WO2008011787A1

WO2008011787A1 - Chip for detecting genetic mutation of cytochrome p450 gene and the use thereof

Info

Publication number: WO2008011787A1
Application number: PCT/CN2007/001853
Authority: WO
Inventors: Haihui Sheng; Huasheng Xiao
Original assignee: Shanghai Biochip Co., Ltd.
Priority date: 2006-07-17
Filing date: 2007-06-12
Publication date: 2008-01-31
Also published as: WO2008011787A8

Abstract

A chip for detecting genetic mutation of cytochrome P450 gene is provided. The chip comprises a solid carrier and probes. The probes can hybridize to the nucleotide sequence and/or its complementary sequence of the cytochrome P450 gene to be detected. Also provided is a method of detecting genetic mutation of cytochrome P450 gene by the chip. The chip can be used to effectively detect the genetic mutation of CYP2C9, CYP2C19, CYP2D6, CYP3A4, CYP3A5 and CYP3A7, and predict the therapeutic efficiency of above 40% of clinical medicaments to realize individuation of medical treatment.

Description

Detection chip for cytochrome P450 gene genetic variation and its application

The invention relates to a medicament gene detection chip, in particular to a detection chip for genetic variation of a cytochrome P450 (CYP450) gene related to human drug metabolism and an application thereof.

Background technique

For a long time, clinicians often take the same drug in the same dose for the same disease according to the clinical trial of the drug, or make slight adjustments to the dose based on clinical experience. Treating the same patient with the same drug often produces a very different effect. Some patients even have serious side effects or no reaction, which not only causes huge waste, but also directly delays the timely treatment of the disease and delays the disease. The annual mortality due to adverse drug reactions and drug-induced diseases and the corresponding health resources are extremely 'astalling'. This individual difference in drugs has become a problem that has plagued clinical drugs and drug manufacturers to develop new drugs. The differences in the response of different individuals to drug therapy can be caused by a variety of factors and produce different corresponding consequences. Among them, 20-95% of drug treatment and effect differences are caused by genetic factors, involving drug target genes, drug metabolizing enzyme genes, drug transport related genes, DNA repair enzyme genes, and some prosthetic synthase genes [Kalow W, Tang BK Endrenyi I. Hypothesis : comparisons of inter- and intra- individual variations can substitute for twin studies in drug research. Pharmacogenetics, .1998, 8 : 283-9. The genetic variation in these genes is the basis for the individual differences in the efficacy of the drug, mainly the genetic variation of single nucleotide polymorphisms (SNPs). Other influencing factors are age, gender, disease, and environmental factors.

The metabolic process of the drug in the body is carried out by various enzymes. The enzymes that catalyze the metabolism of phase I drugs are mainly cytochrome P450 enzymes (CYP). The enzymes with significant genetic polymorphisms include CYP3A4, CYP2D6, CYP2C19, CYP2C9, CYP2C19 and CYP3A5, etc. The enzymes that catalyze the metabolism of phlegm phase drugs mainly include Thiopurine methyltransferase (TPMT), N-acetyltransferase (NAT), glutathione S-transferase (GST), and the like. These enzymes involved in drug metabolism in humans can be divided into at least 30 families [Evans WE, Relling MV. Pharmacogenomics: translating functional genomics into rational therapeutics. Science, 1999, 286: 487-91; Ingelman-Sundberg M, Oscarson M, McLellan RA. Polymorphic human cytochrome P450 enzymes: an opportunity For individualized drug treatment. Trends Pharmacol Sci, 1999, 20: 342-9. The CYP enzyme system is the main enzyme system of metabolic drugs in human body. About 50% of the drugs are mainly metabolized by CYP enzyme system, CYP gene polymorphism and its influence on drug metabolism, and it is also one of the earliest objects of pharmacogenetics research.

In Phase I drug metabolism, approximately 40-45% of the drug is catalyzed by CYP3A4 [Ingelman-Sundberg, M. Pharmacogenetics of cytochrome P450 and its applications in drug therapy: the past, present and future. Trends Pharmacol Sci. 2004 , 25: 193-200.], these drugs include acetaminophen, carbamazepine, and lovastatin: ' ί 肖苯地等. Although many genetic variations have been discovered in the CYP3A4 gene to date, it is more important that functional variability has not been found in the 髙Caucasus and Chinese, and the substrate specificity of CYP3A4 and CYP3A5 overlap. CYP3A4 and CYP3A5 belong to the CYP3A family, and individual differences in CYP3A substrate clearance are mainly due to the polymorphism of CYP3A5. There are many types of drugs that CYP3A5 metabolizes, but the frequency of loss of expression of functional CYP3A5 in the population is high, mainly manifested by polymorphic expression. It is now clear that the loss of CYP3A5 gene expression level is due to a SNP' (A-G)' in the third intron, which causes abnormal cleavage of the CYP3A5 gene, causing early termination of translation, resulting in no Functional CYP3A5 truncated protein [Kuehl P, Zhang J, Lin Y, Lamba J, Assem M, Schuetz J, Watkins PB, Daly A, Wrighton SA, Hall SD, Maurel P, Relling M, Brimer C, Yasuda K, Venkataramanan R, Strom S, Thuramel, Boguski MS, Schuetz E. Sequence diversity in CYP3A promoters and characterization of the genetic basis for polymorphic CYP3A5 expression. Nat Genet, 2001, 27: 383-91. Moreover, abnormal shear products are less stable and are prone to rapid degradation, which may explain why AA homozygous patients have low expression levels of CYP3A5. CYP3A5 with the G allele of this SNP is CYP3A5*3. The frequency of CYP3A5*3 is 88%, 75% and 35% among Caucasians, Asians and Africans.

