CN101067149B - CYP3A detecting chip and its application - Google Patents
CYP3A detecting chip and its application Download PDFInfo
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- CN101067149B CN101067149B CN2006101195534A CN200610119553A CN101067149B CN 101067149 B CN101067149 B CN 101067149B CN 2006101195534 A CN2006101195534 A CN 2006101195534A CN 200610119553 A CN200610119553 A CN 200610119553A CN 101067149 B CN101067149 B CN 101067149B
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Abstract
The present invention relates to gene detecting chip, and discloses one kind of CYP3A detecting chip, which comprises solid carrier and probe. The probe can hybridize with the nucleotide sequence and/or its complementary sequence of the detected CYP3A gene. The present invention also discloses the method of using the chip in detecting CYP3A gene. The CYP3A detecting chip of the present invention may be used in detecting the genetic variation of subtypes CYP3A4, CYP3A5 and CYP3A7 for predicting clinical medicine treatment and individualized treatment.
Description
Technical field
The present invention relates to the medicine gene detecting chip, relate in particular to a kind of CYP3A (cytochrome P450 3A, Cytochrome P450 3A) detection chip and application thereof.
Background technology
CYP3A is the abundantest CYP subfamily of content in human liver and the small intestine, comprises CYP3A4, CYP3A5, CYP3A7 and CYP3A43.CYP3A43 is last certified CYP3A family member, and it is little to be considered to that liver CYP3A is expressed contribution, and the CYP3A of the overwhelming majority is CYP3A4, CYP3A5 and CYP3A7 in human liver and the small intestine.CYP3A4, CYP3A5 and CYP3A7 have participated in about 50% common drug metabolism [Wojnowski L in vivo, Kamdem LK.Clinical implications of CYP3A polymorphisms.Expert Opin Drug Metab Toxicol, 2006,2:171-182], as medicines such as immunosuppressor, chemotherapy of tumors medicine, lipid lowerers, calcium ion channel blockor, tranquillizers.In addition, also participate in the activation of part procarcinogen.Therefore the active individual difference of CYP3A has great effect for the metabolism and the pharmacokinetics of most drug.Though environment and medicine also can cause the active individual difference of CYP3A, yet be influence [the Rodriguez-Antona C that is subjected to inherited genetic factors to a greater extent, Ingelman-Sundberg M.Cytochrome P450 pharmacogenetics and cancer.Oncogene, 2006,25:1679-1691].
Many heritable variations can change the enzymic activity of CYP expression of gene level or its coding, cause the significance of the pharmacokinetics of these enzyme substratess to change, thereby produce serious toxic side effect or cause failing to respond to any medical treatment.Enzymic activity according to P450, the phenotype of individuality can be divided into slow inactivation (poor metabolizer, PM), intermediary metabolism type (intermediate metabolizer, IM), fast metabolic pattern (extensive metabolizer, EM) or ultrafast metabolic pattern (ultrarapid metabolizer, UM).EM is defined as the homozygote individuality of wild-type, carries the individual IM of being of heterozygote of an active allelotrope and an amorphs, and carrying two allelic individualities of defective is PM, and UM carries the active allelotrope of a plurality of copies usually.Therefore PM makes inaxtivation of drug slower, causes plasma drug level too high, be easier to produce toxic side effect, and UM makes the medicine quick inactivating, causes that plasma drug level crosses low and be of no curative effect.Owing to can't carry out gene type, often be difficult to prediction for the therapeutic response of certain drug to most of people.For example, than the EM individuality, routine dose is taken the Cardiovascular Toxicity of Venlafaxine initiation and toxic side effect [the Bertilsson L that tricyclic antidepressants produces, Dahl ML, Dalen P, Al-Shurbaji A.Molecular genetics of CYP2D6:clinical relevance with focus on psychotropicdrugs.Br J Clin Pharmacol, 2002,53:111-122] more common in the PM individuality.When using proton pump inhibitor and amoxycilline Trihydrate bp treatment helicobacter pylori infection, its curative ratio depends on the metabolic phenotype of patient CYP2C19.It is 28.6% that EM patient uses the curative ratio of omeprazole, IM patient is 60%, PM patient is 100%, and this curative effect difference is being used three medicines (proton pump inhibitor, amoxycilline Trihydrate bp and clarithromycin or metronidazole) less [Wilkinson GR.Drug metabolism and variability among patients in drugresponse.N Engl J Med when treating, 2005,352:2211-2221].The patient that the standard care scheme is invalid may be UM, needs escalated dose to cure usually.Obviously, adjusting drug dose according to the classification of genotype-phenotype helps to improve the curative effect of medicine and reduces ADR (adverse drug reaction), the medicine that particularly those therapeutic indexs are low, safety range is narrow.
CYP3A4 is a most important composition among the adult hepatomicrosome CYP, accounts for the 30-40% of CYP total amount, occupies first, and CYP3A4 will be higher than CYP3A5 and CYP3A7 to the specificity of many medicines.So far nearly 40 allelotrope are identified.The frequency of CYP3A4*1B in the Caucasian is 3.6-9.6%, and the frequency among the Black people is 53-67%, does not exist among the Oriental.The susceptibility of CYP3A4*1B and some tumours and progress etc. are relevant, therefore but functional study finds no the dependency between CYP3A4*1B and its substrate pharmacokinetics, infers to think relevant polymorphic close linkages such as the susceptibility of CYP3A4*1B and tumour and progress.CYP3A4 genetic flaw allelotrope has CYP3A4*8, CYP3A4*11, CYP3A4*13, CYP3A4*16 and CYP3A4*17, amorphs has CYP3A4*20, and the enzymic activity of CYP3A4*18A coding is than the CYP3A4.1 height, but these allelic frequencies are all lower.Because most of CYP3A4 allelotrope do not have functional effect, or the very rare significance individual difference that is not enough to cause CYP3A4 genetic expression, the polymorphism of CYP3A4 gene can't be explained the highly variable of its expression.Pregnane X acceptor (pregnane Xreceptor, PXR) generegulation CYP3A4 gene transcription, its polymorphism can cause the change of CYP3A4 expression level, may be one of the CYP3A4 reason of expressing individual difference [Bosch TM, MeijermanI, Beijnen JH, Schellens JH.Genetic polymorphisms of drug-metabolising enzymes and drugtransporters in the chemotherapeutic treatment of cancer.Clin Pharmacokinet, 2006,45:253-285].Another reason may be to have very strong linkage disequilibrium between the heritable variation of CYP3A4 and CYP3A5 gene.
The CYP3A5 expression of gene is the height polymorphism, about 10-20% Caucasian, 33% Japanese and 55% U.S. is African to have the expression level that is easier to detect, CYP3A5 in these people's livers accounts for the 6-99%[Lamba JK of CYP3A total amount, Lin YS, Schuetz EG, Thummel KE.Genetic contribution to variablehuman CYP3A-mediated metabolism.Adv Drug Deliv Rev, 2002,54:1271-1294].It mainly is owing to due to the SNP 6986A＞G (CYP3A5*3) in the 3rd intron, though this SNP can cause unusual shearing, still have a spot of functional protein to be translated, so CYP3A5*3 is the low allelotrope of expressing that the CYP3A5 polymorphism is expressed.The incidence of CYP3A5*3 in Caucasian, Aisa people and African is respectively 88%, 75% and 35%.Other defective allelotrope still has CYP3A5*6, CYP3A5*8, CYP3A5*9, CYP3A5*10 and CYP3A5*11 etc., and wherein CYP3A5*10 and CYP3A5*11 are all chain with SNP 6986A＞G, are in the same haplotype (haplotype).Because CYP3A5*6 and CYP3A5*7 be the frequency higher (10-20%) in African in African and the U.S., is necessary to determine by haplotype analysis whether these 2 SNP are present in the same haplotype with CYP3A5*3.
