of patient calcium antagonists such as nifedipine, felodipines.
According to the synthetic detection probes sequence of the canonical sequence shown in the SEQ ID NO.29-52, synthetic negative according to the canonical sequence shown in the SEQ ID NO.69-80 with reference to probe, synthetic positive according to the canonical sequence shown in the SEQ ID NO.81 with reference to probe, primer according to the synthetic amplified sample of the canonical sequence shown in the SEQ ID NO.96-107, gene chip of Gou Chenging and detection reagent like this, can be used for detecting reactive closely-related TSC (C1784T) with the diuretic antihypertensive medicine, ADRB3 (T727C), SCNN1G_rs5729, SCNN1G_rs5723, these six kinds of transgenations of ENOSA_rs1799983 and GNB3 (C825T) determine that the patient is to hydrochlorothiazide, Furosemide, the individual reaction of diuretic antihypertensive medicines such as bumetanide.
Highly preferred embodiment is, according to the synthetic detection probes sequence of the canonical sequence shown in the SEQ ID NO.1-52, synthetic negative according to the canonical sequence shown in the SEQ ID NO.53-80 with reference to probe, synthetic positive according to the canonical sequence shown in the SEQ ID NO.81 with reference to probe, primer according to the synthetic amplified sample of the canonical sequence shown in the SEQ ID NO.82-107, gene chip of Gou Chenging and detection reagent like this, can be used for detecting closely-related: CYP2C9*3 with multiple hypertension therapeutic drug responsiveness, CYP2C9*13, AGTR1 (A1166C), CYP2D6*10, ADRB1 (C1165G), ACE (I/D), CYP3A5*3, TSC (C1784T), ADRB3 (T727C), SCNN1G_rs5729, SCNN1G_rs5723, ENOSA_rs1799983, the individual reaction of patient to major part hypertension therapeutic medicine commonly used determined in these 13 kinds of transgenations of GNB3 (C825T).
Advantage of the present invention is:
1. the present invention comprehensively, system, high-throughput ground detect and knownly react the closely-related transgenation of individual difference with antihypertensive drug, and can make corresponding adjustment scheme at different pharmaceutical.
2. accurately, sensitive, sudden change detects to said gene specifically.
3. simple and efficient to handle, few to the requirement of sample, human input is few, reduces personal errors greatly, the result difference that can avoid the different experiments personnel operation to bring.
5. compare with PCR-RFLP and sequencing, the present invention pollutes gently, cost is lower.
6. can detect many parts of biological specimens, a plurality of transgenations simultaneously.
7. gene chip of the present invention can be divided into 1 person-portion, 2 person-portions and many person-portions according to the number in point sample zone not to be waited, the present invention is according to the design of preferred 2 person-portions of the needs of practical application, 2 point sample zones promptly are set on substrate, go up required probe respectively, once can detect 2 parts of biological samples.
Description of drawings:
Fig. 1: hypertension drug treatment genes involved detection chip orthographic plan;
Fig. 2: hypertension drug treatment genes involved detection chip scintigram;
Fig. 3: chip A (wild-type probe I+ mutant probe I) scintigram;
Fig. 4: chip B (wild-type probe II+ mutant probe I I) scintigram;
Fig. 5 chip C (wild-type probe III+ mutant probe I II) scintigram;
Fig. 6: hybridization scintigram.
Fig. 7: the hybridization scintigram of chip D and chip E;
Fig. 8: the preparation flow figure of chip detecting system.
Embodiment
Embodiment 1: be used for the preparation of hyperpiesis individual medicine because of the gene chip of sudden change detection
1. the main raw and auxiliary material and the plant and instrument (as shown in table 4) that prepare usefulness
Table 4
| Reagent and plant and instrument | The place of production | Reagent purity/concentration |
| Blank super plain film | Baiao Science and Technology Co. Ltd., Shanghai | / |
| Aminosilane reagents | Acros (U.S.) | 99wt.％ |
| Glutaraldehyde | Acros (U.S.) | 25wt.％ |
| 95% ethanol | Shanghai Chemical Plant | Analytical pure |
| Glacial acetic acid | Shanghai Chemical Plant | Analytical pure |
| NaBH4 | Shanghai Chemical Plant | Analytical pure |
| Na 2HPO 4 | Shanghai Chemical Plant | Analytical pure |
| KH 2PO 4 | Shanghai Chemical Plant | Analytical pure |
| Na 2CO 3 | Shanghai Chemical Plant | Analytical pure |
| NaHCO 3 | Shanghai Chemical Plant | Analytical pure |
| Amido modified oligonucleotide probe | Hunan grand Hao biological medicine company limited | 〉=99% (mass ratio) |
| The Cy5 fluorescent dye primer | Hunan grand Hao biological medicine company limited | 〉=99% (mass ratio) |
| Other primer | Hunan grand Hao biological medicine company limited | 〉=95% (mass ratio) |
| RTaq DNA polymerase | Dalian precious biotechnology company limited | 5u/μl |
| dNTPs | Dalian precious biotechnology company limited | ＞98％2.5mM |
| dUTP | Promega | ＞98％100mM |
| Uridylic glycosylase (UNG) | Promega | ＞98％1u/μl |
| The DNA purification kit | Promega | / |
| The PCR instrument | Germany Biometra T1 Thermocycle | / |
| Dna synthesizer | The EXPEDITE8909 of ABI company | / |
| Point sample instrument | The Germany GeSIM Nano-plotter of company | / |
| Scanner | Axon GenePix4100A | / |
| Desk centrifuge | Germany Heraeus Legent Mach 1.6R | / |
| Low temperature thermostat bath | The last Nereid DKB-2015 of grand company type | / |
| The pure water instrument | Taiwan Ai Kepu AML-1-10-S | / |
2. preparation flow
See Fig. 8
3. detailed preparation process
3.1. substrate is handled: substrate adopts glass medium, and surface treatment mode adopts the aldehyde radical modification, and concrete steps are:
1) selection of blank super plain film: select the super plain film of 76mm * 25mm * 1mm for use, its length and width error is no more than 0.2mm, and thickness error is no more than 0.1mm, does not have damaged, surperficial no marking;
2) super plain film pre-treatment: will surpass plain film and be dipped in the potassium bichromate washing lotion of newly joining, and place after 7 days, and use washed with de-ionized water, and dry;
3) super plain film amination: the ethanolic soln of preparation aminosilane, concentration is respectively 2%, slide is immersed act on 15 minutes in the above-mentioned solution respectively, takes out with deionized water and cleans, and dries;
4) super plain film aldehyde radicalization: the super plain film that above-mentioned amination is handled immerses 5% glutaraldehyde PBS respectively, and (0.2mol/lM pH8.0) in the solution, acts on 30 minutes respectively, and PBS solution cleans and dries;
3.2. the probe preparation: detection probes (comprising wild-type probe and mutant probe) is selected the corresponding sequence in each site of SEQ ID 1～52, selects wherein continuous 14～25 bases arbitrarily, and is synthetic according to method well known to those skilled in the art.Select (comprising negative) the corresponding sequence in each site of SEQ ID53～81 with reference to probe, select wherein continuous 14～25 bases arbitrarily with reference to probe and positive with reference to probe, synthetic according to method well known to those skilled in the art.The present embodiment synthetic all is forward probes, the detection probes in each site and with reference to the actual sequence of probe shown in the sequence of SEQ ID NO.108-124 in the appended sequence table.
Probe synthetic concrete steps are as follows:
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/LNa2CO3/NaHCO3 solution (PH9.0), concentration is 2mmol/L.
3.3. the preparation of gene chip:
By the gene chip plane structure chart that hyperpiesis individual medicine detects because of sudden change that is used for shown in Figure 1, on the substrate of a glass medium, be divided into two gene probe point sample zones, the area in each point sample zone is 7mm*7mm, the gene probe that array in each point sample zone distributes: the wild and mutant probe of the 1st behavior site CYP2C9*3 gene, the 2nd behavior site CYP2C9*3 feminine gender is with reference to probe and positive in probe; Wild and the mutant probe of the 3rd behavior site ADRB1 (G1165C) gene, the 4th behavior site ADRB1 (G1165C) is negative with reference to probe and positive in probe; Wild and the mutant probe of the 5th behavior site AGTR1 (A1166C) gene, the 6th behavior site AGTR1 (A1166C) is negative with reference to probe and positive in probe; Wild and the mutant probe of the 7th behavior site CYP2D6*10 gene, eighth row are that site CYP2D6*10 feminine gender is with reference to probe and positive in probe; Wild and the mutant probe of the 9th behavior site ACE (I/D) gene, the 10th behavior site ACE (I/D) is negative with reference to probe.
3.4. point sample and point sample 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) drying at room temperature was placed 18 hours;
(3) use immediately after the drying at room temperature or be stored in 4 ℃ standby.
3.5. hyperpiesis individual medicine is because of the composition of sudden change chip detecting system
Detection system is mainly by oligonucleotide chip, hybridization solution, washing mother liquor, respectively detect the PCR reaction solution (comprising primer, dNTP, buffer etc.), biological enzyme of gene locus and positively constitute with reference to components such as DNA samples, 4 ℃ of stored refrigerated of oligonucleotide chip, other components are in-20 ℃ of freezing preservations, and wherein the PCR reaction solution needs lucifuge.The pcr amplification primer of each detection site is selected wherein continuous 17～25 bases arbitrarily according to the synthetic canonical sequence of the primer in each site of SEQ ID NO.82～107, and is synthetic by specialized company according to method well known to those skilled in the art.Carry out the Cy5 mark on the primer before present embodiment is chosen in, former primer is as the unrestricted primer (high density primer) of asymmetric PCR, and the actual PCR primer sequence of each detection site is shown in the sequence of SEQ ID NO.125-134 in the sequence table:
Embodiment 2: ((1166A＞C), five kinds of hypertension drugs treatments of CYP2D6*10, ACE (I/D) associated gene mutation detect for 1165G＞C), AGTR1 to CYP2C9*3, ADRB1 with gene chip prepared among the embodiment 1.