Due to the lack of specific antibodies, CYP3A7 - was mistaken for the presence of only fetal liver. Similar to CYP3A5, the expression of CYP3A7 is also polymorphic, and about 10-20% of Caucasians are highly expressed in CYP3A7. In the high expression of CYP3A7, the amount of CYP3A7 accounts for about 9-36% of the total amount of CYP3A, which may be equivalent to or even exceed the amount of CYP3A5 enzyme in CYP3A5 high expression. The major CYP3A7 alleles are CYP3A7*1C, CYP3A7*1B, CTP3A7*2 and CYP3A7*3. CYP3A7*1C and CYP3A7*1B can increase the transcriptional activity of CYP3A7 gene, two It was associated with approximately 67% of Caucasians with high expression of CYP3A7. CYP3A7*2 does not affect gene expression, but its catalytic activity is significantly higher than that of CYP3A7. 1, CYP3A7*2 is 8%, 17%, 28 in whites, Saudi Arabians, Chinese and Tanzanians, respectively. % and 62°/. [Rodriguez-Antona C, Jande M, Rane A, Ingelman-Sundberg M. Identification and phenotype characterization of two CYP3A haplotypes causing different enzymatic capaci ty in fetal livers. Cl in Pharmacol Ther, 2005, 77: 259-270]. The effects of other alleles such as CYP3A7*3 on gene expression and enzyme activity are unclear.

The class of CYP2D6 metabolites is second only to CYP3A4 and is related to the metabolism of approximately 25% of known drugs, including isoquinoxaline, imipramine, clozapine, beta-adrenergic blockers, etc. [Ingelman- Simdberg. Genetic Polymorphisms of cytochrome P450 2D6 (CYP2D6): clinical consequences, evolutionary aspects and functional diversity. Pharmacogenomics J. 2004, 5 : 6-13]. If the patient's CYP2D6 gene is mutated, it will have obvious side effects on the antihypertensive drug, isoquinone. About 3% to 10% of Caucasians lack the activity of the enzyme gene, accounting for about 1% to 2% of the Orientals [Bertilsson L , Dahl ML, Dal en P, Al - Shurbaji A. Molecular genetics of CYP2D6: clinical relevance with focus on psychotropic drugs. Br J Clin Pharmacol. 2002, 53 : 111-22.] ₀ This CYP2D6 enzyme deficiency is called A poor metabolizer (PM), because the drug cannot be metabolized, is prone to side effects, or the prodrug cannot be effectively converted into an active ingredient, resulting in treatment failure. The normal human CTP2D6 gene on both chromosomes 22 is normal, and its metabolism type is extensive metablizer (EM). The intermediate metablizer (IM) is not lacking CYP2D6, but the CYP2D6 activity is lower than normal due to genetic mutation. In the Chinese population, the most common genotype is CYP2D6*10 (due to the change in the 100th nucleotide C-T in the first exon), with a frequency of 51-70% [ Ji L, Pan S , Marti-Jaun J, Hanseler E, Rentsch K, Hersberger M. Single-step assays to analyze CYP2D6 gene polymorphisms in Asians : allele frequencies and a novel *1 B allele in mainland Chinese. Clin Chem. 2002, 48 : 983-8 .]. Ultrarapid metablizer (UM) is often caused by a multiplicity of functional CYP2D6 genes, so patients taking standard doses of CYP2D6 metabolism in I patients will not achieve effective therapeutic effects. UM distribution frequency can range from 2% in the Swedish population to 30% in the Ethiopian population, but in Asian populations The medium frequency is lower. Depending on the individual CYP2D6 metabolite type, it is necessary to adjust the conventional dose to obtain acceptable drug efficacy and safety, which is more clearly studied in psychotropic drugs [Kirchheiner J, Nickchen, Bauer Μ, Wong ML, Licinio J , Roots I, Brockmoller J. Pharmacogenetics of antidepressants and antipsychotics : the contribution of allelic variations to the phenotype of drug response. Mol Psychiatry, 2004, 9: 442 - 473. The CYP2D6 has been found to have more than 80 variants to date, involving single base changes, short fragment insertions/deletions, partial deletions of genes, and even deletions and duplications of entire genes, making it the most polymorphic of the CYP450 supergene family.

CYP2C9 is an important drug-metabolizing enzyme involved in the metabolism of approximately 10% of commonly used drugs in the clinic, as well as in vivo transformation of various endogenous substances [Xie HG, Prasad HC, Kim RB, Stein CM. CYP2C9 allelic variants: Adv Drug Deliv Rev. 2002, 54 : 1257 - 70 ; Lee CR, Goldstein JA, Pieper JA. Cytochrome P450 2C9 polymorphisms : a comprehensive review of the in- vitro and human data. Pharmacogenetics. 2002, 12: 25 Bu 63. ]. Common drugs are warfarin, phenytoin, tolbutamide, phenytoin, imipramine, fluvastatin and some non-inflammatory anti-inflammatory drugs (such as diclofenac, ibuprofen). CYP2C9 has some functional SNPs that can cause significant changes in enzyme activity. In the Caucasian population, CYP2C9*2 and CYP2C9*3 are two common alleles of CYP2C9, located in exon 3 and exon 7, respectively, resulting in 144th arginine cysteine and 359th position. Bright amino acid changes the amino acid. The enzymatic activity of CYP2C9*2 and CYP2C9*3 is lower than that of CYP2C9*1 (wild type), so CYP2C9*2 and CYP2C9*3 homozygous, CYP2C9*2/CYP2C9*3 heterozygotes are slow metabolizing type, accounting for the total number of people. "It is very sensitive to anticoagulant drugs such as warfarin. Compared to people with CYP2C9*1, they only need a small dose. Among the Oriental yellow races, the most common and most common genotype, CYP2C9*3, has a frequency of about 3%, while other genetic variations are rare or absent in the yellow race.

CYP2C19 and CYP2C9 belong to the CYP2C family and are important P450 enzymes. They have obvious genetic polymorphism and can catalyze many clinical drugs, including omeprazole, diazepam, cyclohexalto, and propranolol. At least six defective alleles have been identified. If an individual carries a polymorphic gene of CYP2C19, in the metabolism of omeprazole, the inactivated enzyme causes the blood concentration to be too high, increasing the drug response. People with defects in the CYP2C19 gene are highly sensitive to drugs such as phenytoin and cyclohexene. 12% - 23% of Asians have no CYP2C19 or have very low activity, so these people are slow metabolized, while Caucasians and blacks are only 3% - 5%. CYP2C19 or activity is very low, so these people are fast metabolizing. Among the slow metabolizing individuals in Japan, 70% are CYP2C19*2, followed by CYP2C19*2. CYP2C19*3 is a characteristic genotype of Asians [Nagata K, Yamazoe Y. Genetic polymorphism of human cytochrome p450 involved in drug metabolism. Drug Metab Pharmacokinet. 2002 ; 17 (3) : 167-89. ] ₀ among Japanese About one-fifth of the slow-metabolism type, the proton pump inhibitors omeprazole (live sike) and puer-in-the-vi (the propranolol) are difficult to metabolize in the body. The blood levels of slow metabolizing and fast metabolizing individuals can vary by a factor of seven, which is not difficult to understand why the two drugs have more adverse reactions in the Japanese population.