Because lack specific antibody, CYP3A7 only is mistaken as and exists in the fetus liver always.Similar to CYP3A5, the expression of CYP3A7 also is rendered as polymorphism, and about 10-20% Caucasian is CYP3A7 high expression level person.In CYP3A7 high expression level person, the amount of CYP3A7 accounts for the 9-36% of CYP3A total amount, may be suitable with CYP3A5 high expression level person's CYP3A5 enzyme amount, even surpass.Main CYP3A7 allelotrope has CYP3A7*1C, CYP3A7*1B, CYP3A7*2 and CYP3A7*3.CYP3A7*1C and CYP3A7*1B can improve CYP3A7 gene transcription activity, and both are approximately relevant with 67% Caucasian CYP3A7 high expression level person.CYP3A7*2 does not influence genetic expression, but the catalytic activity of the enzyme of its coding is apparently higher than CYP3A7.1, CYP3A7*2 is white man, Saudi Arabian, the frequency of Chinese and Tanzania's philtrum is respectively 8%, 17%, 28% and 62%[Rodriguez-Antona C, Jande M, Rane A, Ingelman-Sundberg M.Identification and phenotype characterization of two CYP3A haplotypes causingdifferent enzymatic capacity in fetal livers.Clin Pharmacol Ther, 2005,77:259-270].Other allelotrope such as CYP3A7*3 it be unclear that the influence of genetic expression and enzyme activity.
The polymorphism of CYP gene is to cause the one of the main reasons of individuality to the difference of pharmacological agent reaction, therefore if the result of study of pharmacogenetics can be applied to disease treatment, the heritable variation of the CYP gene by detecting the patient, make the clinician obtain patient's genetic information, effective to judge which pharmacological agent, use medicine and dosage targetedly, improve the curative effect of medicine and the toxic side effect of reduction medicine effectively, thereby be expected to significantly reduce medical expense.And for pharmaceutical manufacturer, information according to pharmacogenetics and pharmacogenomics, and the approach of drug target and drug metabolism, select the crowd of suitable clinical trial, to improve the curative effect of medicine to disease treatment, shorten the cycle of the research and development of medicine, improve the clinical approval rate of medicine; According to native's genetic construction, carry out medicaments derivative or similar drug development.
Detect the heritable variation of single CYP gene, because the genotype that institute can somatotype is limited, the practicality that clinical detection is diagnosed is lower.Have only the heritable variation of a plurality of important gene in the set CYP supergene family, somatotype all functions genes involved type can be made accurate prediction to the metabolic phenotype of individuality as far as possible, reaches the purpose of clinical detection diagnosis.And because the height polymorphism of CYP gene, related heritable variation does not wait to hundreds of tens, and obviously traditional detection method is difficult to be applied in the personalized medicine because flux level is low.
Summary of the invention
The technical problem to be solved in the present invention provides a kind of CYP3A detection chip, can effectively detect the heritable variation of somatotype CYP3A4, CYP3A5 and CYP3A7, and the individual metabolic phenotype that obtains thus can assist the doctor in time to find correct therapeutic regimen.For this reason, the present invention also will provide the method for using this chip detection CYP3A gene.
In order to solve the problems of the technologies described above, the present invention is achieved through the following technical solutions:
In one aspect of the invention, provide a kind of CYP3A detection chip, comprised solid phase carrier and probe, nucleotide sequence and/or its complementary sequence of described probe and CYP3A gene to be measured are hybridized.
Solid phase carrier described in the present invention can be selected the known carrier in field for use, as long as described carrier is compatible with described reactant, it is just passable can not influence detected result.Preferably, solid phase carrier selection of the present invention is a kind of in slide, silicon chip, nitrocellulose filter, nylon membrane and the macromolecular material or their arbitrary combination.
Described CYP3A gene to be measured comprises CYP3A4, CYP3A5 and CYP3A7 gene.
Described probe can be DNA, RNA, DNA-RNA mosaic, PNA or derivatives thereof.The length of described probe is hard-core, as long as can finish the function of specific hybrid, combines with purpose nucleotide sequence specificity, and any length can.The length of described probe can be as short as 25,20,15,13 or 10 base length.Equally, the length of described probe can be grown to 60,80,100,150,300 base pairs or longer, even whole gene.Because different probe length has different influences to hybridization efficiency, signal specificity, the length of described probe is 14 base pairs usually at least, the longlyest generally be no more than 30 base pairs, and purpose nucleotide sequence complementary length is between 15-25 the base pair being the best.Self complementary sequence of described probe is most preferably less than 6 base pairs, in order to avoid influence hybridization efficiency.If may also have base mismatch to a certain degree in the described probe, then described probe can add the disadvantageous effect that length is offset base mismatch, to improve hybridization efficiency.
The probe of detection chip of the present invention is DNA, comprise: sequence shown in (a) SEQ IDNO:1～SEQ ID NO:30 of (1) and CYP3A4 gene recombination to be measured, (b) complementary strand of every sequence in the sequence shown in SEQ ID NO:1～SEQ ID NO:30, (c) with the sequence shown in SEQID NO:1～SEQ ID NO:30 in every sequence the sequence of at least 70% homology is arranged;
(2) with sequence shown in (a) SEQ ID NO:31～SEQ ID NO:63 of CYP3A5 gene recombination to be measured, (b) complementary strand of every sequence in the sequence shown in SEQID NO:31～SEQ ID NO:63, (c) with the sequence shown in SEQ ID NO:31～SEQID NO:63 in every sequence the sequence of at least 70% homology is arranged;
(3) with sequence shown in (a) SEQ ID NO:64～SEQ ID NO:109 of CYP3A7 gene recombination to be measured, (b) complementary strand of every sequence in the sequence shown in SEQ ID NO:64～SEQ ID NO:109, (c) with the sequence shown in SEQ ID NO:64～SEQ ID NO:109 in every sequence the sequence of at least 70% homology is arranged.
Preferably, the probe of detection chip of the present invention is selected from sequence shown in SEQ ID NO:1～SEQ ID NO:109.
Described probe sequence can comprise 1～10 base mismatch, preferably, can comprise 1～5 base mismatch, more preferably, can comprise 1～2 base mismatch.
Detection chip of the present invention also comprises at least a contrast probe, and described contrast probe is selected from: negative control probe, positive control probe, hybridization contrast probe and immobilization contrast probe.
Described probe can be fixed on the carrier with several different methods, as point sample method (USA Patent No.5288514, USAPatent No.5556752), light mediated method (USA Patent No.5143854,5384261 and 5561071), paramagnetic particle method (USA Patent No.5541061) etc.Described probe can be fixed on the carrier by the method for chemistry or physics, as being adsorbed on the carrier by ionic linkage, covalent union or other known power, as available UV-crosslinked instrument with as described in probe stationary on carrier.
Described probe can be fixed on the solid phase carrier by connecting arm.Connecting arm can provide one the space is sterically hindered to reduce freely for probe forms double-stranded part, carrying out [the Afanassiev V that helps hybridization, HanemannV, Wolfl S.Preparation of DNA and protein micro arrays on glass slidescoated with an agarose film.Nucleic Acids Res.2000,28:e66; USA Patent No.5556752].Connecting arm is long more, and hybridization efficiency is high more.Typical connecting arm comprises 15～30 functional group length.Connecting arm can be selected the functional group of appropriate form for use, as the mosaic of Poly T (A, C or G), C atom or polyethylene glycol and Poly T (A, C or G), poly-ethanol, poly-cruel, poly-ammonia, cruel and its composition of poly-sulfuric acid.