In August, 2005～2006 year June, the tame front three in refined three hospitals, No.1 Hospital Affiliated to Zhongshan Univ. and Jiangxi Prov. The People's Hospital three hospital carries out clinical verification in Central South University Hunan respectively, detect sample 1073 examples altogether, sample behaviour periphery whole blood, originating is the essential hypertension outpatient service of each city, clinical verification unit place and surrounding area and inpatient and other crowd at random.Clinical verification is operated in clinical verification unit and carries out.The clinical verification method is a counter point, adopts the PCR-RFLP methods of genotyping of routine clinical use and as the direct sequencing of gold standard in contrast, each sample is carried out somatotype detect.Found that: chip method is having splendid performance aspect sensitivity and the specificity, recall rate is more than 99%; Has high coincidence rate between the detected result of the detected result of chip method and sequencing and PCR-RFLP method; The accuracy that chip method detects is higher, CYP2C9 in the sample that all detect, and ADRB1, AGTR1, CYP2D6, the accuracy rate that ACE detects is substantially all more than 99%, and the situation of false retrieval is extremely rare.This illustrates that this hyperpiesis individual medicine is qualified because of the clinical verification of sudden change chip detecting system, can be used for clinical detection.
1. concrete reagent and used instrument are as follows:
1.1. the composition of chip detecting system is as shown in table 5:
Table 5
| 1. oligonucleotide chip | 2 person-portions * 10 slice |
| 2. hybridization solution (6 * SSC) | 200μl |
| 3. wash mother liquor (8%SDS) | 30ml |
| 4.PCR reaction solution 1 | 400μl |
| 5.PCR reaction solution 2 | 400μl |
| 6.PCR reaction solution 3 | 400μl |
| 7.PCR reaction solution 4 | 400μl |
| 8.PCR reaction solution 5 | 400μl |
| 10. enzyme 1 (rTaq, 5u/ μ l) | 20μl |
| 11. enzyme 2 (UNG, 1u/ μ l) | 20μl |
| 12. it is positive with reference to DNA (5 loci gene types are determined) | 20μl |
| 13. positive reference | 30μl |
| 14. siccative | 1 bag |
PCR reaction solution 1～5 is a design synthetic PCR primer among the embodiment 1.
1.2 other instruments and reagent:
PCR instrument, electrophoresis apparatus, hybridization instrument, laser co-focusing chip scanner (passage), ultraviolet spectrophotometer, Promega DNA extraction agent box with 635nm wavelength.
1. concrete testing process is as follows:
2.1 the processing of clinical sample
Gather the peripheric venous blood 2ml of individuality to be detected, use Promega DNA extraction agent box extracting DNA, also the DNA extraction agent of available autogamy carries out extracting, and two kinds of methods are conventionally known to one of skill in the art.DNA concentration should be greater than 200ng/ul, and with UV spectrophotometer measuring A260/A280 ratio, this ratio should be between 1.60～1.80.
2.2PCR amplification
In the Eppendorf of 0.2mL pipe, add PCR reaction solution 18.8 μ l, template DNA 0.8 μ l, and enzyme 1 0.2 μ l, enzyme 2 0.2ul constitute 20.0 μ l reaction systems (utilizing PCR reaction solution 1～5 to be made into A, B, C, D, E 5 tube reaction systems altogether).The pcr amplification program is:
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 the specific segmental method of the PCR method amplification chromogene known technology that has been this area, key wherein is design of primers, the primer sequence that utilizes the present invention to announce, those skilled in the art can rule of thumb or about document determine reaction system and amplification program, independently finishes this operation steps.
2.3 hybridization
The learn from else's experience hybridization solution 7.5 μ l of 43 ℃ of preheatings add (A) (B) (C) (D) each 3 μ l of (E) PCR product, add sun ginseng 1 μ l then, and mixing is drawn 20 μ l mixed solutions, transfers to the point sample zone of chip; Chip is put into the hybridization cabin, and 43 ℃ of water bath heat preservations 2 hours are put in sealing hybridization cabin then into.
Hybridization between amplified production of the present invention and the gene chip is carried out according to the classical way of this area, and persons skilled in the art can be determined the optimum condition of relevant damping fluid, sample concentration, prehybridization temperature, hybridization temperature and time etc. according to experience.
2.4 develop a film
Open the hybridization cabin, take out chip,, place deionized water again, take out, on whizzer instantaneous centrifugal 30 seconds, get rid of residual liquid on the coring sheet in room temperature washing 30 seconds with washings 1 rinsing 30 seconds.
2.5 scanning analysis
(1) dried chip is used the GenePix4100A scanner scanning immediately, PMT is set to 600, and optical maser wavelength is 635nm;
(2) scan image carries out quantitative analysis with 6.0 pairs of scanning results of image analysis software GenePix that scanner carries, and preserves analytical results;
(3) the supporting interpretation of result software of utilization and chip detecting system carries out interpretation of result and arrangement.
(4) print the examining report list.
2. the analytic process to detected result is as follows:
3.1 determine the genotype in each mutational site
As shown in Figure 2, the scanning spectra that obtains by chip scanner is a matrix collection of illustrative plates, walk to the 10th row from the 1st, promptly detect CYP2C9*3, ADRB1 (1165G＞C), the AGTR1 (genotype in 1166A＞C), CYP2D6*10, ACE (I/D) site from top to bottom.The probe distribution situation is as shown in table 6:
Table 6
| Probe location | The 1-5 point | The 6-10 point |
| The 1st row | The CYP2C9 wild-type probe | CYP2C9 mutant probe |
| The 2nd row | The CYP2C9 feminine gender is with reference to probe | Positive reference |
| The 3rd row | The ADRB1 wild-type probe | ADRB1 mutant probe |
| The 4th row | The ADRB1 feminine gender is with reference to probe | Positive reference |
| The 5th row | The AGTR1 wild-type probe | AGTR1 mutant probe |
| The 6th row | The AGTR1 feminine gender is with reference to probe | Positive reference |
| The 7th row | The CYP2D6 wild-type probe | CYP2D6 mutant probe |
| Eighth row | The CYP2D6 feminine gender is with reference to probe | Positive reference |
| The 9th row | The ACE wild-type probe | ACE mutant probe |
| The 10th row | The ACE feminine gender is with reference to probe | The ACE feminine gender is with reference to probe |
Judge idiotype according to the scanning spectra result behind the 1st, 3,5,7, the 9 row probe hybridizations, see table 7 for details:
Table 7
According to detected genotype the medication proposal on adjustments is proposed: as shown in table 8 below
Table 8
Annotate:, different after the losartan process CYP2C9 metabolism with valsartan and irbesartan by its meta-bolites E3174 performance pharmacological effect.
The individuation pharmacological agent gene diagnosis chip detected result of being provided based on Fig. 2 scanning result is as follows:
| Detection site | The somatotype result |
| CYP2D6*10 | MM |
| β-R | WM |
| CYP2C9*3 | WW |
| AT1-R | MM |
| ACE | ID |
The genotyping diagnosis:
1. beta receptor blocking agent (as metoprolol, the promise of card dimension ground etc.): tester's metabolic enzyme activity is low and receptor sensitivity is normal, and the tester is the more responsive type of beta receptor blocking agent.Recommend to use conventional dosage on the low side or routine dose.
2.AT1 receptor antagonist (as losartan, irbesartan etc.): tester's metabolic enzyme activity is normal and acceptor is responsive reduces, and uses the medication of AT1 receptor blocking relatively poor.Recommend to use higher dosage.
3.ACE inhibitor (as captopril etc.): tester ACE is the ID heterozygote, and this tester is more or less the same to the susceptibility of various ACE inhibitor, can be selected according to clinical particular case.
Embodiment 3: the consistence of the gene chip detected result of checking forward probe and reverse probe preparation
The present invention is directed to each site and all can synthesize the probe of one group of forward (comprise detection probes and with reference to probe) and one group of reverse probe (comprise detection probes and with reference to probe), the forward probe in same site and the reverse probe effect when chip detection is identical, prerequisite is that forward probe and corresponding reverse probe are consistent on length and base arrangement formation, each bar probe direction of same detection site should be identical, and different detection site can select synthetic positive or reverse probe to form array at random.The PCR primer in each site carries out mark to preceding primer (F primer) when probe is forward in addition, back primer (R primer) is carried out mark at probe when being reverse.Present embodiment is intended to confirm to use the consistence of gene chip on detected result of forward probe and the preparation of anti-phase probe.
Utilize embodiment 1 described method to prepare chip D, the sequence of its probe and primer is shown in table 8 and table 9; Utilize embodiment 1 described method to prepare chip E equally, its probe is the reverse probe of chip D, and its probe sequence is shown in the sequence of SEQ ID NO.135-150 in the sequence table, and other are with embodiment 1.
Detect respectively with a DNA sample with chip D and chip E, method is with embodiment 2, the chip scanning result who obtains as shown in Figure 7, visible chip D is identical with the detected result of chip E, the forward probe has identical detection effect with corresponding reverse probe, can select at random.
Embodiment 4: the comparison of gene chip of the present invention and similar patent (Chinese patent publication number CN1616675)
Chinese patent publication number CN1616675, open day on May 18th, 2005, the name of innovation and creation is called " individual administration gene type diagnostic chip and manufacture method and methods for using them thereof ", this application case discloses a kind of individual administration gene type diagnostic chip and manufacture method and methods for using them thereof, this diagnosing chip comprises carrying substrates and be the several genes probe that array distributes on substrate, gene probe is at CYP2C9*3, CYP2C19*2, CYP2D6*10, ADRB1 (A145G), ADRB1 (G1165C), these 6 gene mutation site designs relevant of AGTR1 (A1166C) with drug metabolism and effect, being provided with in the gene probe array on the chip with reference to probe, is ACCTTGGAATGTCCACAG with reference to the sequence of probe.Wild-type and two kinds of probes of mutant have been designed in each mutational site.