Polymorphisms in the CYP gene are one of the main causes of differences in the response of individuals to drug therapy. Therefore, if the results of pharmacogenetics can be applied to disease treatment, clinicians can obtain the genetic variation of the patient's CYP gene. The patient's genetic information, in order to determine which drugs are effective, targeted use of drugs and doses, effectively improve the efficacy of the drug and reduce the side effects of the drug, which is expected to significantly reduce medical costs. For pharmaceutical companies, according to the information of pharmacogenetics and pharmacogenomics, as well as the drug target and drug metabolism, select the appropriate clinical trial population to improve the efficacy of drugs on disease treatment, reduce drug inefficiency and toxicity. Side effects, shorten the cycle of research and development of drugs, improve the clinical approval rate of drugs; develop drug derivatives or similar drugs according to the genetic structure of native people.

Since the allelic distribution of CYP genes such as CYP2D6, CYP2C9, CYP2C19, CYP3A4 and CYP3A5 in each population has been studied clearly, and the probability of discovering a new functional genetic variation is very small, it is fully exploitable. The results of existing pharmacogenetics studies can be used to detect functional or marker genetic variation of the CYP gene. The gene chip technology has the characteristics of high throughput, parallelism and micro-chemicalization. It can be used to detect many disease-related genes or drug-effect related genes at the same time, avoiding cumbersome and time-consuming routine genotyping.

Roche partnered with Affymetrix to develop the world's first drug detection chip product for the human drug metabolism gene CYP450 AmpliChip CYP450 [ ling J. Roche' s microarray tests US FDA' s diagnostic policy. Nat Biotechnol. 2003, 21 (9) : 959-60. ] ₀ However, the AmpliChip CYP450 chip can only detect the genotypes of CYP2D6 and CYP2C19, which are only related to about 25% of prescription drug metabolism. Another P450 gene chip is the CodeLink P450 biochip, which was developed by GE Healthcare and uses the allele-specific Primer extension (ASPE) technology. Although the CodeLink P450 biochip can detect 110 polymorphisms of 9 CYP450 genes, it requires 12 A multiplex reaction for DNA preparation, the DNA preparation method is low in level, which is very cumbersome and limits its practical application.

Summary of the invention

The technical problem to be solved by the present invention is to provide a detection chip for genetic variation of cytochrome P450 gene, which can effectively detect genetic variation of CYP2C9, CYP2C19, CYP2D6, CYP3A4, CYP3A5 and CYP3A7, and more than 40% of clinically used drugs. The therapeutic effect is predicted.

The second technical problem to be solved by the present invention is to provide a method for detecting genetic variation of the cytochrome P450 gene using the above chip.

In order to solve the above technical problem, the present invention is achieved by the following technical solutions:

In one aspect of the invention, a detection chip for genetic variation of a cytochrome P450 gene is provided, comprising a solid phase carrier and a probe, and the nucleotide sequence of the cytochrome P450 gene to be tested and/or its complement Hybridization is carried out.

The solid phase carrier in the present invention may be selected from a carrier known in the art as long as the carrier is compatible with the reactant, and the detection result is not affected. Preferably, the solid phase carrier of the present invention is selected from the group consisting of a slide, a silicon wafer, a nitrocellulose membrane, a nylon membrane, and a polymer material, or any combination thereof.

The cytochrome P450 gene to be tested includes the CYP2D6, CYP2C19, CYP2C9, CYP3A4, CYP3A5 and CYP3A7 genes.

The probe may be DNA, RNA, DNA-RNA chimera, PNA or a derivative thereof. The length of the probe is not limited as long as the function of specific hybridization can be accomplished, and specific binding to the nucleotide sequence of interest can be performed in any length. The probe can be as short as 25, 20, 15, 13 or 10 bases in length. Also, the length of the probe can be as long as 60, 80, 100, 150, 300 base pairs or longer, or even the entire gene. Since different probe lengths have different effects on hybridization efficiency and signal specificity, the length of the probe is usually at least 14 base pairs, and the longest is generally no more than 30 base pairs, and the nucleotide sequence of interest Complementary lengths are optimal between 15 and 25 base pairs. Preferably, the probe has a self-complementary sequence of less than 4 base pairs to avoid affecting hybridization efficiency. If a certain degree of mismatched base is likely to be present in the probe, the probe can be increased in length to counteract the adverse effects of mismatched bases to increase hybridization efficiency.

The probe of the detection chip of the present invention is DNA, comprising: (1) hybridization with the CYP2D6 gene to be tested (a) SEQ ID NO: 1 to SEQ ID NO: 76, (b) SEQ ID NO: 1 to SEQ ID N0: the complementary strand of each sequence in the sequence shown by 76, (c) and each sequence in the sequence shown in SEQ ID NO: 1 to SEQ ID NO: 76 a sequence having at least 70% homology;

(2) (a) the sequence shown in SEQ ID NO: 77 to SEQ ID NO: 9 which hybridizes with the CYP2C19 gene to be tested, and (b) each of the sequences shown in SEQ ID NO: 77 to SEQ ID NO: 94 a complementary strand, (c) a sequence having at least 70% homology to each of the sequences set forth in SEQ ID NO: 77 to SEQ ID NO: 94;

(3) (a) a sequence complementary to the CYP2C9 gene to be tested (a) a sequence of SEQ ID NO: 95 to SEQ ID NO: 112, (b) a complementary strand of each of the sequences of SEQ ID NO: 95 to SEQ ID NO: 112, c) a sequence having at least 70% homology to each of the sequences set forth in SEQ ID NO: 95 to SEQ ID NO: 112;