Described probe or connecting arm are fixed on the solid phase carrier by link molecule.Probe stationary can be passed through the realization of C-C key to carrier, for example, and the voltalef surface; Or better use siloxane bond (glass or silicon-dioxide use when making upholder).The siloxane bond bonding can be by upholder and link molecule Trichloromonosilane base or radical reaction such as trialkoxysilyl finish.Aminoalkyl group silane, light basic alkyl silane, 2 one light ethyl one aminopropyl triethoxysilanes, light ethyl one aminopropyl triethoxysilane or light propyl-triethoxysilicane all are surface adsorption groups of great use.
Described probe can be modified, and modifying method can be 5 '-NH
2Modification, 5 '-SH modify, 5 '-Poly T (A, C or G) modifies, 5 '-biotin modification, 3 '-NH
2Modification, 3 '-SH modification, 3 '-Poly T (A, C or G) modification and 3 '-biotin modification etc.
Described probe can have one or several, even is all through mark, and that described mark comprises is fluorescein-labelled, biotin labeling, radioelement mark, enzyme labelling and FRET (fluorescence resonance energy transfer) mark.
In another aspect of this invention, provide a kind of method that said chip detects the CYP3A gene of using, comprised the steps:
(1) carries the probe of hybridizing at surface of solid phase carriers point with nucleotide sequence and/or its complementary sequence of CYP3A gene to be measured;
(2) nucleic acid of extracting CYP3A gene to be measured;
(3) the purpose nucleotide sequence of preparation CYP3A gene to be measured;
(4) the purpose nucleotide sequence of markers step (3);
(5) be loaded under the condition that the probe on the solid phase carrier hybridizes with the described point of step (1) being suitable for, add purpose nucleotide sequence, and make it react the enough time through mark;
(6) result of detection hybridization.
In another aspect of this invention, also provide a kind of method that said chip detects the CYP3A gene of using, comprised the steps:
(1) nucleotide sequence of mark and CYP3A gene to be measured and/or its complementary sequence probe of hybridizing;
(2) nucleic acid of extracting CYP3A gene to be measured;
(3) the purpose nucleotide sequence of preparation CYP3A gene to be measured;
(4) carry the described purpose nucleotide sequence of step (3) at surface of solid phase carriers point;
(5) be loaded under the condition that the purpose nucleotide sequence on the solid phase carrier hybridizes with the described point of step (4) being suitable for, add probe, and make it react the enough time through mark;
(6) result of detection hybridization.
Appropriate samples comprises described in the method for the present invention: come from any tissue that contains nucleic acid of human and animal's blood, saliva, hair and other and from the sample of plant and environment product (as earth or water).
The nucleic acid of sample can be extracted from sample with any suitable method.For example, sample nucleic acid can extract from sample with magnetic bead, and a lot of biotech firms can provide nucleic acid extractive test kit, as Qiagen, and Invitron etc.
Because development of technology can directly increase with the cell that contains nucleic acid in the target sample without the nucleic acid extracting now.The cell that contains nucleic acid of target sample can be extracted from sample with any suitable method.For example, the cell that contains nucleic acid in the target sample can be separated from sample with magnetic bead.
The preparation of described purpose nucleotide sequence can comprise the step of amplification, directly increases with the cell that contains nucleic acid in the isolating target sample, and also available extractive target nucleic acid directly increases.As from whole blood, separating white corpuscle, directly make template, amplification purpose nucleotide sequence with isolating white corpuscle or with the extractive nucleic acid of whole blood with magnetic bead.Strand that amplification obtains or double-stranded DNA or RNA can contain fluorescence or biotin labeling, and the DNA of mark or RNA can not purifiedly be directly used in hybridization.With the preferred target nucleotide molecule of chip hybridization described in the present invention be single stranded nucleic acid molecule, after double chain acid molecule is handled through sex change etc. also in chip hybridization of the present invention.
The purpose nucleotide sequence can use any suitable amplification method to carry out enrichment, as: polymerase chain reaction (polymerase chain reaction, PCR), multiplex PCR, ligase chain reaction (ligase chainreaction, LCR), rolling circle amplification (rolling cycle amplification, RCA), based on the amplification of nucleotide sequence (nucleic acid sequence-based amplification, NASBA), strand displacement amplification (stranddisplacement amplification, SDA) and the amplification of transcriptive intermediate (transcription medicatedamplification, TMA) etc.
In one embodiment of the invention, the pcr amplification method is adopted in the preparation of purpose nucleotide sequence, and this method the primer contains nucleotide chain or its complementary strand of sequence shown in SEQ ID NO:110～SEQ ID NO:143.
As long as enough to produce specific hybrid, described purpose nucleotide sequence length is also unrestricted on the upstream and downstream direction for the length of the purpose nucleotide sequence that is used to hybridize.Suitable purpose nucleotide sequence length from 30 to 200 base pair length.The target nucleotide sequences of hybridization usefulness is long or too short, can influence hybridization efficiency, and makes the target nucleotide sequences of hybridization on probe be easy to be eluted, and causes fluorescent signal to cross weak or lose.Long dna fragmentation, available DNase I carry out fragmentation to be handled, and perhaps adds the Brdurd of suitable proportion in reaction system, and the PCR product is handled with ura DNA glycosidase then, the fragmentation dna fragmentation.As for RNA, treatment process is comparatively simple, directly gets final product with high salt and pyroprocessing.
But the hybridization homology between described probe and the purpose nucleotide sequence also can be non-homogeneous.
Described probe or purpose nucleotide sequence all are suitable for mark.Probe is at the synthetic mark of introducing, and the purpose nucleotide sequence can be introduced mark in amplification, and perhaps mark is introduced with suitable method in the amplification back.
Suitable mark comprises fluorescent mark, labelled with radioisotope, chromophoric group, twinkler, FRET, enzyme, vitamin H or the aglucon of special combination part is arranged.
The hybridization of the inventive method can be carried out in any hybridization solution, as contains the hybridization solution of SSPE and tensio-active agent.Hybridization solution can contain the SSPE of any concentration, for example 1～10 * SSPE.Also can use any suitable tensio-active agent, as Triton-100, SDS, SLS (sarcosyl), CTAB (six alkyl trimethyl ammonium bromides) etc.The tensio-active agent that any concentration also can be arranged in the hybridization solution is as 0.01～1% (w/w).Described hybridization can be carried out under any suitable temperature, and as 25 ℃～65 ℃, described hybridization time is 2 minutes～18 hours.Can change rigorous degree, the hybridization specificity of hybridization conditions to improve or to reduce hybridization.
The washing of described results of hybridization before detection can be used any suitable washings, and this washings can contain the tensio-active agent of 0～3% (w/w), washs sustainable reasonable time, as 1～30 minute.Can wash with the washings of room temperature, or the preheating after scouring, as 42 ℃.Available different washings successively washs.
Hybridization between described probe and the purpose nucleotide sequence can adopt any known method to detect.According to the difference of mark, can select suitable detection method for use, for example fluorescently-labeled probe or purpose nucleotide sequence can detect with laser scanner or luminoscope, and the probe of radioelement mark or purpose nucleotide sequence can detect with radioautograph.
Overall hybridization specificity can be determined with any suitable standard, for example can determine by the method that Chinese patent CN1566366A describes.
The positive signal of chip can be determined with any suitable standard, for example can determine by the method that Chinese patent CN1566366A describes, or determine greater than the method for average negative control probe and 3 times of standard deviation sums with hybridization signal.