We utilize embodiment 1 described method to prepare chip F, according to the synthetic detection probes sequence of the canonical sequence shown in the SEQ ID NO.1-52, synthetic negative according to the canonical sequence shown in the SEQ ID NO.53-80 with reference to probe, synthetic positive according to the canonical sequence shown in the SEQ ID NO.81 with reference to probe, primer according to the synthetic amplified sample of the canonical sequence shown in the SEQ ID NO.82-107, gene chip of Gou Chenging and detection reagent like this, can be used for detecting closely-related: CYP2C9*3 with multiple hypertension therapeutic drug responsiveness, CYP2C9*13, AGTR1 (A1166C), CYP2D6*10, ADRB1 (C1165G), ACE (I/D), CYP3A5*3, TSC (C1784T), ADRB3 (T727C), SCNN1G_rs5729, SCNN1G_rs5723, ENOSA_rs1799983, the individual reaction of patient to major part hypertension therapeutic medicine commonly used determined in these 13 kinds of transgenations of GNB3 (C825T).
According to method and step that patent CN1616675 specification sheets is described, we have manufactured experimently chip G, and the sequence of its probe is shown in the sequence of SEQ ID NO.151-163 in the sequence table, and primer sequence is shown in the sequence of SEQ ID NO.164-175 in the sequence table.
Detect respectively with a DNA sample with chip F and chip G, method is with embodiment 2, and the detection in Gene Mutation result who obtains is as shown in table 9, and it is as shown in table 10 that scheme is adjusted in the medication that draws thus.
Table 9
| The site | Chip F genotype detection result | Chip G genotype detection result |
| CYP2C9*3 | CYP2C9*1/*1 wild-type homozygote | CYP2C9*1/*1 wild-type homozygote |
| CYP2C9*13 | CYP2C9*1/*13 mutant heterozygote | Do not have |
| AGTR1(A1166C) | AGTR1(1166)AA | AGTR1(1166)AA |
| CYP2D6*10 | CYP2D6*1/*10 mutant heterozygote | CYP2D6*1/*10 mutant heterozygote |
| ADRB1(C1165G) | ADRB1(1165)CC | ADRB1(1165)CC |
| ADRB1(A145G) | Do not have | ADRB1(145)AA |
| ACE(I/D) | ACE(DD) | ACE(DD) |
| CYP3A5*3 | CYP3A5*1/*3 | Do not have |
| TSC(C1784T) | TSC(1784)CT | Do not have |
| ADRB3(T727C) | ADRB3(727)CC | Do not have |
| SCNN1G_rs5729 | SCNN1G_rs5729 wild-type homozygote | Do not have |
| SCNN1G_rs5723 | CNN1G_rs5723 wild-type homozygote | Do not have |
| ENOSA_rs1799983 | ENOSA_rs1799983 wild-type homozygote | Do not have |
| GNB3(C825T) | GNB3(825)CC | Do not have |
| CYP2C19*2 | Do not have | CYP2C19*1/*2 mutant heterozygote |
Table 10
| Medicament categories | Chip F medication proposal on adjustments | Chip G medication proposal on adjustments |
| Angiotensin II receptor antagonists | Losartan is recommended to use higher dosage, valsartan and irbesartan are recommended to use dosage on the low side. | Losartan, valsartan, irbesartan all recommend to use routine dose. |
| The beta-2 adrenoceptor retarding agent | The beta-2 adrenoceptor retarding agent is recommended to use than low dosage. | The beta-2 adrenoceptor retarding agent is recommended to use than low dosage. |
| Angiotensin converting enzyme inhibitor | Recommend to use benazepril, fosinopril, do not recommend to use enalapril, imidapril. | There is not the suggestion of recommendation. |
| Calcium antagonist | Calcium antagonists such as amlodipine, nifedipine are recommended to use dosage on the low side. | There is not the suggestion of recommendation. |
| The diuretic antihypertensive medicine | Diuretic antihypertensive medicines such as hydrochlorothiazide, Furosemide are recommended to use higher dosage. | There is not the suggestion of recommendation. |
Can see by table 10, have bigger difference aspect the medication of adjustment hypertension according to the gene chip F of technical scheme preparation of the present invention and according to the gene chip G that patent CN1616675 specification sheets prepares.Chip F can detect reactive closely-related 13 transgenations with five class hypertension therapeutic medicines commonly used; Chip G only can detect 6 transgenations, wherein CYP2C19*2 and hypertension therapeutic medicine are irrelevant, ADRB1 (A145G) is basic in Chinese not to distribute, there is not the meaning of detection, remaining 4 detection site are only relevant with two class medicines, wherein the reactive closely-related sudden change with angiotensin II receptor antagonists has only detected CYP2C9*3 and AGTR1 (A1166C), and the site CYP2C9*13 important for another one then do not detect.Based on above reason, when carrying out the medication adjustment according to detected result, chip F compares with chip G, chip F can be contained five all class hypertension therapeutic medicines, wherein the medication of angiotensin converting enzyme inhibitor, calcium antagonist and diuretic antihypertensive medicine be to use chip G can not adjust, chip F is more comprehensive to the medication proposal on adjustments of angiotensin II receptor antagonists in addition.Chip F has also removed two sites not detecting meaning on the chip G, has saved preparation cost.
In sum, gene detecting chip of the present invention is better than the gene chip that patent CN1616675 discloses.
Sequence table
Individual Applicant
---------------------
Street：
City：
State：
Country：
PostalCode：
PhoneNumber：
FaxNumber：
EmailAddress：
＜110〉LastName: grand Hao
＜110〉FirstName: week
<110>MiddleInitial：
<110>Suffix：
Application Project
--------------------
＜120〉Title: hyperpiesis individual medicine is because of the sudden change chip detecting system
<130>AppFileReference：121
<140>CurrentAppNumber：
<141>CurrentFilingDate：_ _ _ _-_ _-_ _
<211>Length：41
SequenceName：1
SequenceDescription:CYP2C9*3 forward wild-type probe canonical sequence
<212>Type：DNA
<213>OrganismName：Homo sapiens
<400>PreSequenceString：tgcacgaggt ccagagatac attgaccttc tccaccagcc t
41
<211>Length：41
SequenceName：2
The reverse wild-type probe canonical sequence of SequenceDescription:CYP2C9*3
<212>Type：DNA
<213>OrganismName：Homo sapiens
<400>PreSequenceString：aggctggtgg agaaggtcaa tgtatctctg gacctcgtgc a
41
<211>Length：41
SequenceName：3
SequenceDescription:CYP2C9*3 forward mutation type probe canonical sequence
<212>Type：DNA
<213>OrganismName：Homo sapiens
<400>PreSequenceString：tgcacgaggt ccagagatac cttgaccttc tccaccagcc t
41
<211>Length：41
SequenceName：4
SequenceDescription:CYP2C9*3 inverse transition type probe canonical sequence
<212>Type：DNA
<213>OrganismName：Homo sapiens
<400>PreSequenceString：aggctggtgg agaaggtcaa ggtatctctg gacctcgtgc a
41
<211>Length：41
SequenceName：5
SequenceDescription:CYP2C9*13 forward wild-type probe canonical sequence
<212>Type：DNA
<213>OrganismName：Homo sapiens
<400>PreSequenceString：gtttgaccct catcactttc cggatgaagg tggcaatttt a
41
<211>Length：41
SequenceName：6
The reverse wild-type probe canonical sequence of SequenceDescription:CYP2C9*13
<212>Type：DNA
<213>OrganismName：Homo sapiens
<400>PreSequenceString：taaaattgcc accttcatcc ggaaagtgat gagggtcaaa c
41
<211>Length：41
SequenceName：7
SequenceDescription:CYP2C9*13 forward mutation type probe canonical sequence
<212>Type：DNA
<213>OrganismName：Homo sapiens
<400>PreSequenceString：gtttgaccct catcactttc tggatgaagg tggcaatttt a
41
<211>Length：41
SequenceName：8
SequenceDescription:CYP2C9*13 inverse transition type probe canonical sequence
<212>Type：DNA
<213>OrganismName：Homo sapiens
<400>PreSequenceString：taaaattgcc accttcatcc agaaagtgat gagggtcaaa c
41
<211>Length：41
SequenceName：9
SequenceDescription:AGTR1 (A1166C) forward wild-type probe canonical sequence
<212>Type：DNA
<213>OrganismName：Homo sapiens
<400>PreSequenceString：cacttcacta ccaaatgagc attagctact tttcagaatt g
41
<211>Length：41
SequenceName：10
SequenceDescription:AGTR1 (A1166C) is the wild-type probe canonical sequence oppositely
<212>Type：DNA
<213>OrganismName：Homo sapiens
<400>PreSequenceString：caattctgaa aagtagctaa tgctcatttg gtagtgaagt g
41
<211>Length：41
SequenceName：11
SequenceDescription:AGTR1 (A1166C) forward mutation type probe canonical sequence
<212>Type：DNA
<213>OrganismName：Homo sapiens
<400>PreSequenceString：cacttcacta ccaaatgagc cttagctact tttcagaatt g
41
<211>Length：41
SequenceName：12
SequenceDescription:AGTR1 (A1166C) inverse transition type probe canonical sequence
<212>Type：DNA
<213>OrganismName：Homo sapiens