(4) (a) the sequence of SEQ ID NO: 113 to SEQ ID NO: 142 which hybridizes with the CYP3A gene to be tested, and (b) each sequence of the sequence of SEQ ID NO: 113 to SEQ ID NO: 142 a complementary strand, (c) a sequence having at least 70% homology to each of the sequences set forth in SEQ ID NO: 113 to SEQ ID NO: 142;

(5) (a) the sequence shown in SEQ ID NO: 143 to SEQ ID NO: 201, (b) the sequence of each of the sequences shown in SEQ ID NO: 143 to SEQ ID NO: 201, which hybridizes to the CYP3A5 gene to be tested. a complementary strand, (c) a sequence having at least 70% homology to each of the sequences set forth in SEQ ID NO: 143 to SEQ ID NO: 201;

(6) (a) the sequence shown in SEQ ID NO: 202 to SEQ ID NO: 247, (b) each of the sequences shown in SEQ ID NO: 202 to SEQ ID NO: 247, which hybridizes to the CYP3A7 gene to be tested. A complementary strand, (c) a sequence having at least 70% homology to each of the sequences set forth in SEQ ID NO: 202 to SEQ ID NO: 247.

Preferably, the probe of the detection chip of the present invention is selected from the sequences shown in SEQ ID NO: 1 to SEQ ID NO: 247.

The probe sequence may comprise from 1 to 10 mismatch bases, preferably from 1 to 5 mismatch bases, and more preferably from 1 to 2 mismatch bases.

The test chip of the present invention further comprises at least one control probe selected from the group consisting of: a negative control probe, a positive control probe, a hybridization control probe, and an immobilized control probe.

The probe may be immobilized on a carrier by a variety of methods, such as a spotting method (USA Patent No. 5288514, USA Patent No. 5556752), a light-mediated method (USA Patent No. 5143854, 5384261 and 5561071), a magnetic bead method. (USA Patent No. 5541061) and the like. The probe may be immobilized on a carrier by chemical or physical means, such as by ion bonding, covalent bonding or other known forces on the carrier. Above, the probe can be fixed to the carrier by an ultraviolet cross-linker.

The probe can be immobilized on a solid support via a connecting arm. The linker arm provides a free space for the probe to form a double-stranded portion to reduce steric hindrance and facilitate the hybridization reaction [Afanassiev V, HanemannV, Wolfl S. Preparation of DNA and protein micro arrays on glass slides coated with An agarose film. Nucleic Acids Res. 2000, 28: e66; USA Patent No. 5556752]. The longer the connecting arm, the higher the hybridization efficiency. A typical connecting arm includes 15 to 30 functional group lengths. The connecting arm may be selected from a suitable functional group such as Poly T (A, C or G), C atom or a chimera of polyethylene glycol with Poly T (A, C or G), polyethanol, poly, Polyamide, polysulfate and its composition.

The probe may be modified, and the modification method may be 5'-NH ₂ modification, 5'-SH modification, 5'-Poly T (A, C or G) modification, 5, - biotin modification, 3, - NH ₂ modification, 3, -SH modification, 3'-Poly T (A, C or G) modification and 3'-biotin modification.

The probe may have one or several, or even all, labels, including fluorescein labels, biotin labels, radioactive element labels, enzyme labels, and fluorescent resonance energy transfer labels.

In another aspect of the invention, a method for detecting genetic variation of a cytochrome P450 gene using the above-described chip is provided, comprising the steps of:

(1) extracting a nucleic acid of a cytochrome P450 gene to be tested from a suitable sample;

(2) Preparation of the nucleotide sequence of the cytochrome P450 gene to be tested;

(3) labeling the nucleotide sequence of interest of step (2);

(4) selecting the chip of claim 1, adding a labeled nucleotide sequence of interest under conditions suitable for hybridization with the selected chip, and allowing the reaction to be sufficiently time;

(5) The result of detecting the hybridization reaction.

Suitable samples as described in the methods of the invention include: blood, saliva, hair and any other nucleic acid containing tissue from humans and animals, as well as samples from plants and environmental products such as soil or water.

The nucleic acid of the sample can be extracted from the sample by any suitable method. For example, sample nucleic acids can be extracted from samples using magnetic beads, and many biotech companies can provide nucleic acid extraction kits such as Qiagen, Invitron, and others.

Due to advances in technology, it is now possible to directly use nucleic acid-containing cells in target samples for amplification without nucleic acid extraction. The nucleic acid-containing cells of the target sample can be extracted from the sample by any suitable method. For example, nucleic acid-containing cells in a target sample can be separated from the sample by magnetic beads.

The preparation of the nucleotide sequence of interest may comprise the step of amplifying, directly amplifying the nucleic acid-containing cells in the isolated target sample, or directly amplifying the extracted target nucleic acid. If the white blood cells are separated from the whole blood by magnetic beads, the nucleic acid sequence of interest is amplified directly using the isolated white blood cells or the nucleic acid extracted by whole blood as a template. The amplified single-stranded or double-stranded DNA or RNA may contain a fluorescent or biotin label, and the labeled DNA or RNA may be directly used for hybridization without purification. The preferred target nucleotide molecule to be hybridized to the chip of the present invention is a single-stranded nucleic acid molecule which is also hybridized to the chip of the present invention after being subjected to denaturation or the like.

The nucleotide sequence of interest can be enriched using any suitable amplification method, such as: polymerase chain reaction

(polymerase chain reaction, PCR), multiplex PCR, ligase chain reaction (LCR), rolling cycle amplification (RCA), nucleic acid sequence-based amplification (NASA) ), strand displacement amplification (SDA) and transcription-mediated amplification

( transcription medicated amplification, TMA) and so on. The TMA method preferably uses the T7 promoter.