The copy number that the present invention also can be used for goal gene detects.The quantity that is incorporated into the purpose nucleotide sequence on the chip probe can be detected, and relevant with the copy number of goal gene.Quantity according to the nucleotide sequence of the crt gene that contains the known copy number can be carried out quantitatively the copy number of goal gene.Fluorescence can be surveyed with photomultiplier CCD or laser scanning.
CYP3A detection chip of the present invention and application thereof are compared with traditional detection method, and CYP3A GeneScreen cubing is had high-throughput, advantages such as collimation and traceization, make the medical worker in time grasp a large amount of genetic information of patient, instruct patient's rational use of drug, realize personalized medicine.For pharmaceutical manufacturer, CYP3A detection chip of the present invention can make it select suitable clinical trial crowd, thereby improves curative effect of medication, reduces the inefficiency and the toxic side effect of medicine, shortens the R﹠D cycle of medicine.
Description of drawings
Accompanying drawing is the results of hybridization figure of the CYP3A detection chip of the embodiment of the invention 1.
Embodiment
The present invention is further detailed explanation below in conjunction with drawings and Examples.
Embodiment 1 is reverse hybridized
1. the preparation of gene chip
(1) probe dissolving
Every probe TE solution dilution, final concentration is 10mM.With concentration be the probe of 10mM and PBS solution that concentration is 200mM in the medium volume mixture of 384 orifice plates, seal 384 orifice plates with adhesive sheet, vibration is 2 minutes under the room temperature, and is centrifugal ,-20 ℃ of preservations are used in order to point sample.
(2) point sample
The probe that designs and synthesizes in advance is downloaded on the solid phase carrier sheet base of materials such as slide, silicon chip by contact point sample or ink jet type point of sample.The sheet base adopts Cell Associates CSS-100 aldehyde radical sheet base, the point sample instrument of Ominigrid 100 models of GeneMachine company, humidity: 65-75% (being as the criterion) with FullMoon sheet base, temperature is a point sample under 25 ℃ the condition, the form of arranging of probe point sample is as shown in table 1, after point sample finishes, place half an hour after, chip is taken out, and drying at room temperature is preserved.
Table 1 probe is arranged
| 3A4-15603C | 3A4-15603G | 3A4-15603T | 3A4-13908G | 3A4-13908A | 3A4-13908T | 3A4-15615T | 3A4-15615C | 3A4-15615A |
| 3A4-20070T | 3A4-20070C | 3A4-20070A | 3A4-20230G | 3A4-20230A | 3A4-20230T | 3A4-21867C | 3A4-21867T | 3A4-21867A |
| 3A4-21896C | 3A4-21896T | 3A4-21896A | 3A4-22026C | 3A4-22026T | 3A4-22026A | 3A4-25890N | 3A4-25890A | 3A4-25890T |
| 3A4/-392A | 3A4/-392G | 3A4/-392T | 3A5-3775A | 3A5-3775G | 3A5-3775T | 3A5-27289C | 3A5-27289A | 3A5-27289T |
| 3A5-3699C | 3A5-3699T | 3A5-3699A | 3A5-6986G | 3A5-6986A | 3A5-6986T | 3A5-12952T | 3A5-12952C | 3A5-12952A |
| 3A5-14665A | 3A5-14665G | 3A5-14665T | 3A5-14690G | 3A5-14690A | 3A5-14690T | 3A5-19386G | 3A5-19386A | 3A5-19386T |
| 3A5-27132N | 3A5-27132T | 3A5-27132A | 3A5-31611T | 3A5-31611C | 3A5-31611A | 3A5-29753T | 3A5-29753c | 3A5-29753g |
| 3A7/-314C | 3A7/-314T | 3A7/-314G | 3A7-26041C | 3A7-26041G | 3A7-26041A | 3A7/-232A | 3A7/-232C | 3A7/-232G |
| 3A7/-91G | 3A7/-91A | 3A7/-91C | 3A7/-4％ | 3A7/-49A | 3A7/-49T | 3A7/-262T | 3A7/-262A | 3A7/-262G |
| 3A7-4011N | 3A7-4011T | 3A7-4011G | 3A7/-291G | 3A7/-291G*1C | 3A7/-291T | 3A7/-291T*1C | 3A7/-291A | 3A7/-284T |
| 3A7/-284T*1C | 3A7/-284A | 3A7/-284A*1C | 3A7/-284C | 3A7/-282T | 3A7/-282T*1C | 3A7/-282C | 3A7/-282C*1C | 3A7/-282A |
| 3A7/-281A | 3A7/-281A*1C | 3A7/-281T | 3A7/-281T*1C | 3A7/-281G | 3A7/-270T | 3A7/-270T*1C | 3A7/-270G | 3A7/-270G*1C |
| 3A7/-270T | Hybridize negative probe | Hybridize negative probe | Hybridize positive probe | Hybridize positive probe | CY3 | CY3 | Position probe | Position probe |
2. the processing of testing sample and mark
(1) amplification of human gene group DNA's extracting and goal gene
Employing FlexiGene DNA Kit (250) (concrete steps are as follows for QIAGEN, Cat.No.51206) genomic dna in the test kit extracting human peripheral:
1) the periphery whole blood mixing of ACD (Citric Acid 8g/L, Sodium Citrate 22g/L, glucose 24.5g/L) anti-freezing is got 1ml and go into the 15ml centrifuge tube, add 2.5ml damping fluid FG1, fully put upside down mixing;
2) 2000g is centrifugal 5 minutes, supernatant discarded.Left standstill 2 minutes;
3) add 500ul damping fluid FG2/QIAGEN proteolytic enzyme mix reagent, mixing immediately is until original sediment completely dissolve;
4) 65 ℃ of water-baths are 10 minutes, 40 times/minute at the uniform velocity vibrations;
5) add the abundant mixing of 500ul 100% Virahol, until visible DNA flocks occurring;
6) 3000g is centrifugal 5 minutes, and supernatant discarded was inverted 2 minutes;
7) add 500ul 70% ethanol, vibration;
8) 3000g is centrifugal 5 minutes, and supernatant discarded was inverted about 1 hour, no longer included the globule in pipe;
9) add 200ul damping fluid FG3 dissolving DNA, vibration gently, dissolving is spent the night;
10) extracting being dissolved good DNA moves in the 1.5ml centrifuge tube that autoclaving crosses, get 1ul and carry out electrophoresis (1% sepharose, 0.5 * TBE, EB, 80MV, 1.5 hour electrophoresis), at FR-200 ultraviolet and visible analytical equipment photographs photo, and contrast marker (Lambda DNA/EcoRI+HindIII) carries out quantitatively.
Carry out the amplification of purpose nucleotide sequence with the primer (SEQ ID NO:110～SEQ ID NO:121) of CYP3A4, the primer (SEQ IDNO:122～SEQ ID NO:137) of CYP3A5 and the primer (SEQ ID NO:138～SEQ ID NO:143) of CYP3A7.Pcr amplification carries out with 30 μ l reaction systems, reaction system is concentration 0.16 μ M, the genomic dna 10ng of 0.3mM dNTP, 10mM Tris-HCl, 50mM KCl, 2mM MgCl2,20%Q solution (Qiagen), upstream and downstream primer, Taq enzyme 0.6U (Takara).Use Touch-down PCR response procedures [Don RH, Cox PT, Wainwright BJ, Baker K, Mattick JS. ' Touchdown ' PCRto circumvent spurious priming during gene amplification.Nucleic Acids Res.1991,19:4008]: 94 ℃ of sex change 5min; 94 ℃ of sex change 40s, 64 ℃ of annealing 1min, each circulation reduces by 0.5 ℃, and 72 ℃ are extended 50s, totally 10 circulations; 94 ℃ of sex change 40s then, 59 ℃ of annealing 40s, 72 ℃ are extended 50s, totally 30 circulations; Last 72 ℃ are extended 5min.PCR finishes the back and detects amplification with 1.5% sepharose.