<400>PreSequenceString：caattctgaa aagtagctaa ggctcatttg gtagtgaagt g
41
<211>Length：41
SequenceName：13
SequenceDescription:CYP2D6*10 forward wild-type probe canonical sequence
<212>Type：DNA
<213>OrganismName：Homo sapiens
<400>PreSequenceString：aacgctgggc tgcacgctac ccaccaggcc ccctgccact g
41
<211>Length：41
SequenceName：14
The reverse wild-type probe canonical sequence of SequenceDescription:CYP2D6*10
<212>Type：DNA
<213>OrganismName：Homo sapiens
<400>PreSequenceString：cagtggcagg gggcctggtg ggtagcgtgc agcccagcgt t
41
<211>Length：41
SequenceName：15
SequenceDescription:CYP2D6*10 forward mutation type probe canonical sequence
<212>Type：DNA
<213>OrganismName：Homo sapiens
<400>PreSequenceString：aacgctgggc tgcacgctac tcaccaggcc ccctgccact g
41
<211>Length：41
SequenceName：16
SequenceDescription:CYP2D6*10 inverse transition type probe canonical sequence
<212>Type：DNA
<213>OrganismName：Homo sapiens
<400>PreSequenceString：cagtggcagg gggcctggtg agtagcgtgc agcccagcgt t
41
<211>Length：41
SequenceName：17
SequenceDescription:ADRB1 (C1165G) forward wild-type probe canonical sequence
<212>Type：DNA
<213>OrganismName：Homo sapiens
<400>PreSequenceString：acttccgcaa ggccttccag ggactgctct gctgcgcgcg c
41
<211>Length：41
SequenceName：18
SequenceDescription:ADRB1 (C1165G) is the wild-type probe canonical sequence oppositely
<212>Type：DNA
<213>OrganismName：Homo sapiens
<400>PreSequenceString：gcgcgcgcag cagagcagtc cctggaaggc cttgcggaag t
41
<211>Length：41
SequenceName：19
SequenceDescription:ADRB1 (C1165G) forward mutation type probe canonical sequence
<212>Type：DNA
<213>OrganismName：Homo sapiens
<400>PreSequenceString：acttccgcaa ggccttccag cgactgctct gctgcgcgcg c
41
<211>Length：41
SequenceName：20
SequenceDescription:ADRB1 (C1165G) inverse transition type probe canonical sequence
<212>Type：DNA
<213>OrganismName：Homo sapiens
<400>PreSequenceString：gcgcgcgcag cagagcagtc gctggaaggc cttgcggaag t
41
<211>Length：40
SequenceName：21
SequenceDescription:ACE (I/D) forward wild-type probe canonical sequence
<212>Type：DNA
<213>OrganismName：Homo sapiens
<400>PreSequenceString：tttgtatttt tagtagagac ggggtttcac cgttttagcc
40
<211>Length：40
SequenceName：22
SequenceDescription:ACE (I/D) is the wild-type probe canonical sequence oppositely
<212>Type：DNA
<213>OrganismName：Homo sapiens
<400>PreSequenceString：ggctaaaacg gtgaaacccc gtctctacta aaaatacaaa
40
<211>Length：40
SequenceName：23
SequenceDescription:ACE (I/D) forward mutation type probe canonical sequence
<212>Type：DNA
<213>OrganismName：Homo sapiens
<400>PreSequenceString：tagacctgct gcctattcac tcagttttat gtggtttcgc
40
<211>Length：40
SequenceName：24
SequenceDescription:ACE (I/D) inverse transition type probe canonical sequence
<212>Type：DNA
<213>OrganismName：Homo sapiens
<400>PreSequenceString：gcgaaaccac ataaaactga gtgaataggc agcaggtcta
40
<211>Length：41
SequenceName：25
SequenceDescription:CYP3A5*3 forward wild-type probe canonical sequence
<212>Type：DNA
<213>OrganismName：Homo sapiens
<400>PreSequenceString：aagagctctt ttgtctttca gtatctcttc cctgtttgga c
41
<211>Length：41
SequenceName：26
The reverse wild-type probe canonical sequence of SequenceDescription:CYP3A5*3
<212>Type：DNA
<213>OrganismName：Homo sapiens
<400>PreSequenceString：gtccaaacag ggaagagata ctgaaagaca aaagagctct t
41
<211>Length：41
SequenceName：27
SequenceDescription:CYP3A5*3 forward mutation type probe canonical sequence
<212>Type：DNA
<213>OrganismName：Homo sapiens
<400>PreSequenceString：aagagctctt ttgtctttca atatctcttc cctgtttgga c
41
<211>Length：41
SequenceName：28
SequenceDescription:CYP3A5*3 inverse transition type probe canonical sequence
<212>Type：DNA
<213>OrganismName：Homo sapiens
<400>PreSequenceString：gtccaaacag ggaagagata ttgaaagaca aaagagctct t
41
<211>Length：41
SequenceName：29
SequenceDescription:TSC (C1784T) forward wild-type probe canonical sequence
<212>Type：DNA
<213>OrganismName：Homo sapiens
<400>PreSequenceString：cagtgggctg gatgcagaga cgccgtccct agcaccccta c
41
<211>Length：41
SequenceName：30
SequenceDescription:TSC (C1784T) is the wild-type probe canonical sequence oppositely
<212>Type：DNA
<213>OrganismName：Homo sapiens
<400>PreSequenceString：gtaggggtgc tagggacggc gtctctgcat ccagcccact g
41
<211>Length：41
SequenceName：31
SequenceDescription:TSC (C1784T) forward mutation type probe canonical sequence
<212>Type：DNA
<213>OrganismName：Homo sapiens
<400>PreSequenceString：cagtgggctg gatgcagaga tgccgtccct agcaccccta c
41
<211>Length：41
SequenceName：32
SequenceDescription:TSC (C1784T) inverse transition type probe canonical sequence
<212>Type：DNA
<213>OrganismName：Homo sapiens
<400>PreSequenceString：gtaggggtgc tagggacggc atctctgcat ccagcccact g
41
<211>Length：41
SequenceName：33
SequenceDescription:ADRB3 (T727C) forward wild-type probe canonical sequence
<212>Type：DNA
<213>OrganismName：Homo sapiens
<400>PreSequenceString：ggtctggagt ctcggagtcc aggcgatggc cacgatgacc a
41
<211>Length：41
SequenceName：34
SequenceDescription:ADRB3 (T727C) is the wild-type probe canonical sequence oppositely
<212>Type：DNA
<213>OrganismName：Homo sapiens
<400>PreSequenceString：tggtcatcgt ggccatcgcc tggactccga gactccagac c
41
<211>Length：41
SequenceName：35
SequenceDescription:ADRB3 (T727C) forward mutation type probe canonical sequence
<212>Type：DNA
<213>OrganismName：Homo sapiens
<400>PreSequenceString：ggtctggagt ctcggagtcc gggcgatggc cacgatgacc a
41
<211>Length：41
SequenceName：36
SequenceDescription:ADRB3 (T727C) inverse transition type probe canonical sequence
<212>Type：DNA
<213>OrganismName：Homo sapiens
<400>PreSequenceString：tggtcatcgt ggccatcgcc cggactccga gactccagac c
41
<211>Length：41
SequenceName：37
SequenceDescription:SCNN1G_rs5729 forward wild-type probe canonical sequence
<212>Type：DNA
<213>OrganismName：Homo sapiens
<400>PreSequenceString：tggtggagat cagagtgccg aggtggaggt ctgggactat g
41
<211>Length：41
SequenceName：38
The reverse wild-type probe canonical sequence of SequenceDescription:SCNN1G_rs5729
<212>Type：DNA
<213>OrganismName：Homo sapiens
<400>PreSequenceString：catagtccca gacctccacc tcggcactct gatctccacc a
41
<211>Length：41
SequenceName：39
SequenceDescription:SCNN1G_rs5729 forward mutation type probe canonical sequence
<212>Type：DNA
<213>OrganismName：Homo sapiens
<400>PreSequenceString：tggtggagat cagagtgccg tggtggaggt ctgggactat g
41
<211>Length：41
SequenceName：40
SequenceDescription:SCNN1G_rs5729 inverse transition type probe canonical sequence
<212>Type：DNA
<213>OrganismName：Homo sapiens
<400>PreSequenceString：catagtccca gacctccacc acggcactct gatctccacc a
41
<211>Length：41
SequenceName：41
SequenceDescription:SCNN1G_rs5723 forward wild-type probe canonical sequence
<212>Type：DNA
<213>OrganismName：Homo sapiens
<400>PreSequenceString：acccagatgc tggatgagct gtgaggcagg gttgagaaga c
41
<211>Length：41
SequenceName：42
The reverse wild-type probe canonical sequence of SequenceDescription:SCNN1G_rs5723
<212>Type：DNA
<213>OrganismName：Homo sapiens
<400>PreSequenceString：gtcttctcaa ccctgcctca cagctcatcc agcatctggg t
41
<211>Length：41
SequenceName：43
SequenceDescription:SCNN1G_rs5723 forward mutation type probe canonical sequence
<212>Type：DNA
<213>OrganismName：Homo sapiens
<400>PreSequenceString：acccagatgc tggatgagct ctgaggcagg gttgagaaga c
41
<211>Length：41
SequenceName：44
SequenceDescription:SCNN1G_rs5723 inverse transition type probe canonical sequence
<212>Type：DNA
<213>OrganismName：Homo sapiens
<400>PreSequenceString：gtcttctcaa ccctgcctca gagctcatcc agcatctggg t
41
<211>Length：41
SequenceName：45
SequenceDescription:ENOSA_rs1799983 forward wild-type probe canonical sequence
<212>Type：DNA
<213>OrganismName：Homo sapiens
<400>PreSequenceString：ctgctgcagg ccccagatga gcccccagaa ctcttccttc t
41
<211>Length：41
SequenceName：46
The reverse wild-type probe canonical sequence of SequenceDescription:ENOSA_rs1799983
<212>Type：DNA
<213>OrganismName：Homo sapiens
<400>PreSequenceString：agaaggaaga gttctggggg ctcatctggg gcctgcagca g
41
<211>Length：41
SequenceName：47
SequenceDescription:ENOSA_rs1799983 forward mutation type probe canonical sequence
<212>Type：DNA
<213>OrganismName：Homo sapiens
<400>PreSequenceString：ctgctgcagg ccccagatga tcccccagaa ctcttccttc t
41
<211>Length：41
SequenceName：48
SequenceDescription:ENOSA_rs1799983 inverse transition type probe canonical sequence
<212>Type：DNA
<213>OrganismName：Homo sapiens
<400>PreSequenceString：agaaggaaga gttctggggg atcatctggg gcctgcagca g