As long as the nucleotide sequence of interest for hybridization is of sufficient length to produce specific hybridization, the length of the nucleotide sequence of interest is not limited in the upstream and downstream directions. Suitable nucleotide sequences of interest are from 30 to 200 base pairs in length. The target nucleotide sequence for hybridization is too long or too short, which affects the efficiency of hybridization and makes the target nucleotide sequence hybridized on the probe easy to elute, resulting in a weak or missing fluorescent signal. Longer DNA fragments can be fragmented with DNase I, or an appropriate ratio of deoxyuracil can be added to the reaction system, and the PCR product can be treated with uracil DNA glycosidase to fragment the DNA fragment. As for RNA, the treatment method is relatively simple, and it can be directly treated with high salt and sputum temperature.

In one embodiment of the present invention, the nucleotide sequence of interest is prepared by a PCR amplification method, and the primer used in the method comprises (a) SEQ ID NO: 248 to SEQ; [nucleotides of the sequence shown by D NO: 311 Chain, (b) SEQ ID NO: 248 to SEQ ID NO: 311 The complementary strand of each sequence in the sequence shown, (c) and each sequence in the sequence shown in SEQ ID NO: 248 to SEQ ID NO: 311 A sequence with at least 70% homology.

Preferably, the primer sequences of the present invention are the nucleotide strands of the sequences set forth in SEQ ID NO: 248 to SEQ ID NO: 311.

The hybridization between the probe and the nucleotide sequence of interest may be homologous or non-homologous.

The nucleotide sequence of interest is suitable for labeling, can be introduced into the amplification, or used after amplification A suitable method introduces a label.

The nucleotide sequence of the target includes fluorescent label, radioisotope label, chromophore, illuminant,

FRET, an enzyme, biotin or a ligand with a specific binding ligand.

Hybridization of the method of the invention can be carried out in any hybridization solution, such as a hybridization solution containing SSPE and a surfactant. The hybridization solution can contain any concentration of SSPE, such as 1 to 10 X SSPE. Any suitable surfactant such as Triton-100, SDS, SLS (sodium lauryl sarcosinate), CTAB (hexaalkyltrimethylammonium bromide), or the like can also be used. 01〜1% (w/w)。 The hybridization solution may also have any concentration of surfactant, such as 0.01 to 1% (w / w). The hybridization can be carried out at any suitable temperature, such as from 25 ° C to 65 ° C, and the hybridization time is from 5 minutes to 18 hours. Hybridization conditions can be altered to increase or decrease the stringency and hybrid specificity of the hybridization.

The result of the hybridization may be any washing liquid before the test, and the washing liquid may contain 0 to 3% (w/w) of the surfactant, and the washing may be carried out for a suitable period of time, for example, 1 to 30 minutes. It can be washed with a room temperature wash or after preheating, such as 42 °C. Washing can be carried out in succession with different washing solutions.

Hybridization between the probe and the nucleotide sequence of interest can be detected by any known method. Depending on the label, a suitable detection method can be used. For example, a fluorescently labeled probe or a nucleotide sequence of interest can be detected by a laser scanner or a fluorometer, and a radioactive element-labeled probe or a nucleotide sequence of interest can be irradiated. Development detection.

The overall hybridization specificity can be determined by any suitable standard, for example, as determined by the method described in Chinese Patent No. CN1566366A.

The positive signal of the chip can be determined by any suitable standard, for example, by the method described in Chinese Patent No. CN1566366A, or by the method in which the hybridization signal is greater than the sum of the average background noise and 3 times the standard deviation.

The invention is also applicable to copy number detection of a gene of interest. The number of nucleotide sequences of interest bound to the chip probe can be detected and correlated with the copy number of the gene of interest. The copy number of the gene of interest can be quantified based on the number of nucleotide sequences of the control gene containing the known copy number. Fluorescence energy is detected by photomultiplier CCD or laser scanning.

The detection chip for the genetic variation of the cytochrome P450 gene of the invention and the application thereof have the advantages of high throughput, parallelism and trace amount for gene screening compared with the conventional detection method, and can effectively classify CYP2C9, CYP2C19, CYP2D6. Genetic variation of CYP3A4, CYP3A5 and CYP3A7, enabling medical services In a short period of time, the personnel can grasp a large amount of genetic information of the patient, find a correct medication plan in time, and guide the patient to use the medicine rationally.

DRAWINGS

1 is a diagram showing the result of electrophoresis detection after PCR amplification of a single pair of primers according to Example 2 of the present invention; FIG. 2 is a diagram showing the results of electrophoresis detection after multiplex PCR amplification according to Example 2 of the present invention; Figure 2 is a diagram showing the results of electrophoresis detection after fragmentation of the PCR product; Figure 4 is a diagram showing the result of hybridization of the detection chip of Example 3 of the present invention;

Fig. 5 is a view showing the result of hybridization of the detecting chip of Example 4 of the present invention.

detailed description

The present invention will be further described in detail below with reference to the accompanying drawings and embodiments.

Example 1 Preparation of Gene Chip

Probe dissolution

Each probe was diluted with TE solution to a final concentration of 10 mM. Mix a probe with a concentration of 10 with a 200 mM PBS solution in a medium volume in a 384-well plate, seal the 384-well plate with a sticker, shake at room temperature for 2 minutes, centrifuge, and store at -20 °C. Use.

2. Spotting

The pre-designed and synthesized probes are loaded onto a solid-phase carrier substrate of a material such as a slide or a silicon wafer by contact spotting or ink jet spotting. The base is based on Cell Associates CSS-100 aldehyde base, GeneMachine's Ominigrid 100 model, and the humidity is 65 -75% (based on the FullMoon base). The temperature is 25 点. The format is 1 X 3, and each matrix is 8X 18. The layout of the probe is as shown in Table 1. After the sample is finished, after half an hour, the chip is taken out and stored at room temperature.