When carrying out multi-PRC reaction, the primer of sequence shown in SEQ ID NO:110～SEQ ID NO:143 to be put into a reaction system increase, system is 50 μ l.The reaction system of multiplex PCR is: every kind of dNTP 0.3 μ mol/L, Tricine-KOH (PH8.7) 40mmol/L, KCl 16mmol/L, MgCl
23.5mmol/L, BSA 3.75 μ g/ml, every primer 2 μ mol/L, DNA 80ng and 2.2 * Titanium Taq archaeal dna polymerase (ClontechLaboratories Inc., USA).Multi-PRC reaction condition: 95 ℃ of sex change 3min; 95 ℃ of sex change 30s, 66 ℃ of annealing 2.5min, 68 ℃ are extended 4min, totally 40 circulations; Last 68 ℃ prolong 10min.Behind the pcr amplification, get 3 μ l PCR reaction product and do agarose gel electrophoresis.These PCR products can be used for following hybridization step after treatment.
(2) PCR product purification and fragmentation
All PCR products of each sample mix, usefulness QIAquick PCR Purification Kit (concrete steps are as follows for Qiagen, Cat.No.28106) purifying:
1) the damping fluid PB of 5 times of PCR product volumes of adding, mixing need not to remove paraffin oil or kerosene;
2) the centrifugal post of QIAquick being positioned over the 2ml that test kit provides collects in the pipe;
3) sample is added in the centrifugal post of QIAquick, centrifugal 30-60s is with in conjunction with DNA;
4) outwell 2ml and collect centrifugal liquid in the pipe, the centrifugal post of QIAquick is put in the former collection pipe;
5) 0.75ml damping fluid PE is joined in the centrifugal post of QIAquick wash centrifugal 30-60s;
6) outwell 2ml and collect centrifugal liquid in the pipe, the centrifugal post of QIAquick is put in the former collection pipe centrifugal 1min;
7) the centrifugal post of QIAquick is positioned in the centrifugal pipe of autoclaved 1.5ml;
8) add 50 μ l damping fluid EB (10mM Tris-Cl, pH 8.5) or H in QIAquick film central authorities
2O is with eluted dna, and centrifugal 1min in order to improve the concentration of DNA, can only add 30 μ l elution buffers in QIAquick film central authorities, leaves standstill 1min, and is centrifugal then.
The PCR of purifying after the concentration, carries out fragmentation with DNase I after measured.The reaction system of fragmentation comprises: 30 μ l purified pcr products (10 μ g), 10 * DNase I damping fluid of 4 μ l, the DNase I of 0.12 μ l, the ddH of 5.88 μ l
2O.Reaction conditions is that 37 ℃ of temperature are bathed 5min, 95 ℃ of 15min then.Product behind the fragmentation runs 4% sepharose, guarantees that most fragments is in 30-200 base pair.
(3) fluorescein-labelled
Utilize deoxynucleotidyl transferase to carry out fluorescein-labelled at 3 ' end, 40 μ l reaction systems of mark comprise: 25 μ l fragmentation PCR products, 5 * deoxynucleotidyl transferase damping fluid of 8 μ l, the CY3-N6-ddCTP of 1 μ l (1mM), the deoxynucleotidyl transferase of 3 μ l (20U/ μ l), the ddH of 3 μ l
2O.Reaction conditions is that 37 ℃ of temperature are bathed 120min, then 95 ℃ of heating 15min.
3. hybridization, washing and result detect
95 ℃ of sex change 10min of fluorescently-labeled PCR product place on ice immediately, are used for hybridization, hybridization 20 μ l systems comprise: fluorescein-labeled PCR product 15 μ l, 20 * SSPE, 1.2 μ l, 1%Triton 0.2 μ l, 10 * Denhandts, 0.9 μ l, methane amide 0.5 μ l, ddH
2O 2.2 μ l.Reaction conditions is that 48 ℃ of temperature are bathed 120min, use 1 * lavation buffer solution I (5 * SSC then in succession, 0.1%SDS), 1 * lavation buffer solution II (2 * SSC, 0.1%SDS) and 1 * lavation buffer solution III (1 * SSC) respectively washs 10min at 42 ℃, washs 0.5min with ddH2O at last.
Chip after the washing after drying, scans (also can with other laser scanner) with GenePix 4000B confocal laser scanner.The results of hybridization that chip after the scanning hybridization obtains as shown in drawings, handle image with GenePix Pro again and obtain data file, then the data file is analyzed the genotype that just can obtain goal gene, the somatotype result is CYP3A4, CYP3A5 and CYP3A7.
The hybridization of embodiment 2 forwards
1.CYP3A4, CYP3A5 and CYP3A7 gene amplification and chip preparation
With CYP3A4, CYP3A5 and CYP3A7 primer (SEQ ID NO:110～SEQ ID NO:143) amplification gene.Pcr amplification carries out with 100 μ l reaction systems, and reaction system is 0.3mM dNTP, 10mM Tris-HCl, 50mMKCl, 2mM MgCl
2, 20%Q solution (Qiagen), 0.01 μ MSHV-F, 0.2 μ M SHV-R, 100ng genomic dna, 3U Ex Taq enzyme (Takara).Loop parameter: 94 ℃ of sex change 5min; 94 ℃ of sex change 40s, 65 ℃ of annealing 1min, 72 ℃ are extended 1min, totally 40 circulations; Last 72 ℃ are extended 5min.PCR product QIAquick PCR Purification Kit (Qiagen, Cat.No.28106) purifying.The PCR production concentration of purifying is adjusted to 400ng/ μ l.
With concentration be the PCR product of 400ng/ μ l and 100% DMSO in the medium volume mixture of 384 orifice plates, seal 384 orifice plates with adhesive sheet, vibration is 2 minutes under the room temperature, and is centrifugal.The PCR product of 200ng/ μ l is downloaded on the solid phase carrier sheet base of materials such as slide, silicon chip by contact point sample or ink jet type point of sample.Adopt the point sample instrument of Ominigrid 100 models of GeneMachine company, humidity: 65-75% (being as the criterion) with FullMoon sheet base, temperature is a point sample under 25 ℃ the condition, point sample finish place half an hour after, chip is put in the saturated aqueous common salt box, and 37 ℃ of aquations are spent the night, and take out next day, 600mJ is crosslinked, and the crosslinked chip that finishes can use.
2. chip hybridization
The hybridization system comprises: the fluorescently-labeled oligonucleotide probe of 2nM, 1.2 μ l, 20 * SSPE, 0.2 μ l1%Triton, 0.9 μ l, 10 * Denhandts, 0.5 μ l methane amide, 2.2 μ l ddH
2O.Reaction conditions is that 50 ℃ of temperature were bathed 2 hours, use in succession then 1 * lavation buffer solution I (5 * SSC, 0.1%SDS), 1 * lavation buffer solution II (2 * SSC, 0.1%SDS) and 1 * lavation buffer solution III (1 * SSC) respectively washs 10min at 42 ℃, uses ddH at last
2O washing 10 seconds.Chip after the washing after drying, scans (also can with other laser scanner) with GenePix 4000B confocal laser scanner.