41
<211>Length：41
SequenceName：49
SequenceDescription:GNB3 (C825T) forward wild-type probe canonical sequence
<212>Type：DNA
<213>OrganismName：Homo sapiens
<400>PreSequenceString：atctgcacgg gctcggatga cgcttcctgc cgcttgtttg a
41
<211>Length：41
SequenceName：50
SequenceDescription:GNB3 (C825T) is the wild-type probe canonical sequence oppositely
<212>Type：DNA
<213>OrganismName：Homo sapiens
<400>PreSequenceString：tcaaacaagc ggcaggaagc gtcatccgag cccgtgcaga t
41
<211>Length：41
SequenceName：51
SequenceDescription:GNB3 (C825T) forward mutation type probe canonical sequence
<212>Type：DNA
<213>OrganismName：Homo sapiens
<400>PreSequenceString：atctgcacgg gctcggatga tgcttcctgc cgcttgtttg a
41
<211>Length：41
SequenceName：52
SequenceDescription:GNB3 (C825T) inverse transition type probe canonical sequence
<212>Type：DNA
<213>OrganismName：Homo sapiens
<400>PreSequenceString：tcaaacaagc ggcaggaagc atcatccgag cccgtgcaga t
41
<211>Length：41
SequenceName：53
SequenceDescription:CYP2C9*3 forward feminine gender is with reference to the probe canonical sequence
<212>Type：DNA
<213>OrganismName：Homo sapiens
<400>PreSequenceString：tgcacgaggt ccagagatac tttgaccttc tccaccagcc t
41
<211>Length：41
SequenceName：54
SequenceDescription:CYP2C9*3 is oppositely negative with reference to the probe canonical sequence
<212>Type：DNA
<213>OrganismName：Homo sapiens
<400>PreSequenceString：aggctggtgg agaaggtcaa agtatctctg gacctcgtgc a
41
<211>Length：41
SequenceName：55
SequenceDescription:CYP2C9*13 forward feminine gender is with reference to the probe canonical sequence
<212>Type：DNA
<213>OrganismName：Homo sapiens
<400>PreSequenceString：gtttgaccct catcactttc aggatgaagg tggcaatttt a
41
<211>Length：41
SequenceName：56
SequenceDescription:CYP2C9*13 is oppositely negative with reference to the probe canonical sequence
<212>Type：DNA
<213>OrganismName：Homo sapiens
<400>PreSequenceString：taaaattgcc accttcatcc tgaaagtgat gagggtcaaa c
41
<211>Length：41
SequenceName：57
SequenceDescription:AGTR1 (A1166C) forward feminine gender is with reference to the probe canonical sequence
<212>Type：DNA
<213>OrganismName：Homo sapiens
<400>PreSequenceString：cacttcacta ccaaatgagc gttagctact tttcagaatt g
41
<211>Length：41
SequenceName：58
SequenceDescription:AGTR1 (A1166C) is oppositely negative with reference to the probe canonical sequence
<212>Type：DNA
<213>OrganismName：Homo sapiens
<400>PreSequenceString：caattctgaa aagtagctaa cgctcatttg gtagtgaagt g
41
<211>Length：41
SequenceName：59
SequenceDescription:CYP2D6*10 forward feminine gender is with reference to the probe canonical sequence
<212>Type：DNA
<213>OrganismName：Homo sapiens
<400>PreSequenceString：aacgctgggc tgcacgctac acaccaggcc ccctgccact g
41
<211>Length：41
SequenceName：60
SequenceDescription:CYP2D6*10 is oppositely negative with reference to the probe canonical sequence
<212>Type：DNA
<213>OrganismName：Homo sapiens
<400>PreSequenceString：cagtggcagg gggcctggtg tgtagcgtgc agcccagcgt t
41
<211>Length：41
SequenceName：61
SequenceDescription:ADRB1 (C1165G) forward feminine gender is with reference to the probe canonical sequence
<212>Type：DNA
<213>OrganismName：Homo sapiens
<400>PreSequenceString：acttccgcaa ggccttccag tgactgctct gctgcgcgcg c
41
<211>Length：41
SequenceName：62
SequenceDescription:ADRB1 (C1165G) is oppositely negative with reference to the probe canonical sequence
<212>Type：DNA
<213>OrganismName：Homo sapiens
<400>PreSequenceString：gcgcgcgcag cagagcagtc actggaaggc cttgcggaag t
41
<211>Length：40
SequenceName：63
SequenceDescription:ACE (I/D) forward wild-type feminine gender is with reference to the probe canonical sequence
<212>Type：DNA
<213>OrganismName：Homo sapiens
<400>PreSequenceString：tttgtatttt tagtagatac ggtgtttcac cgttttagcc
40
<211>Length：40
SequenceName：64
The reverse wild-type feminine gender of SequenceDescription:ACE (I/D) is with reference to the probe canonical sequence
<212>Type：DNA
<213>OrganismName：Homo sapiens
<400>PreSequenceString：ggctaaaacg gtgaaacacc gtatctacta aaaatacaaa
40
<211>Length：40
SequenceName：65
SequenceDescription:ACE (I/D) forward mutation type feminine gender is with reference to the probe canonical sequence
<212>Type：DNA
<213>OrganismName：Homo sapiens
<400>PreSequenceString：tagacctgat gcctattcac tcagttttat ttggtttcgc
40
<211>Length：40
SequenceName：66
SequenceDescription:ACE (I/D) inverse transition type feminine gender is with reference to the probe canonical sequence
<212>Type：DNA
<213>OrganismName：Homo sapiens
<400>PreSequenceString：gcgaaaccaa ataaaactga gtgaataggc atcaggtcta
40
<211>Length：41
SequenceName：67
SequenceDescription:CYP3A5*3 forward feminine gender is with reference to the probe canonical sequence
<212>Type：DNA
<213>OrganismName：Homo sapiens
<400>PreSequenceString：aagagctctt ttgtctttca ttatctcttc cctgtttgga c
41
<211>Length：41
SequenceName：68
SequenceDescription:CYP3A5*3 is oppositely negative with reference to the probe canonical sequence
<212>Type：DNA
<213>OrganismName：Homo sapiens
<400>PreSequenceString：gtccaaacag ggaagagata atgaaagaca aaagagctct t
41
<211>Length：41
SequenceName：69
SequenceDescription:TSC (C1784T) forward feminine gender is with reference to the probe canonical sequence
<212>Type：DNA
<213>OrganismName：Homo sapiens
<400>PreSequenceString：cagtgggctg gatgcagaga agccgtccct agcaccccta c
41
<211>Length：41
SequenceName：70
SequenceDescription:TSC (C1784T) is oppositely negative with reference to the probe canonical sequence
<212>Type：DNA
<213>OrganismName：Homo sapiens
<400>PreSequenceString：gtaggggtgc tagggacggc ttctctgcat ccagcccact g
41
<211>Length：41
SequenceName：71
SequenceDescription:ADRB3 (T727C) forward feminine gender is with reference to the probe canonical sequence
<212>Type：DNA
<213>OrganismName：Homo sapiens
<400>PreSequenceString：ggtctggagt ctcggagtcc tggcgatggc cacgatgacc a
41
<211>Length：41
SequenceName：72
SequenceDescription:ADRB3 (T727C) is oppositely negative with reference to the probe canonical sequence
<212>Type：DNA
<213>OrganismName：Homo sapiens
<400>PreSequenceString：tggtcatcgt ggccatcgcc aggactccga gactccagac c
41
<211>Length：41
SequenceName：73
SequenceDescription:SCNN1G_rs5729 forward feminine gender is with reference to the probe canonical sequence
<212>Type：DNA
<213>OrganismName：Homo sapiens
<400>PreSequenceString：tggtggagat cagagtgccg cggtggaggt ctgggactat g
41
<211>Length：41
SequenceName：74
SequenceDescription:SCNN1G_rs5729 is oppositely negative with reference to the probe canonical sequence
<212>Type：DNA
<213>OrganismName：Homo sapiens
<400>PreSequenceString：catagtccca gacctccacc gcggcactct gatctccacc a
41
<211>Length：41
SequenceName：75
SequenceDescription:SCNN1G_rs5723 forward feminine gender is with reference to the probe canonical sequence
<212>Type：DNA
<213>OrganismName：Homo sapiens
<400>PreSequenceString：acccagatgc tggatgagct ttgaggcagg gttgagaaga c
41
<211>Length：41
SequenceName：76
SequenceDescription:SCNN1G_rs5723 is oppositely negative with reference to the probe canonical sequence
<212>Type：DNA
<213>OrganismName：Homo sapiens
<400>PreSequenceString：gtcttctcaa ccctgcctca aagctcatcc agcatctggg t
41
<211>Length：41
SequenceName：77
SequenceDescription:ENOSA_rs1799983 forward feminine gender is with reference to the probe canonical sequence
<212>Type：DNA
<213>OrganismName：Homo sapiens
<400>PreSequenceString：ctgctgcagg ccccagatga tcccccagaa ctcttccttc t
41
<211>Length：41
SequenceName：78
SequenceDescription:ENOSA_rs1799983 is oppositely negative with reference to the probe canonical sequence
<212>Type：DNA
<213>OrganismName：Homo sapiens
<400>PreSequenceString：agaaggaaga gttctggggg atcatctggg gcctgcagca g
41
<211>Length：41
SequenceName：79
SequenceDescription:GNB3 (C825T) forward feminine gender is with reference to the probe canonical sequence
<212>Type：DNA
<213>OrganismName：Homo sapiens
<400>PreSequenceString：atctgcacgg gctcggatga agcttcctgc cgcttgtttg a
41
<211>Length：41
SequenceName：80
SequenceDescription:GNB3 (C825T) is oppositely negative with reference to the probe canonical sequence
<212>Type：DNA
<213>OrganismName：Homo sapiens
<400>PreSequenceString：tcaaacaagc ggcaggaagc ttcatccgag cccgtgcaga t
41
<211>Length：19
SequenceName：81
SequenceDescription: positive with reference to the probe canonical sequence
<212>Type：DNA
<213>OrganismName：Homo sapiens
<400>PreSequenceString：cattcaacaa gttacgact
19
<211>Length：34
SequenceName：82