Table 1 Probe arrangement

Example 2 Processing and Labeling of Samples to be Tested

1. Human genome DNA extraction refers to amplification of the target gene

The genomic DNA in human peripheral blood was extracted using the FlexiGene DNA Kit (250) (QIAGEN, Cat. No. 51206) kit. The specific steps are as follows:

(1) Mixing ACD (8 g/L of decanoic acid, 22 g/L of sodium citrate, 22.5 g/L of glucose), and condensing the whole blood of the whole blood into a 15 ml centrifuge tube, and adding 2. 5 ral buffer FG1 , fully reverse the hook;

(2) Centrifuge at 2000g for 5 minutes, discard the supernatant. Allow to stand for 2 minutes;

(3) Add 500ul of buffer FG2/QIAGEN protease mixing reagent and mix immediately until the original precipitate disappears completely;

(4) Water bath at 65 °C for 10 minutes, 40 times / minute at constant speed;

(5) Add 500 ul of 100% isopropanol and mix well until visible D A flocculent precipitate appears;

(6) Centrifuge at 3000g for 5 minutes, discard the supernatant and invert for 2 minutes;

(7) Add 500ul of 70% ethanol and shake it;

(8) Centrifuge at 3000g for 5 minutes, discard the supernatant and invert for about 1 hour until there is no more water in the tube;

(9) Add 200 ul of buffer FG3 to dissolve the DNA, gently shake and dissolve overnight; (10) Transfer the extracted DNA into an autoclaved 1. 5ηύ centrifuge tube and take lul for electrophoresis (1% agarose gel, 0.5 XTBE, EB, 80MV, 1. 5 hours electrophoresis) Photographs were taken on the FR-200 UV and Visa Analyzer and quantified against the marker (Lambda DNA/EcoRI+Hindlll).

The primers for amplification of the CYP2D6, CYP2C19, CYP2C9 and CYP3A5 genes are the nucleotide strands of the sequences shown in SEQ ID NO: 248 to SEQ ID NO: 277. PCR amplification was carried out using a 30 μΐ reaction system, the reaction system was 0.3 mM dNTP, 10 mM Tris-HC1, 50 mM KC1, 2 mM MgC12, 20% Q solution (Qiagen), upstream and downstream primer concentration 0. 16幽, genomic DNA 10 ng, Taq enzyme 0. 6 U (Takara). Application of Touch-down PCR Reaction Program [Don RH, Cox PT, Wainwright BJ, Baker K, Mat tick JS. 'Touchdown' PCR to circumvent spurious priming during gene amplification. Nucleic Acids Res. 1991, 19 : 4008]: 94°C Denaturation for 5 min; denaturation at 94 °C for 40 s, annealing at 64 °C for 1 min, reduction of 0.5 °C for each cycle, 50 s for 72 s, for 10 cycles; then denaturation at 94 °C for 40 s, 59 Annealing at °C for 40 s, extension at 72 °C for 50 s for 30 cycles; finally extending at 72 °C for 5 min. After the end of PCR, the amplification results were detected with a 1.5% agarose gel. The results of the PCR products are shown in Figure 1. The names and lengths of the PCR products in Figure 1 are from left to right:

CYP2D6P1 is 1184 bp, CYP2C9P1 is 365 bp, CYPA5P3 is 567 bp, CYP3A5P2 is 639 bp, CYP3A5P1 is 690 bp, CYP2C19P2 is 400 bp, CYP2D6P3 is 671 bp, CYP3A5P4 is 393 bp, CYP3A5P5 is 510 bp, CYP3A5P7 is 668 bp, CYP2D6P4 is 517 bp, CYP2D6P2 is 1497 bp, CYP3A5P6 is 473 bp. CYP2C19P1 is 1732b and CYP2C9P2 is 914 bp, which is consistent with the theoretical value.

When the multiplex PCR reaction is carried out, primers of the sequences shown in SEQ ID NO: 248 to SEQ ID NO: 277 are placed in a reaction system for amplification, and the system is 50 μl. The multiplex PCR reaction system is: each dNTP 0. 3 μπιοΙ/L, Tricine - K0H (pH 8.7) 40 mmol/L, C1 16 mmol/L, MgCl ₂ 3. 5 ramol/L, BSA 3. 75 4g /ml, 2Wnol/L per primer, DNA 80ng and 2. 2 X Titanium Taq DNA polymerase (Clontech Laboratories Inc., USA). Multiplex PCR reaction conditions: %. C denaturation 3 min; 95 Ό denaturation 30 s, 66 ° C annealing 2. 5 min, 68 ° C extension 4 min, a total of 40 cycles; the last 68 Ό extended 10 min ₀ PCR amplification, take 3 μΐ PCR reaction product for agarose Gel electrophoresis, the electrophoresis results are shown in Figure 2. These PCR products are processed and used in the following hybridization steps.

2. PCR product purification and fragmentation All PCR products of each sample were mixed and purified using QIAquick PGR Purification Kit (Qiagen, Cat. No. 28106). The specific steps are as follows:

(1) Add 5 times the volume of the PCR product buffer PB, mix the hook, no need to remove paraffin oil or kerosene;

(2) Place the QIAquick spin column in the 2 ml collection tube provided by the kit;

(3) Add the sample to a QIAquick spin column and centrifuge for 30-60 s to bind the DNA;

(4) Pour 2 ml of the centrifuged liquid from the collection tube and place the QIAquick spin column on the original collection tube.

(5) Add 0. 75 ml buffer PE to the QIAquick spin column for washing, centrifugation 30- 60

S ;

(6) Pour 2 ml of the centrifuged liquid from the collection tube, place the QIAquick spin column into the original collection tube, and centrifuge for 1 min;

(7) Place the QIAquick spin column in an autoclaved 1. 5 ml centrifuge tube;

(8) Add 50 ΐ buffer ΕΒ (10 mM Tris-Cl, pH 8.5) or 0 to the center of the QIAquick membrane to remove DNA and centrifuge for 1 min. To increase the DNA concentration, add only 30 in the center of the QIAquick membrane. The μΐ elution buffer was allowed to stand for 1 min and then centrifuged.

The purified PCR was assayed for concentration and fragmented with DNase I. The fragmented reaction system consists of: Purification of PCR product 10 4g (30 μΐ)

10 X DNase I Buffer 4 μΐ

40 μι system

DNase I 0. 12 μΐ

, ddH ₂ 0 5. 88 μΐ

The reaction conditions were a 37 ° C warm bath for 5 min, then 95 ° C for 15 min. The fragmented product was run on a 5% agarose gel, and the electrophoresis results are shown in Figure 3. .