Sequence table
＜110〉Shanghai Biochip Co., Ltd
＜120〉CYP3A detection chip and application thereof
<130>NP-11108
<160>143
<170>PatentIn?version?3.3
<210>1
<211>21
<212>DNA
＜213〉artificial sequence
<400>1
catgtgatca?ctagcacatc?a 21
<210>2
<211>21
<212>DNA
＜213〉artificial sequence
<400>2
catgtgatca?gtagcacatc?a 21
<210>3
<211>21
<212>DNA
＜213〉artificial sequence
<400>3
catgtgatca?ttagcacatc?a 21
<210>4
<211>22
<212>DNA
＜213〉artificial sequence
<400>4
aagagattac?gatcattgct?gt 22
<210>5
<211>23
<212>DNA
＜213〉artificial sequence
<400>5
gaagagatta?caatcattgc?tgt 23
<210>6
<211>23
<212>DNA
＜213〉artificial sequence
<400>6
gaagagatta?ctatcattgc?tgt 23
<210>7
<211>21
<212>DNA
＜213〉artificial sequence
<400>7
agcacatcat?ttggagtgaa?c 21
<210>8
<211>21
<212>DNA
＜213〉artificial sequence
<400>8
tagcacatca?tctggagtga?a 21
<210>9
<211>21
<212>DNA
＜213〉artificial sequence
<400>9
agcacatcat?atggagtgaa?c 21
<210>10
<211>17
<212>DNA
＜213〉artificial sequence
<400>10
gtccgatctg?gagctcg 17
<210>11
<211>16
<212>DNA
＜213〉artificial sequence
<400>11
gtccgatccg?gagctc 16
<210>12
<211>17
<212>DNA
＜213〉artificial sequence
<400>12
gtccgatcag?gagctcg 17
<210>13
<211>19
<212>DNA
＜213〉artificial sequence
<400>13
gagtggatgg?tacatggag 19
<210>14
<211>21
<212>DNA
＜213〉artificial sequence
<400>14
tgagtggatg?atacatggag?a 21
<210>15
<211>21
<212>DNA
＜213〉artificial sequence
<400>15
tgagtggatg?ttacatggag?a 21
<210>16
<211>22
<212>DNA
＜213〉artificial sequence
<400>16
gtgaatgaaa?cgctcagatt?at 22
<210>17
<211>22
<212>DNA
＜213〉artificial sequence
<400>17
ggtgaatgaa?atgctcagat?ta 22
<210>18
<211>22
<212>DNA
＜213〉artificial sequence
<400>18
ggtgaatgaa?aagctcagat?ta 22
<210>19
<211>19
<212>DNA
＜213〉artificial sequence
<400>19
gctatgagac?ttgagaggg 19
<210>20
<211>21
<212>DNA
＜213〉artificial sequence
<400>20
tgctatgaga?tttgagaggg?t 21
<210>21
<211>21
<212>DNA
＜213〉artificial sequence
<400>21
tgctatgaga?attgagaggg?t 21
<210>22
<211>20
<212>DNA
＜213〉artificial sequence
<400>22
aagttcctcc?ctgaaaggta 20
<210>23
<211>21
<212>DNA
＜213〉artificial sequence
<400>23
aagttcctcc?ttgaaaggta?c 21
<210>24
<211>21
<212>DNA
＜213〉artificial sequence
<400>24
aagttcctcc?atgaaaggta?c 21
<210>25
<211>21
<212>DNA
＜213〉artificial sequence
<400>25
aaccagaaaa?acccgttgtt?c 21
<210>26
<211>21
<212>DNA
＜213〉artificial sequence
<400>26
accagaaaaa?acccgttgtt?c 21
<210>27
<211>21
<212>DNA
＜213〉artificial sequence
<400>27
accagaaaaa?tcccgttgtt?c 21
<210>28
<211>18
<212>DNA
＜213〉artificial sequence
<400>28
acaagggcaa?gagagagg 18
<210>29
<211>17
<212>DNA
＜213〉artificial sequence
<400>29
caagggcagg?agagagg 17
<210>30
<211>18
<212>DNA
＜213〉artificial sequence
<400>30
acaagggcat?gagagagg 18
<210>31
<211>20
<212>DNA
＜213〉artificial sequence
<400>31
gttttgtcct?atcgtcaggt 20
<210>32
<211>19
<212>DNA
＜213〉artificial sequence
<400>32
tttgtcctgt?cgtcaggtg 19
<210>33
<211>20
<212>DNA
＜213〉artificial sequence
<400>33
gttttgtcct?ttcgtcaggt 20
<210>34
<211>22
<212>DNA
＜213〉artificial sequence
<400>34
gtgattccaa?cttatgctct?tc 22
<210>35
<211>23
<212>DNA
＜213〉artificial sequence
<400>35
ggtgattcca?aattatgctc?ttc 23
<210>36
<211>23
<212>DNA
＜213〉artificial sequence
<400>36
ggtgattcca?atttatgctc?ttc 23
<210>37
<211>19
<212>DNA
＜213〉artificial sequence
<400>37
tatgggaccc?gtacacatg 19
<210>38
<211>19
<212>DNA
＜213〉artificial sequence
<400>38
tatgggacct?gtacacatg 19
<210>39
<211>19
<212>DNA
＜213〉artificial sequence
<400>39
tatgggacca?gtacacatg 19
<210>40
<211>27
<212>DNA
＜213〉artificial sequence
<400>40
cttttgtctt?tcagtatctc?ttccctg 27
<210>41
<211>27
<212>DNA
＜213〉artificial sequence
<400>41
cttttgtctt?tcaatatctc?ttccctg 27
<210>42
<211>27
<212>DNA
＜213〉artificial sequence
<400>42
cttttgtctt?tcattatctc?ttccctg 27
<210>43
<211>21
<212>DNA
＜213〉artificial sequence
<400>43
ctcaaggagg?tatgaaaata?a 21
<210>44
<211>21
<212>DNA
＜213〉artificial sequence
<400>44
ctcaaggagg?catgaaaata?a 21
<210>45
<211>21
<212>DNA
＜213〉artificial sequence
<400>45
ctcaaggagg?aatgaaaata?a 21
<210>46
<211>27
<212>DNA
＜213〉artificial sequence
<400>46
ctcaacaatc?cacaagaccc?ctttgtg 27
<210>47
<211>26
<212>DNA
＜213〉artificial sequence
<400>47
tcaacaatcc?acgagacccc?tttgtg 26
<210>48
<211>27
<212>DNA
＜213〉artificial sequence
<400>48
ctcaacaatc?cactagaccc?ctttgtg 27
<210>49
<211>25
<212>DNA
＜213〉artificial sequence
<400>49
ggagagcact?aagaagttcc?taaaa 25
<210>50
<211>25
<212>DNA
＜213〉artificial sequence
<400>50
ggagagcact?aaaaagttcc?taaaa 25
<210>51
<211>25
<212>DNA
＜213〉artificial sequence
<400>51
ggagagcact?aataagttcc?taaaa 25
<210>52
<211>20
<212>DNA
＜213〉artificial sequence
<400>52
ggagattgat?gcagttttgc 20
<210>53
<211>20
<212>DNA
＜213〉artificial sequence
<400>53
ggagattgat?acagttttgc 20
<210>54
<211>20
<212>DNA
＜213〉artificial sequence
<400>54
ggagattgat?tcagttttgc 20
<210>55
<211>17
<212>DNA
＜213〉artificial sequence
<400>55
cctatgatgc?cgtggta 17
<210>56
<211>19
<212>DNA
＜213〉artificial sequence
<400>56
cctatgatgt?ccgtggtac 19
<210>57
<211>19
<212>DNA
＜213〉artificial sequence
<400>57
cctatgatga?ccgtggtac 19
<210>58
<211>23
<212>DNA
＜213〉artificial sequence
<400>58
tattctaagg?atttctactt?tgg 23
<210>59
<211>23
<212>DNA
＜213〉artificial sequence
<400>59
tattctaagg?acttctactt?tgg 23
<210>60
<211>23
<212>DNA
＜213〉artificial sequence
<400>60
tattctaagg?aattctactt?tgg 23
<210>61
<211>20
<212>DNA
＜213〉artificial sequence
<400>61
gcatgaggtt?tgctctcatg 20
<210>62
<211>19
<212>DNA
＜213〉artificial sequence
<400>62
gcatgaggtc?tgctctcat 19
<210>63
<211>19
<212>DNA
＜213〉artificial sequence
<400>63
gcatgaggtg?tgctctcat 19
<210>64
<211>19
<212>DNA
＜213〉artificial sequence
<400>64
cttggactcc?ccagtaaca 19
<210>65
<211>20
<212>DNA
＜213〉artificial sequence
<400>65
ccttggactc?tccagtaaca 20
<210>66
<211>19
<212>DNA
＜213〉artificial sequence
<400>66
cttggactcg?ccagtaaca 19
<210>67
<211>22
<212>DNA
＜213〉artificial sequence
<400>67
attaactcaa?tggaggtcag?tg 22
<210>68
<211>22
<212>DNA
＜213〉artificial sequence
<400>68
attaactcaa?aggaggtcag?tg 22
<210>69
<211>21
<212>DNA
＜213〉artificial sequence
<400>69
attaactcaa?gggaggtcag?t 21
<210>70
<211>22
<212>DNA
＜213〉artificial sequence
<400>70
gtgtgtgatt?atttgccaac?tg 22
<210>71
<211>21
<212>DNA
＜213〉artificial sequence
<400>71
gtgtgtgatt?ctttgccaac?t 21
<210>72
<211>21
<212>DNA
＜213〉artificial sequence
<400>72
gtgtgtgatt?gtttgccaac?t 21
<210>73
<211>17
<212>DNA