Primer (F primer) canonical sequence before the SequenceDescription:CYP2C9*3
<212>Type：DNA
<213>OrganismName：Homo sapiens
<400>PreSequenceString：taagtttgtt tctcctacac tgcaactcca tgtt
34
<211>Length：35
SequenceName：83
Primer behind the SequenceDescription:CYP2C9*3 (R primer) canonical sequence
<212>Type：DNA
<213>OrganismName：Homo sapiens
<400>PreSequenceString：tgccattttt ctccttttcc atcagttttt acttg
35
<211>Length：34
SequenceName：84
Primer (F primer) canonical sequence before the SequenceDescription:CYP2C9*13
<212>Type：DNA
<213>OrganismName：Homo sapiens
<400>PreSequenceString：acagatgctg aaacaggcac atgcacacct acca
34
<211>Length：35
SequenceName：85
Primer behind the SequenceDescription:CYP2C9*13 (R primer) canonical sequence
<212>Type：DNA
<213>OrganismName：Homo sapiens
<400>PreSequenceString：aagcatacaa atacaatgaa aatatcatgc taaat
35
<211>Length：34
SequenceName：86
The preceding primer of SequenceDescription:AGTR1 (A1166C) (F primer) canonical sequence
<212>Type：DNA
<213>OrganismName：Homo sapiens
<400>PreSequenceString：cacgctttcc taccgcccct cagataatgt aagc
34
<211>Length：35
SequenceName：87
SequenceDescription:AGTR1 (A1166C) back primer (R primer) canonical sequence
<212>Type：DNA
<213>OrganismName：Homo sapiens
<400>PreSequenceString：agctttagaa aagtcggttc agtccacata atgca
35
<211>Length：35
SequenceName：88
Primer (F primer) canonical sequence before the SequenceDescription:CYP2D6*10
<212>Type：DNA
<213>OrganismName：Homo sapiens
<400>PreSequenceString：ttcacccacc atccatgttt gcttctggta gggga
35
<211>Length：36
SequenceName：89
Primer behind the SequenceDescription:CYP2D6*10 (R primer) canonical sequence
<212>Type：DNA
<213>OrganismName：Homo sapiens
<400>PreSequenceString：aggagcccat ttggtagtga ggcaggtatg gggcta
36
<211>Length：35
SequenceName：90
The preceding primer of SequenceDescription:ADRB1 (C1165G) (F primer) canonical sequence
<212>Type：DNA
<213>OrganismName：Homo sapiens
<400>PreSequenceString：tgggcttcga gttcacctgc tatcggtctt aagta
35
<211>Length：35
SequenceName：91
SequenceDescription:ADRB1 (C1165G) back primer (R primer) canonical sequence
<212>Type：DNA
<213>OrganismName：Homo sapiens
<400>PreSequenceString：catcatgggc gtcttcacgc tctgctggct gccct
35
<211>Length：34
SequenceName：92
The preceding primer of SequenceDescription:ACE (I/D) (F primer) canonical sequence
<212>Type：DNA
<213>OrganismName：Homo sapiens
<400>PreSequenceString：ttgtaagggg agctcagaga atttcagagc tgga
34
<211>Length：36
SequenceName：93
SequenceDescription:ACE (I/D) back primer (R primer) canonical sequence
<212>Type：DNA
<213>OrganismName：Homo sapiens
<400>PreSequenceString：gtaagccact gctggagacc actcccatcc tttctc
36
<211>Length：34
SequenceName：94
Primer (F primer) canonical sequence before the SequenceDescription:CYP3A5*3
<212>Type：DNA
<213>OrganismName：Homo sapiens
<400>PreSequenceString：gtaatccata cccctagttg tacgacacac agca
34
<211>Length：35
SequenceName：95
Primer behind the SequenceDescription:CYP3A5*3 (R primer) canonical sequence
<212>Type：DNA
<213>OrganismName：Homo sapiens
<400>PreSequenceString：gatttacctg ccttcaattt ttcactgacc taata
35
<211>Length：34
SequenceName：96
The preceding primer of SequenceDescription:TSC (C1784T) (F primer) canonical sequence
<212>Type：DNA
<213>OrganismName：Homo sapiens
<400>PreSequenceString：catcagtcat ctcgtggctg ggccggctgt caaa
34
<211>Length：34
SequenceName：97
SequenceDescription:TSC (C1784T) back primer (R primer) canonical sequence
<212>Type：DNA
<213>OrganismName：Homo sapiens
<400>PreSequenceString：ttgaactcct aagctcaagc aatccaccca cctt
34
<211>Length：33
SequenceName：98
The preceding primer of SequenceDescription:ADRB3 (T727C) (F primer) canonical sequence
<212>Type：DNA
<213>OrganismName：Homo sapiens
<400>PreSequenceString：caccctggcg cccaataccg ccaacaccag tgg
33
<211>Length：34
SequenceName：99
SequenceDescription:ADRB3 (T727C) back primer (R primer) canonical sequence
<212>Type：DNA
<213>OrganismName：Homo sapiens
<400>PreSequenceString：cggcaccacc aggagtccca tcaccaggtc ggct
34
<211>Length：35
SequenceName：100
Primer (F primer) canonical sequence before the SequenceDescription:SCNN1G_rs5729
<212>Type：DNA
<213>OrganismName：Homo sapiens
<400>PreSequenceString：accttgggcc ggtaacttaa tctttctgtg cctca
35
<211>Length：35
SequenceName：101
Primer behind the SequenceDescription:SCNN1G_rs5729 (R primer) canonical sequence
<212>Type：DNA
<213>OrganismName：Homo sapiens
<400>PreSequenceString：tgccagccct gaagctggag ccgtttgtga ataaa
35
<211>Length：34
SequenceName：102
Primer (F primer) canonical sequence before the SequenceDescription:SCNN1G_rs5723
<212>Type：DNA
<213>OrganismName：Homo sapiens
<400>PreSequenceString：tctgggggca cccgatccat gtccttagac catg
34
<211>Length：36
SequenceName：103
Primer behind the SequenceDescription:SCNN1G_rs5723 (R primer) canonical sequence
<212>Type：DNA
<213>OrganismName：Homo sapiens
<400>PreSequenceString：tagacgatga cctacccact ttcaactctg ctttgc
36
<211>Length：34
SequenceName：104
Primer (F primer) canonical sequence before the SequenceDescription:ENOSA_rs1799983
<212>Type：DNA
<213>OrganismName：Homo sapiens
<400>PreSequenceString：tcagggatgg ccccctccat cccacccagt caat
34
<211>Length：34
SequenceName：105
Primer behind the SequenceDescription:ENOSA_rs1799983 (R primer) canonical sequence
<212>Type：DNA
<213>OrganismName：Homo sapiens
<400>PreSequenceString：cctgggtggt cacggagacc cagccaatga ggga
34
<211>Length：34
SequenceName：106
The preceding primer of SequenceDescription:GNB3 (C825T) (F primer) canonical sequence
<212>Type：DNA
<213>OrganismName：Homo sapiens
<400>PreSequenceString：agttgaagtc gtcgtagcca gcgaatagta ggcg
34
<211>Length：35
SequenceName：107
SequenceDescription:GNB3 (C825T) back primer (R primer) canonical sequence
<212>Type：DNA
<213>OrganismName：Homo sapiens
<400>PreSequenceString：tggagctgtc aggtgggagg cagagggcgg gagag
35
<211>Length：19
SequenceName：108
SequenceDescription: embodiment one CYP2C9*3 wild-type probe sequence
<212>Type：DNA
<213>OrganismName：Homo sapiens
<400>PreSequenceString：ccagagatac attgacctt
19
<211>Length：19
SequenceName：109
SequenceDescription: embodiment one CYP2C9*3 mutant probe sequence
<212>Type：DNA
<213>OrganismName：Homo sapiens
<400>PreSequenceString：ccagagatac cttgacctt
19
<211>Length：19
SequenceName：110
SequenceDescription: embodiment one CYP2C9*3 feminine gender is with reference to probe sequence
<212>Type：DNA
<213>OrganismName：Homo sapiens
<400>PreSequenceString：ccagagatac tttgacctt
19
<211>Length：17
SequenceName：111
SequenceDescription: embodiment one AGTR1 (A1166C) wild-type probe sequence
<212>Type：DNA
<213>OrganismName：Homo sapiens
<400>PreSequenceString：agtagctaat gctcatt
17
<211>Length：17
SequenceName：112
SequenceDescription: embodiment one AGTR1 (A1166C) mutant probe sequence
<212>Type：DNA
<213>OrganismName：Homo sapiens
<400>PreSequenceString：agtagctaag gctcatt
17
<211>Length：17
SequenceName：113
SequenceDescription: embodiment one AGTR1 (A1166C) is negative with reference to probe sequence
<212>Type：DNA
<213>OrganismName：Homo sapiens
<400>PreSequenceString：agtagctaac gctcatt
17
<211>Length：17
SequenceName：114
SequenceDescription: embodiment one CYP2D6*10 wild-type probe sequence
<212>Type：DNA
<213>OrganismName：Homo sapiens
<400>PreSequenceString：tgcacgctac ccaccag
17
<211>Length：17
SequenceName：115
SequenceDescription: embodiment one CYP2D6*10 mutant probe sequence
<212>Type：DNA
<213>OrganismName：Homo sapiens
<400>PreSequenceString：tgcacgctac tcaccag
17
<211>Length：17
SequenceName：116
SequenceDescription: embodiment one CYP2D6*10 feminine gender is with reference to probe sequence
<212>Type：DNA
<213>OrganismName：Homo sapiens
<400>PreSequenceString：tgcacgctac acaccag
17
<211>Length：17
SequenceName：117
SequenceDescription: embodiment one ADRB1 (C1165G) wild-type probe sequence
<212>Type：DNA
<213>OrganismName：Homo sapiens
<400>PreSequenceString：gccttccagg gactgct
17
<211>Length：17
SequenceName：118
SequenceDescription: embodiment one ADRB1 (C1165G) mutant probe sequence
<212>Type：DNA
<213>OrganismName：Homo sapiens
<400>PreSequenceString：gcct tccagc ggactgc
17
<211>Length：17
SequenceName：119
SequenceDescription: embodiment one ADRB1 (C1165G) is negative with reference to probe sequence
<212>Type：DNA
<213>OrganismName：Homo sapiens
<400>PreSequenceString：gccttccagt gactgct
17
<211>Length：22
SequenceName：120
SequenceDescription: embodiment one ACE (I/D) wild-type probe sequence
<212>Type：DNA