3. Fluorescein labeling

Fluorescein labeling was performed at the 3' end using a deoxynucleotidyl transferase. The labeled reaction system included: Fragmentation PCR product 25 μΐ

5 X deoxynucleotidyl transferase buffer 8 μΐ

40 ΐ system CY3-N6-ddCTP ( 1 raM) 1 μΐ

Deoxynucleotidyl transferase (20 U ) 3 μΐ

ddH ₂ 0 3 μΐ The reaction conditions were a 37 ° C warm bath for 120 min and then heated at 95 ° C for 15 min.

Example 3 Hybridization, washing and results detection

The fluorescently labeled PCR product was denatured at 95 ° C for 10 min and immediately placed on ice for hybridization. The hybrid reaction system included:

f fluorescein-labeled PCR product is μΐ

20 X SSPE 1. 2 μΐ

1% Triton 0. 2 μΐ

20 μΐ system

10 X Denhandts 0. 9 μΐ

Formamide 0. 5 μΐ

Dd3⁄40 2. 2 μΐ

The reaction conditions were 48 ° C for 120 min, followed by IX Wash Buffer I (5 X SSC, 0.1% SDS), IX Wash Buffer II (2 X SSC, 0.1% SDS) and IX Wash Buffer. 5分钟。 The hydrazine ( IX SSC) was washed at 42 ° C for 10 min, and finally washed with ddH20 0. 5 min.

The washed chip, after drying, is scanned with a GenePix 4000B confocal laser scanner (other laser scanners can also be used). The hybridization results of the scanned hybridization chip are shown in Fig. 4. The image is processed by GenePix Pro, and then the data file is analyzed to obtain the genotype of the target gene. The typing result shown in Fig. 4 is CYP2D6*10, CYP2C19*1, CYP2C9*1 and CYP3A5*3.

Example 4 Preparation and Application of CYP3A Gene Chip

1. Preparation of gene chip is the same as in embodiment 1

After the pre-designed and synthesized probe is dissolved, it is carried on a solid-phase carrier substrate of a material such as a slide glass or a silicon wafer by contact spotting or ink jet spotting. The layout of the probe spotting is shown in Table 2. After the spotting is completed, after placing it for half an hour, the chip is taken out and stored at room temperature. Table 2 Probe arrangement

2. Processing and marking of the sample to be tested

(1) The procedure of human genome DNA extraction is the same as in Example 2.

(2) Amplification of the target gene

The primers for amplification of the CYP3A4, CYP3A5 and CYP3A7 genes are the nucleotide strands of the sequences shown in SEQ ID NO: 278 to SEQ ID NO: 311. PCR amplification was carried out using a 30 μΐ reaction system, the reaction system was 0.3 mM dNTP, 10 raM Tris-HC1, 50 mM KC1, 2 mM MgC12, 20% Q solution (Qiagen), upstream and downstream primer concentration 0. 16 M, genomic DNA lOng, Taq enzyme 0·6 U (Takara). Application of Touch-down PCR Reaction Program [Don RH, Cox PT, Wainwright BJ, Baker K, Mattick JS. 'Touchdown' PCR to circumvent spurious priming during gene amplification. Nucleic Acids Res. 1991, 19 : 4008]: 94 °C denaturation 5 min; denaturation at 94 °C for 40 s, annealing at 64 °C for 1 min, each cycle decreased by 0.5 °C, 72 °C for 50 s for 10 cycles; then denaturation at 94 °C for 40 s, 59 °C Annealing for 40 s, extending at 72 °C for 50 s for 30 cycles; finally extending at 72 °C for 5 min. After the end of PCR, the amplification results were measured using a 1.5% agarose gel.

When the multiplex PCR reaction is carried out, the primers of the sequences shown in SEQ ID NO: 278 to SEQ ID NO: 311 are placed in a reaction system for amplification, and the system is 50 μl. The multiplex PCR reaction system was: each dNTP 0. 3 Mmol/L, Tricine-K0H (pH 7.8) 40 mmol/L, KC1 16 leg ol/L, MgCl ₂ 3. 5 mmol/L, BSA 3. 75 μ _§ /ιη1 , 2Mmol/L per primer, 80 ng DNA and 2. 2 X Titanium Taq DNA polymerase (Clontech Laboratories Inc., USA). Multiplex PCR reaction conditions: 95 °C change Sex 3 rain; denaturation at 95 °C for 30 s, annealing at 66 °C for 2.5 min, extension at 68 °C for 4 min, a total of 40 cycles; and finally extending at 68 °C for 10 min. After PCR amplification, 3 μΐ PCR reaction product was taken for agarose gel electrophoresis. These PCR products are processed and used in the following hybridization steps.

(3) Purification and fragmentation of PCR products

All PCR products of each sample were mixed and purified using QIAquick PCR Purification Kit CQiagen, Cat. No. 28106), and the specific procedure was the same as in Example 2.

The purified PCR was assayed for concentration and fragmented with DNase I. The fragmented reaction system consisted of: 30 ΐ purified PCR product (10 μg), 4 μΐ 10X DNase I buffer, 0.12 μΐ DNase I, 5.88 ΐ dd 0. The reaction conditions were a 37 ° C warm bath for 5 min, then 95 ° C for 15 min. The fragmented product runs on a 4% agarose gel, ensuring that the vast majority of the fragments are between 30 and 200 base pairs.

(4) Fluorescein labeling

Fluorescein labeling at the 3' end using deoxynucleotidyl transferase, the labeled 40 μΐ reaction system consists of: 25 μΐ fragmented PCR product, 8 μΐ 5 X deoxynucleotidyl transferase buffer, 1 μΐ of CY3 - Ν6- ddCTP (1 mM), 3 μΐ of deoxynucleotidyl transferase (20 UM), 3 μΐ of dd 0. The reaction conditions were a 37 ° C warm bath for 120 min and then heated at 95 ° C for 15 min.