＜213〉artificial sequence
<400>73
ctgtgcaggg?caggaaa 17
<210>74
<211>18
<212>DNA
＜213〉artificial sequence
<400>74
ctgtgcagag?caggaaag 18
<210>75
<211>17
<212>DNA
＜213〉artificial sequence
<400>75
ctgtgcagcg?caggaaa 17
<210>76
<211>18
<212>DNA
＜213〉artificial sequence
<400>76
agcagcacgc?tgctgaaa 18
<210>77
<211>18
<212>DNA
＜213〉artificial sequence
<400>77
cagcagcaca?ctgctgaa 18
<210>78
<211>18
<212>DNA
＜213〉artificial sequence
<400>78
cagcagcact?ctgctgaa 18
<210>79
<211>19
<212>DNA
＜213〉artificial sequence
<400>79
agtactggac?agagcctga 19
<210>80
<211>19
<212>DNA
＜213〉artificial sequence
<400>80
agtactggag?agagcctga 19
<210>81
<211>19
<212>DNA
＜213〉artificial sequence
<400>81
agtactggaa?agagcctga 19
<210>82
<211>21
<212>DNA
＜213〉artificial sequence
<400>82
ctgccttttt?tgggaaatgc?t 21
<210>83
<211>22
<212>DNA
＜213〉artificial sequence
<400>83
ctgccttttt?ttgggaaatg?ct 22
<210>84
<211>21
<212>DNA
＜213〉artificial sequence
<400>84
tgcctttttt?ggggaaatgc?t 21
<210>85
<211>24
<212>DNA
＜213〉artificial sequence
<400>85
tgattgagtt?gtgtatgatt?ctac 24
<210>86
<211>23
<212>DNA
＜213〉artificial sequence
<400>86
tgattgagtt?gtgtatgata?cct 23
<210>87
<211>26
<212>DNA
＜213〉artificial sequence
<400>87
tgattgagtt?gtttatgatt?ctacat 26
<210>88
<211>24
<212>DNA
＜213〉artificial sequence
<400>88
tgattgagtt?gtttatgata?cctc 24
<210>89
<211>26
<212>DNA
＜213〉artificial sequence
<400>89
tgattgagtt?gtatatgatt?ctacat 26
<210>90
<211>27
<212>DNA
＜213〉artificial sequence
<400>90
agttgtgtat?gattctacat?agaatat 27
<210>91
<211>27
<212>DNA
＜213〉artificial sequence
<400>91
agttgtttat?gattcctcat?agaatat 27
<210>92
<211>27
<212>DNA
＜213〉artificial sequence
<400>92
agttgtgtat?gatactacat?agaatat 27
<210>93
<211>27
<212>DNA
＜213〉artificial sequence
<400>93
agttgtttat?gatacctcat?agaatat 27
<210>94
<211>24
<212>DNA
＜213〉artificial sequence
<400>94
gttgtgtatg?atcctacata?gaat 24
<210>95
<211>27
<212>DNA
＜213〉artificial sequence
<400>95
gttgtgtatg?attctacata?gaatatt 27
<210>96
<211>28
<212>DNA
＜213〉artificial sequence
<400>96
ttgtttatga?tactacatag?aatatgaa 28
<210>97
<211>27
<212>DNA
＜213〉artificial sequence
<400>97
ttgtgtatga?ttccacatag?aatatta 27
<210>98
<211>27
<212>DNA
＜213〉artificial sequence
<400>98
ttgtttatga?taccacatag?aatatga 27
<210>99
<211>27
<212>DNA
＜213〉artificial sequence
<400>99
gttgtgtatg?attcaacata?gaatatt 27
<210>100
<211>28
<212>DNA
＜213〉artificial sequence
<400>100
ttgtgtatga?ttctacatag?aatattaa 28
<210>101
<211>27
<212>DNA
＜213〉artificial sequence
<400>101
tgtttatgat?accacataga?atatgaa 27
<210>102
<211>28
<212>DNA
＜213〉artificial sequence
<400>102
ttgtgtatga?ttcttcatag?aatattaa 28
<210>103
<211>27
<212>DNA
＜213〉artificial sequence
<400>103
tgtttatgat?acctcataga?atatgaa 27
<210>104
<211>27
<212>DNA
＜213〉artificial sequence
<400>104
tgtgtatgat?tctgcataga?atattaa 27
<210>105
<211>25
<212>DNA
＜213〉artificial sequence
<400>105
tacatagaat?attaactcaa?tggag 25
<210>106
<211>25
<212>DNA
＜213〉artificial sequence
<400>106
ctcatagaat?attaactcaa?aggag 25
<210>107
<211>24
<212>DNA
＜213〉artificial sequence
<400>107
acatagaata?tgaactcaat?ggag 24
<210>108
<211>24
<212>DNA
＜213〉artificial sequence
<400>108
tcatagaata?tgaactcaaa?ggag 24
<210>109
<211>25
<212>DNA
＜213〉artificial sequence
<400>109
tacatagaat?ataaactcaa?tggag 25
<210>110
<211>23
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
＜223〉primer
<400>110
cccacacaaa?tacatcccag?gac 23
<210>111
<211>22
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
＜223〉primer
<400>111
cccagcatgg?agcagtaagt?ga 22
<210>112
<211>23
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
＜223〉primer
<400>112
tgcatgcata?gaggaaggat?ggt 23
<210>113
<211>22
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
＜223〉primer
<400>113
tgatgacagg?gtttgtgaca?gg 22
<210>114
<211>22
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
＜223〉primer
<400>114
gtggcatgag?gaggagtgga?tg 22
<210>115
<211>22
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
＜223〉primer
<400>115
gggaagtggt?gaggaggcat?tt 22
<210>116
<211>22
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
＜223〉primer
<400>116
ccgaatgctt?cccaccttca?ta 22
<210>117
<211>25
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
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<400>117
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<211>23
<212>DNA
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<221>misc_feature
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<212>DNA
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<220>
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<221>misc_feature
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<220>
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<212>DNA
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<210>131
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<212>DNA
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<211>25
<212>DNA
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<210>133
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<212>DNA
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<211>25
<212>DNA
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<221>misc_feature
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<211>25
<212>DNA
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<210>136
<211>24
<212>DNA
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Claims (9)
1. CYP3A detection chip, comprise solid phase carrier and probe, it is characterized in that, the nucleotide sequence of described probe and CYP3A gene to be measured and/or its complementary sequence are hybridized, described CYP3A gene to be measured comprises CYP3A4, CYP3A5 and CYP3A7 gene, described probe is DNA, and its sequence is:
(1) with sequence shown in (a) SEQ ID NO:1～SEQ ID NO:30 of CYP3A4 gene recombination to be measured, (b) complementary strand of every sequence in the sequence shown in SEQID NO:1～SEQ ID NO:30;
(2) with sequence shown in (a) SEQ ID NO:31～SEQ ID NO:63 of CYP3A5 gene recombination to be measured, (b) complementary strand of every sequence in the sequence shown in SEQID NO:31～SEQ ID NO:63;
(3) with sequence shown in (a) SEQ ID NO:64～SEQ ID NO:109 of CYP3A7 gene recombination to be measured, (b) complementary strand of every sequence in the sequence shown in SEQID NO:64～SEQ ID NO:109.