<213>OrganismName：Homo sapiens
<400>PreSequenceString：ttagtagaga cggggtttca cc
22
<211>Length：22
SequenceName：121
SequenceDescription: embodiment one ACE (I/D) wild-type feminine gender is with reference to probe sequence
<212>Type：DNA
<213>OrganismName：Homo sapiens
<400>PreSequenceString：ttagtagata cggtgtttca cc
22
<211>Length：32
SequenceName：122
SequenceDescription: embodiment one ACE (I/D) mutant probe sequence
<212>Type：DNA
<213>OrganismName：Homo sapiens
<400>PreSequenceString：cctgctgcct attcactcag ttttatgtgg tt
32
<211>Length：32
SequenceName：123
SequenceDescription: embodiment one ACE (I/D) mutant feminine gender is with reference to probe sequence
<212>Type：DNA
<213>OrganismName：Homo sapiens
<400>PreSequenceString：cctgatgcct attcactcag ttttatttgg tt
32
<211>Length：19
SequenceName：124
SequenceDescription: embodiment one positive is with reference to probe sequence
<212>Type：DNA
<213>OrganismName：Homo sapiens
<400>PreSequenceString：cattcaacaa gttacgact
19
<211>Length：18
SequenceName：125
Primer (F primer) sequence before SequenceDescription: embodiment one CYP2C9*3
<212>Type：DNA
<213>OrganismName：Homo sapiens
<400>PreSequenceString：aacatggagt tgcagtgt
18
<211>Length：17
SequenceName：126
Primer behind SequenceDescription: embodiment one CYP2C9*3 (R primer) sequence
<212>Type：DNA
<213>OrganismName：Homo sapiens
<400>PreSequenceString：tccttttcca tcagttt
17
<211>Length：18
SequenceName：127
The preceding primer of SequenceDescription: embodiment one AGTR1 (A1166C) (F primer) sequence
<212>Type：DNA
<213>OrganismName：Homo sapiens
<400>PreSequenceString：ttcctaccgc ccctcaga
18
<211>Length：19
SequenceName：128
SequenceDescription: embodiment one AGTR1 (A1166C) back primer (R primer) sequence
<212>Type：DNA
<213>OrganismName：Homo sapiens
<400>PreSequenceString：aaaagtcggt tcagtccac
19
<211>Length：17
SequenceName：129
Primer (F primer) sequence before SequenceDescription: embodiment one CYP2D6*10
<212>Type：DNA
<213>OrganismName：Homo sapiens
<400>PreSequenceString：caccatccat gtttgct
17
<211>Length：23
SequenceName：130
Primer behind SequenceDescription: embodiment one CYP2D6*10 (R primer) sequence
<212>Type：DNA
<213>OrganismName：Homo sapiens
<400>PreSequenceString：ccatttggta gtgaggcagg tat
23
<211>Length：17
SequenceName：131
The preceding primer of SequenceDescription: embodiment one ADRB1 (C1165G) (F primer) sequence
<212>Type：DNA
<213>OrganismName：Homo sapiens
<400>PreSequenceString：tgggcttcga gttcacc
17
<211>Length：18
SequenceName：132
SequenceDescription: embodiment one ADRB1 (C1165G) back primer (R primer) sequence
<212>Type：DNA
<213>OrganismName：Homo sapiens
<400>PreSequenceString：tcatgggcgt cttcacgc
18
<211>Length：22
SequenceName：133
The preceding primer of SequenceDescription: embodiment one ACE (I/D) (F primer) sequence
<212>Type：DNA
<213>OrganismName：Homo sapiens
<400>PreSequenceString：gtggccatca cattcgtcag at
22
<211>Length：20
SequenceName：134
SequenceDescription: embodiment one ACE (I/D) back primer (R primer) sequence
<212>Type：DNA
<213>OrganismName：Homo sapiens
<400>PreSequenceString：ctggagacca ctcccatcct
20
<211>Length：19
SequenceName：135
SequenceDescription: embodiment three CYP2C9*3 wild-type probe sequences
<212>Type：DNA
<213>OrganismName：Homo sapiens
<400>PreSequenceString：aaggtcaatg tatctctgg
19
<211>Length：19
SequenceName：136
SequenceDescription: embodiment three CYP2C9*3 mutant probe sequences
<212>Type：DNA
<213>OrganismName：Homo sapiens
<400>PreSequenceString：aaggtcaagg tatctctgg
19
<211>Length：19
SequenceName：137
SequenceDescription: embodiment three CYP2C9*3 feminine genders are with reference to probe sequence
<212>Type：DNA
<213>OrganismName：Homo sapiens
<400>PreSequenceString：aaggtcaaag tatctctgg
19
<211>Length：17
SequenceName：138
SequenceDescription: embodiment three AGTR1 (A1166C) wild-type probe sequence
<212>Type：DNA
<213>OrganismName：Homo sapiens
<400>PreSequenceString：aatgagcatt agctact
17
<211>Length：17
SequenceName：139
SequenceDescription: embodiment three AGTR1 (A1166C) mutant probe sequence
<212>Type：DNA
<213>OrganismName：Homo sapiens
<400>PreSequenceString：aatgagcctt agctact
17
<211>Length：17
SequenceName：140
SequenceDescription: embodiment three AGTR1 (A1166C) are negative with reference to probe sequence
<212>Type：DNA
<213>OrganismName：Homo sapiens
<400>PreSequenceString：aatgagcgtt agctact
17
<211>Length：17
SequenceName：141
SequenceDescription: embodiment three CYP2D6*10 wild-type probe sequences
<212>Type：DNA
<213>OrganismName：Homo sapiens
<400>PreSequenceString：tgcacgctac gcaccag
17
<211>Length：17
SequenceName：142
SequenceDescription: embodiment three CYP2D6*10 mutant probe sequences
<212>Type：DNA
<213>OrganismName：Homo sapiens
<400>PreSequenceString：tgcacgctac acaccag
17
<211>Length：17
SequenceName：143
SequenceDescription: embodiment three CYP2D6*10 feminine genders are with reference to probe sequence
<212>Type：DNA
<213>OrganismName：Homo sapiens
<400>PreSequenceString：tgcacgctac tcaccag
17
<211>Length：17
SequenceName：144
SequenceDescription: embodiment three ADRB1 (C1165G) wild-type probe sequence
<212>Type：DNA
<213>OrganismName：Homo sapiens
<400>PreSequenceStr ing：agcagtccct ggaaggc
17
<211>Length：17
SequenceName：145
SequenceDescription: embodiment three ADRB1 (C1165G) mutant probe sequence
<212>Type：DNA
<213>OrganismName：Homo sapiens
<400>PreSequenceString：agcagtcgct ggaaggc
17
<211>Length：17
SequenceName：146
SequenceDescription: embodiment three ADRB1 (C1165G) are negative with reference to probe sequence
<212>Type：DNA
<213>OrganismName：Homo sapiens
<400>PreSequenceString：agcagtcact ggaaggc
17
<211>Length：22
SequenceName：147
SequenceDescription: embodiment three ACE (I/D) wild-type probe sequence
<212>Type：DNA
<213>OrganismName：Homo sapiens
<400>PreSequenceString：ggtgaaaccc cgtctctact aa
22
<211>Length：22
SequenceName：148
SequenceDescription: embodiment three ACE (I/D) wild-type feminine gender is with reference to probe sequence
<212>Type：DNA
<213>OrganismName：Homo sapiens
<400>PreSequenceString：ggtgaaacac cgtatctact aa
22
<211>Length：32
SequenceName：149
SequenceDescription: embodiment three ACE (I/D) mutant probe sequence
<212>Type：DNA
<213>OrganismName：Homo sapiens
<400>PreSequenceString：aaccacataa aactgagtga ataggcagca gg
32
<211>Length：31
SequenceName：150
SequenceDescription: embodiment three ACE (I/D) mutant feminine gender is with reference to probe sequence
<212>Type：DNA
<213>OrganismName：Homo sapiens
<400>PreSequenceString：aaccaaataa aactgagtga ataggcatca g
31
<211>Length：20
SequenceName：151
SequenceDescription: embodiment four CYP2C9*3 wild-type probe sequences
<212>Type：DNA
<213>OrganismName：Homo sapiens
<400>PreSequenceString：cagagataca ttgaccttct
20
<211>Length：20
SequenceName：152
SequenceDescription: embodiment four CYP2C9*3 mutant probe sequences
<212>Type：DNA
<213>OrganismName：Homo sapiens
<400>PreSequenceString：cagagatacc ttgaccttct
20
<211>Length：22
SequenceName：153
SequenceDescription: embodiment four AGTR1 (A1166C) wild-type probe sequence
<212>Type：DNA
<213>OrganismName：Homo sapiens
<400>PreSequenceString：accaaatgag cattagctac tt
22
<211>Length：22
SequenceName：154
SequenceDescription: embodiment four AGTR1 (A1166C) mutant probe sequence
<212>Type：DNA
<213>OrganismName：Homo sapiens
<400>PreSequenceString：accaaatgag ccttagctac tt
22
<211>Length：22
SequenceName：155
SequenceDescription: embodiment four CYP2D6*10 wild-type probe sequences
<212>Type：DNA
<213>OrganismName：Homo sapiens
<400>PreSequenceString：gctgcacgct acccaccagg cc
22
<211>Length：22
SequenceName：156
SequenceDescription: embodiment four CYP2D6*10 mutant probe sequences
<212>Type：DNA
<213>OrganismName：Homo sapiens
<400>PreSequenceString：gctgcacgct actcaccagg cc
22
<211>Length：22
SequenceName：157
SequenceDescription: embodiment four ADRB1 (A145G) wild-type probe sequence
<212>Type：DNA
<213>OrganismName：Homo sapiens
<400>PreSequenceString：cgccagcgaa agccccgagc cg
22
<211>Length：22
SequenceName：158
SequenceDescription: embodiment four ADRB1 (A145G) mutant probe sequence
<212>Type：DNA
<213>OrganismName：Homo sapiens
<400>PreSequenceString：cgccagcgaa ggccccgagc cg
<211>Length：22
SequenceName：159
SequenceDescription: embodiment four ADRB1 (C1165G) wild-type probe sequence
<212>Type：DNA
<213>OrganismName：Homo sapiens
<400>PreSequenceString：aggccttcca gggactgctg cg
22
<211>Length：22
SequenceName：160