3. Hybridization, washing and results detection

The fluorescently-labeled PCR product was denatured for 10 min and immediately placed on ice for hybridization. The hybridization reaction 20 l system included: fluorescein-labeled PCR product 15 μΐ, 20 X SSPE 1.2 μΐ, 1% Triton 0.2 μΐ, 10 X Denhandts 0.9 μ1, formamide 0.5 μ1, dd3⁄40 2.2 μ1. The reaction conditions were 48 ° C warm bath for 120 min, followed by 1 X Wash Buffer I (5X SSC, 0.1% SDS), IX Wash Buffer II (2X SSC, 0.1% SDS) and IX Wash Buffer ΠΙ (IX SSC) Each was washed at 42 ° C for 10 min and finally washed with ddH20 for 0.5 min.

The washed chip was scanned with a GenePix 4000B confocal laser scanner (other laser scanners were also available). The hybridization results obtained by scanning the hybridized chip are shown in Fig. 5. The image is obtained by processing the image with GenePix Pro, and then the data file is analyzed to obtain the genotype of the target gene, and the result of the typing is CYP3A4, CYP3A5 and CYP3A7.

Claims

Rights request

A detection chip for genetic variation of a cytochrome P450 gene, comprising a solid phase carrier and a probe, characterized in that the probe hybridizes with a nucleotide sequence of a cytochrome P450 gene to be tested and/or a complement thereof.

The test chip according to claim 1, wherein the cytochrome P450 gene to be tested comprises CYP2D6, CYP2C19, CYP2C9, CYP3A4, CYP3A5 and CYP3A7 genes.

The test chip according to claim 1 or 2, wherein the probe is DNA, RNA, DNA-RNA chimera, PNA or a derivative thereof.

4. The detecting chip according to claim 3, wherein the probe is a DM, comprising:

(1) (a) the sequence shown in SEQ ID NO: 1 to SEQ ID NO: 76, and (b) each sequence in the sequence shown in SEQ ID NO: 1 to SEQ ID NO: 76, which hybridizes to the CYP2D6 gene to be tested. a complementary strand, (c) a sequence having at least 70% homology to each of the sequences set forth in SEQ ID NO: 1 to SEQ ID NO: 76;

(2) (a) the sequence shown in SEQ ID NO: 77 to SEQ ID NO: 94, (b) each of the sequences shown in SEQ ID NO: 77 to SEQ ID NO: 94, which hybridizes to the CYP2C19 gene to be tested. a complementary strand, (c) a sequence having at least 70% homology to each of the sequences set forth in SEQ ID NO: 77 to SEQ ID NO: 94;

(3) (a) the sequence shown in SEQ ID NO: 95 to SEQ ID NO: 112, (b) the sequence of each of the sequences shown in SEQ ID NO: 95 to SEQ ID NO: 112, which hybridizes to the CYP2C9 gene to be tested. a complementary strand, (c) a sequence having at least 70% homology to each of the sequences set forth in SEQ ID NO: 95 to SEQ ID NO: 112;

(4) (a) the sequence of SEQ ID NO: 113 to SEQ ID NO: 142 which hybridizes with the CYP3A4 gene to be tested, and (b) each sequence of the sequence of SEQ ID NO: 113 to SEQ ID NO: 142 a complementary strand, (c) a sequence having at least 70% homology to each of the sequences set forth in SEQ ID NO: 113 to SEQ ID NO: 142;

(5) (a) the sequence of SEQ ID NO: 143 to SEQ ID NO: 201, (b) the sequence of each of the sequences shown in SEQ ID NO: 143 to SEQ ID NO: 201, which hybridizes to the CYP3A5 gene to be tested. a complementary strand, (c) a sequence having at least 70% homology to each of the sequences set forth in SEQ ID NO: 143 to SEQ ID NO: 201;

(6) (a) the sequence shown in SEQ ID NO: 202 to SEQ ID NO: 247, (b) each of the sequences shown in SEQ ID NO: 202 to SEQ ID NO: 247, which hybridizes to the CYP3A7 gene to be tested. Complementary strand, (c) a sequence with at least 70% homology to each of the sequences set forth in SEQ ID NO: 202 to SEQ ID NO: 247 Column.

The detection chip according to claim 4, wherein the probe is selected from the group consisting of: SEQ ID NO: 1 to SEQ ID NO: 247.

6. The test chip according to claim 1, further comprising at least one control probe selected from the group consisting of: a negative control probe, a positive control probe, a hybridization control probe, and a fixation Control probe.

The detecting chip according to claim 1 or 6, wherein the probe is provided with a connecting arm, and the probe can be modified.

The detecting chip according to claim 1 or 6, wherein the probe is labeled, and the labeling comprises: a fluorescein label, a biotin label, an enzyme label, a radioactive element label or a fluorescence resonance energy. Transfer tag.

9. The test chip according to claim 1, wherein the solid phase carrier material comprises one of a glass sheet, a silicon wafer, a nitrocellulose membrane, a nylon membrane, and a ruthenium molecular material, or any combination thereof.

10. A method for detecting a genetic variation of a cytochrome P450 gene using the chip of claim 1, comprising the steps of:

(1) extracting the nucleic acid of the cytochrome P450 gene to be tested from a suitable sample;

(3) labeling the nucleotide sequence of interest of step (2);

(5) The result of detecting the hybridization reaction.

The method according to claim 10, wherein the nucleotide sequence of interest in the step (2) is single-stranded or double-stranded DMA or RNA, preferably single-stranded DNA or RNA.

12. The method according to claim 10, wherein the step (2) of preparing the nucleotide sequence of interest comprises a PCR amplification reaction, and the primer used in the reaction comprises (a) SEQ ID NO: 248 to SEQ ID. The nucleotide chain of the sequence of N0:311, (b) the complementary strand of each sequence in the sequence of SEQ ID NO: 248 to SEQ ID NO: 311, (c) and SEQ ID NO: 248 to SEQ ID N0 A sequence of at least 70% homology to each of the sequences shown in 311.

13. The method according to claim 12, wherein the primer sequence is SEQ ID Nucleotide chain of the sequence of N0:248~SEQ ID NO:311.

14. The method according to claim 10, wherein the labeling of the nucleotide sequence of interest of step (3) comprises using a fluorescein label, a biotin label, a radioactive element label or an enzyme label.

The method according to claim 10, wherein the hybridization temperature in the step (4) is from 25 ° C to 65 ° C, and the hybridization time is from 5 minutes to 18 hours.