2. detection chip as claimed in claim 1 is characterized in that described probe can be provided with connecting arm, and described probe can be modified.
3. detection chip as claimed in claim 1 is characterized in that, described solid phase carrier selection is a kind of in slide, silicon chip, nitrocellulose filter, nylon membrane and the macromolecular material or their arbitrary combination.
4. an application rights requires the method for 1 described chip detection CYP3A gene, it is characterized in that, comprises the steps:
(1) carries the described probe of claim 1 at surface of solid phase carriers point;
(2) nucleic acid of extracting CYP3A gene to be measured;
(3) the purpose nucleotide sequence of preparation CYP3A gene to be measured, the preparation of described purpose nucleotide sequence comprises pcr amplification reaction, this reaction the primer is nucleotide chain or its complementary strand of sequence shown in SEQ ID NO:110～SEQ ID NO:143;
(4) the purpose nucleotide sequence of markers step (3);
(5) be loaded under the condition that the probe on the solid phase carrier hybridizes with the described point of step (1) being suitable for, add purpose nucleotide sequence, and make it react the enough time through mark;
(6) result of detection hybridization.
5. method as claimed in claim 4 is characterized in that, the mark of the described purpose nucleotide sequence of step (4) adopts and comprises fluorescein-labelled, biotin labeling, radioelement mark or enzyme labelling.
6. method as claimed in claim 4 is characterized in that, the hybridization temperature in the described step (5) is 25 ℃～65 ℃, and hybridization time is 2 minutes～18 hours.
7. an application rights requires the method for 1 described chip detection CYP3A gene, it is characterized in that, comprises the steps:
(1) the described probe of mark claim 1;
(2) nucleic acid of extracting CYP3A gene to be measured:
(3) the purpose nucleotide sequence of preparation CYP3A gene to be measured, the preparation of described purpose nucleotide sequence comprises pcr amplification reaction, this reaction the primer is nucleotide chain or its complementary strand of sequence shown in SEQ ID NO:110～SEQ ID NO:143;
(4) carry the described purpose nucleotide sequence of step (3) at surface of solid phase carriers point;
(5) be loaded under the condition that the purpose nucleotide sequence on the solid phase carrier hybridizes with the described point of step (4) being suitable for, add probe, and make it react the enough time through mark;
(6) result of detection hybridization.
8. method as claimed in claim 7 is characterized in that, the described probe mark of step (1) adopts and comprises fluorescein-labelled, biotin labeling, FRET (fluorescence resonance energy transfer) mark, radioelement mark or enzyme labelling.
9. method as claimed in claim 7 is characterized in that, the hybridization temperature in the described step (5) is 25 ℃～65 ℃, and hybridization time is 2 minutes～18 hours.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2006101195534A CN101067149B (en) | 2006-12-13 | 2006-12-13 | CYP3A detecting chip and its application |
| PCT/CN2007/001853 WO2008011787A1 (en) | 2006-07-17 | 2007-06-12 | Chip for detecting genetic mutation of cytochrome p450 gene and the use thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2006101195534A CN101067149B (en) | 2006-12-13 | 2006-12-13 | CYP3A detecting chip and its application |
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| Publication Number | Publication Date |
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| CN101067149A CN101067149A (en) | 2007-11-07 |
| CN101067149B true CN101067149B (en) | 2010-12-29 |
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| CN2006101195534A Active CN101067149B (en) | 2006-07-17 | 2006-12-13 | CYP3A detecting chip and its application |
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Families Citing this family (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101812524B (en) * | 2010-04-09 | 2012-08-22 | 广州益善生物技术有限公司 | Specific primer, liquid phase chip and detection method for CYP3A5 gene SNP (Single Nucleotide Polymorphism) detection |
| JP2012196209A (en) * | 2011-03-09 | 2012-10-18 | Arkray Inc | Probe for detecting polymorphism in cyp3a gene, method for detecting polymorphism, method for evaluating medicine efficacy and reagent kit for detecting polymorphism |
| CN104293920B (en) * | 2014-09-17 | 2016-05-04 | 武汉康录生物技术有限公司 | A kind of kit and using method thereof for fast detecting mankind VKORC1 and CYP2C9 gene pleiomorphism |
| CN104212904B (en) * | 2014-09-17 | 2016-05-04 | 武汉康录生物技术有限公司 | A kind of kit and using method thereof for fast detecting mankind CYP2C19 gene pleiomorphism |
| JP2016192940A (en) * | 2015-04-01 | 2016-11-17 | 東洋鋼鈑株式会社 | PROBE FOR DETECTING CYP3A4*1b AND PROBE FOR DETECTING CYP3A5*3 |
| CN105274221A (en) * | 2015-10-14 | 2016-01-27 | 北京晋祺生物科技有限公司 | CYP3A5*3 detection kit |
| CN110093415A (en) * | 2019-04-30 | 2019-08-06 | 上海百傲科技股份有限公司 | Detect method, kit, primer pair and the probe of CYP3A5 gene |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1544652A (en) * | 2003-10-27 | 2004-11-10 | 上海第二医科大学附属第九人民医院 | Miniaturized oligonucleotide chip for sort diagnosis of head and neck squamous cell carcinoma |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1544652A (en) * | 2003-10-27 | 2004-11-10 | 上海第二医科大学附属第九人民医院 | Miniaturized oligonucleotide chip for sort diagnosis of head and neck squamous cell carcinoma |
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Assignee: Shanghai Biotechnology Corporation Assignor: Shanghai Biochip Co., Ltd Contract record no.: 2011310000186 Denomination of invention: CYP3A detecting chip and its application Granted publication date: 20101229 License type: Exclusive License Open date: 20071107 Record date: 20110905 |
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Owner name: SHANGHAI BIOTECHNOLOGY CORPORATION Free format text: FORMER OWNER: SHANGHAI BIOCHIP CO., LTD Effective date: 20130819 |
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Effective date of registration: 20130819 Address after: 201203 Shanghai city Pudong New Area Zhangjiang libing Road No. 151 Patentee after: Shanghai Biotechnology Corporation Address before: 201203 Shanghai city Pudong New Area Zhangjiang libing Road No. 151 Patentee before: Shanghai Biochip Co., Ltd |