SequenceDescription: embodiment four ADRB1 (C1165G) mutant probe sequence
<212>Type：DNA
<213>OrganismName：Homo sapiens
<400>PreSequenceString：aggccttcca gcgactgctg cg
22
<211>Length：21
SequenceName：161
SequenceDescription: embodiment four CYP2C19*2 wild-type probe sequences
<212>Type：DNA
<213>OrganismName：Homo sapiens
<400>PreSequenceString：attatttccc gggaacccat a
21
<211>Length：21
SequenceName：162
SequenceDescription: embodiment four CYP2C19*2 mutant probe sequences
<212>Type：DNA
<213>OrganismName：Homo sapiens
<400>PreSequenceString：attatttccc aggaacccat a
21
<211>Length：18
SequenceName：163
SequenceDescription: embodiment four positives are with reference to probe sequence
<212>Type：DNA
<213>OrganismName：Homo sapiens
<400>PreSequenceString：accttggaat gtccacag
18
<211>Length：20
SequenceName：164
Primer (F primer) sequence before SequenceDescription: embodiment four CYP2C9*3
<212>Type：DNA
<213>OrganismName：Homo sapiens
<400>PreSequenceString：tgcacgaggt ccagaggtac
20
<211>Length：21
SequenceName：165
Primer behind SequenceDescript ion: embodiment four CYP2C9*3 (R primer) sequence
<212>Type：DNA
<213>OrganismName：Homo sapiens
<400>PreSequenceString：aaacatggag ttgcagtgta g
21
<211>Length：23
SequenceName：166
The preceding primer of SequenceDescription: embodiment four AGTR1 (A1166C) (F primer) sequence
<212>Type：DNA
<213>OrganismName：Homo sapiens
<400>PreSequenceString：ttgaggttga gtgacatgtt cga
23
<211>Length：21
SequenceName：167
SequenceDescription: embodiment four AGTR1 (A1166C) back primer (R primer) sequence
<212>Type：DNA
<213>OrganismName：Homo sapiens
<400>PreSequenceString：cggttcagtc cacataatgc a
21
<211>Length：23
SequenceName：168
Primer (F primer) sequence before SequenceDescription: embodiment four CYP2D6*10
<212>Type：DNA
<213>OrganismName：Homo sapiens
<400>PreSequenceString：ccatttggta gtgaggcagg tat
23
<211>Length：23
SequenceName：169
Primer behind SequenceDescription: embodiment four CYP2D6*10 (R primer) sequence
<212>Type：DNA
<213>OrganismName：Homo sapiens
<400>PreSequenceString：caccatccat gtttgcttct ggt
23
<211>Length：20
SequenceName：170
The preceding primer of SequenceDescription: embodiment four ADRB1 (A145G) (F primer) sequence
<212>Type：DNA
<213>OrganismName：Homo sapiens
<400>PreSequenceString：ccgggcttct ggggtgttcc
20
<211>Length：22
SequenceName：171
SequenceDescript ion: embodiment four ADRB1 (A145G) back primer (R primer) sequence
<212>Type：DNA
<213>OrganismName：Homo sapiens
<400>PreSequenceString：ggcgaggtga tggcgaggta gc
22
<211>Length：20
SequenceName：172
The preceding primer of SequenceDescription: embodiment four ADRB1 (C1165G) (F primer) sequence
<212>Type：DNA
<213>OrganismName：Homo sapiens
<400>PreSequenceString：catcatgggc gtcttcacgc
20
<211>Length：20
SequenceName：173
SequenceDescription: embodiment four ADRB1 (C1165G) back primer (R primer) sequence
<212>Type：DNA
<213>OrganismName：Homo sapiens
<400>PreSequenceString：tgggcttcga gttcacctgc
20
<211>Length：21
SequenceName：174
Primer (F primer) sequence before SequenceDescription: embodiment four CYP2C19*2
<212>Type：DNA
<213>OrganismName：Homo sapiens
<400>PreSequenceString：cagagcttgg catattgtat c
21
<211>Length：22
SequenceName：175
Primer behind SequenceDescription: embodiment four CYP2C19*2 (R primer) sequence
<212>Type：DNA
<213>OrganismName：Homo sapiens
<400>PreSequenceString：gtaaacacac aactagtcaa tg
22
Claims (10)
1. one kind is used to detect the gene chip of hyperpiesis individual medicine because of sudden change, comprise solid support, be fixed on the gene probe on the solid support in order and be used for the segmental PCR primer of amplified sample mutator gene, gene probe comprises the oligonucleotide probe that detects the different genes site mutation, positive in probe and negative with reference to probe, it is characterized in that wherein said gene probe and PCR primer all are at ACE (I/D), one or two gene mutation site among the CYP3A5*3 is or/and at CYP2C9*3, CYP2C9*13, AGTR1 (A1166C), CYP2D6*10, ADRB1 (C1165G), TSC (C1784T), ADRB3 (T727C), SCNN1G_rs5729, SCNN1G_rs5723, ENOSA_rs1799983, two or more gene mutation site designs among the GNB3 (C825T).
2. the gene chip that is used to detect hyperpiesis individual medicine because of sudden change according to claim 1 is characterized in that described gene probe and PCR primer are all at following mutational site or Sites Combination design: ACE (I/D); CYP3A5*3; The combination of CYP2C9*3, CYP2C9*13 and AGTR1 (A1166C); The combination of CYP2D6*10 and ADRB1 (C1165G); The combination of TSC (C1784T), ADRB3 (T727C), SCNN1G_rs5729, SCNN1G_rs5723, ENOSA_rs1799983 and GNB3 (C825T).
3. the gene chip that is used to detect hyperpiesis individual medicine because of sudden change according to claim 1 is characterized in that described gene probe and PCR primer all are the unitized design at CYP2C9*3, CYP2C9*13, AGTR1 (A1166C), CYP2D6*10, ADRB1 (C1165G), ACE (I/D), CYP3A5*3, TSC (C1784T), ADRB3 (T727C), SCNN1G_rs5729, SCNN1G_rs5723, these 13 mutational sites of ENOSA_rs1799983, GNB3 (C825T).
4. according to claim 1, the 2 or 3 described gene chips that are used for hyperpiesis individual medicine because of the sudden change detection, the sequence oligonucleotide probe that it is characterized in that the different mutational sites of described detection comprises forward wild-type probe, forward mutation type probe, reverse wild-type probe, inverse transition type probe; And corresponding feminine gender comprises that with reference to probe sequence the forward feminine gender is with reference to probe and oppositely negative with reference to probe; By any continuous 14～25 based compositions in the corresponding sequence of SEQID NO.1--80, each bar probe in same site is being consistent aspect sequence length and the correspondence position on reference sequences.
5. according to claim 1, the 2 or 3 described gene chips that are used for hyperpiesis individual medicines because of the sudden change detection, it is characterized in that the described positive that is fixed on the solid support with reference to probe sequence is: CATTCAACAAGTTACGACT.
6. according to claim 1, the 2 or 3 described gene chips that are used for hyperpiesis individual medicine because of the sudden change detection, it is characterized in that the described segmental PCR primer sequence of amplified sample mutator gene that is used for is selected from any continuous 17～25 based compositions in the SEQ ID NO.82--107 sequence.
7. the described hyperpiesis individual medicine that is used for of one of claim 1 to 6 can be applicable to detect CYP2C9*3, CYP2C9*13 because of the gene chip that sudden change detects, AGTR1 (A1166C), CYP2D6*10, ADRB1 (C1165G), ACE (I/D), CYP3A5*3, TSC (C1784T), ADRB3 (T727C), SCNN1G_rs5729, SCNN1G_rs5723, the transgenation of ENOSA_rs1799983 and GNB3 (C825T).
8. the described detection of claim 2 is used to detect the gene chip of hyperpiesis individual medicine because of sudden change, detects following mutational site or Sites Combination design: ACE (I/D); CYP3A5*3; The combination of CYP2C9*3, CYP2C9*13 and AGTR1 (A1166C); The combination of CYP2D6*10 and ADRB1 (C1165G); The combination of TSC (C1784T), ADRB3 (T727C), SCNN1G_rs5729, SCNN1G_rs5723, ENOSA_rs1799983 and GNB3 (C825T) is respectively applied for the medication of instructing angiotensin converting enzyme inhibitor; Instruct the medication of calcium antagonist; Instruct the medication of angiotensin II receptor antagonists; Instruct the medication of beta-blocker; Instruct the medication of diuretic antihypertensive medicine.
9. the described detection of claim 3 is used to detect the gene chip of hyperpiesis individual medicine because of sudden change, detect following CYP2C9*3, CYP2C9*13, AGTR1 (A1166C), CYP2D6*10, ADRB1 (C1165G), ACE (I/D), CYP3A5*3, TSC (C1784T), ADRB3 (T727C), SCNN1G_rs5729, SCNN1G_rs5723, ENOSA_rs1799983, the unitized design in these 13 mutational sites of GNB3 (C825T) is used in reference to the emissary vein AECI, calcium antagonist, angiotensin II receptor antagonists, beta-blocker, the comprehensive medication of the main antihypertensive drug of this five class of diuretic antihypertensive medicine.
10. a test kit includes the described gene chip of one of claim 1-6.
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