RU2716589C1 - Method of determining polymorphic markers in cyp2c19 and cyp2d6 genes for determining individual sensitivity to antidepressants - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to the field of biotechnology. Functional activity of cytochrome CYP2C19 and CYP2D6 cytoplasm are largely due to polymorphism of their coding genes. Presence of nucleotide substitutions in certain loci leads to formation of different (from the point of view of metabolic rate) phenotypes of these enzymes (normal, slow, intermediate, fast). Thus, it is possible to predict the phenotype based on the genotype. Genetic testing of CYP2C19 and CYP2D6 in prescribing selective serotonin reuptake inhibitors (SSRIs) and tricyclic antidepressants (TCAs) is included in the Recommendations of the Consortium of Clinical Pharmacogenetics Implementation (CPIC, USA) (Hicks J, Sangkuhl K, Swen J et al; Clinical pharmacogenetics implementation consortium guideline (CPIC) for CYP2D6 and CYP2C19 genotypes and dosing of tricyclic antidepressants: 2016 update. Clin Pharmacol Ther. 2017 Jul; 102(1):37-44, Hicks JK, Bishop JR, Sangkuhl K et al; Clinical Pharmacogenetics Implementation Consortium (CPIC) Guideline for CYP2D6 and CYP2C19 Genotypes and Dosing of Selective Serotonin Reuptake Inhibitors, Clin Pharmacol Ther. 2015 Aug; 98(2): 127-34). Given method enables detecting the following polymorphic markers in the genes: CYP2C19 *1/*2/*3/*17 (rs4244285, rs4986893, rs12248560) and CYP2D6 *1/*3/*4/*10/*41 (rs35742686, rs3892097, rs1065852, rs28371725). Determination of gene polymorphism enables to select the preparation and its dose effectively when prescribing antidepressant therapy.

EFFECT: invention makes it possible to perform analysis in short term and with low material costs.

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Description

FIELD OF THE INVENTION

The invention relates to the field of pharmacogenetics and personalized medicine, and relates to a method for determining genetic markers that determine individual sensitivity to antidepressants (SSRIs and TCAs) using the polymerase chain reaction (PCR) and subsequent hybridization with an oligonucleotide biological microchip (biochip).

The use of pharmacogenetic testing using the present invention will allow you to individually select an antidepressant and its dose, reduce the risk of unwanted drug reactions and prescribe an ineffective drug.

State of the art

Modern methods for determining gene polymorphism are usually based on PCR with various variations, while the design of reactions and methods for detecting results vary significantly and depend on specific tasks.

Currently, there are a number of methods for the analysis of polymorphic markers in the CYP2C19 and CYP2D6 genes (various alleles of these genes). The spectrum of the analyzed markers varies somewhat.

B1,

K, Bachofer J, Steimer W. Optimized strategy for rapid cytochrome P450 2D6 genotyping by real-time long PCR. Clin Chem. 2003 Oct; 49(10): 1624-31].Allele-specific PCR followed by gel electrophoresis is an inexpensive method that does not require highly qualified personnel. A significant drawback of this method is the formulation of a large number of parallel reactions (for each polymorphic locus at least 2 reactions per sample) and their separation using gel electrophoresis, which significantly reduces the value of the method in routine clinical diagnostics [Orita M., Iwahana N., Kanazawa N., Sekya T. Detection of polymorphism of human DNA by gel electrophoresis as single cell conformation polymorphism. Proc. Natl. Acad Sci. 1989. 86: 2766-2770. Gaudet M, Fara AG, Beritognolo I, Sabatti M. Allele-specific PCR in SNP genotyping. Methods Mol Biol. 2009; 578: 415-24,

B1,

K, Bachofer J, Steimer W. Optimized strategy for rapid cytochrome P450 2D6 genotyping by real-time long PCR. Clin Chem. 2003 Oct; 49 (10): 1624-31].

PCR followed by Sanger sequencing is rarely used in routine practice, mainly used to verify analyzes. The method is based on conducting termination reactions (with the addition of terminating 2 ', 3'-dideoxynucleoside triphosphates) and separation of the products of these reactions using capillary electrophoresis. The method is accurate, informative, allows you to determine the complete nucleotide sequence of the studied fragment, allows you to detect de novo mutations. However, it has a number of significant drawbacks: a separate amplification reaction is required for each analyzed locus, the duration and complexity of the analysis, the use of expensive equipment and the presence of highly qualified personnel [Xiong Y, Wang M, Fang K, Xing Q, Feng G, Shen L, He L, Qin S. A systematic genetic polymorphism analysis of the CYP2C9 gene in four different geographical Han populations in mainland China. Genomics 2011 May; 97 (5): 277-81].

Allele-specific restriction analysis is based on PCR followed by a restriction reaction, during which, using restriction enzymes, specific DNA cleavage is carried out at specific sites. Analysis of restriction products is carried out by electrophoresis. Moreover, for the analysis of each mutation, a separate PCR followed by a restriction reaction is required, which makes this method time-consuming and increases the probability of error. However, this method does not require highly qualified personnel and expensive equipment [Ota M, Fukushima H, Kulski JK, Inoko H. Single nucleotide polymorphism detection by polymerase chain reaction-restriction fragment length polymorphism. Nat Protoc. 2007; 2 (11): 2857-64. Yuce-Artun N, Baskak B, Ozel-Kizil ET, Ozdemir H, Uckun Z, Devrimci-Ozguven H, Suzen HS. Influence of CYP2B6 and CYP2C19 polymorphisms on sertraline metabolism in major depression patients. Int J Clin Pharm. 2016 Apr; 38 (2): 388-94.].

Various methods based on real-time PCR (RT-PCR) are widely used to analyze genetic polymorphism. Based on this approach, a large number of test systems are being developed. For RT-PCR, a mixture containing an intercalating fluorescent dye SYBRGreen / EVAGreen, which interacts with double-stranded DNA, or an allele-specific TaqMan oligonucleotide carrying both a fluorescent label and a quencher [Fudio S, Borobia AM, Pinana E, Ramirez, is used Tabares B, Guerra P, Carcas A, Frias J. Evaluation of the influence of sex and CYP2C19 and CYP2D6 polymorphisms in the disposition of citalopram. Eur J Pharmacol. 2010 Jan 25; 626 (2-3): 200-4.]. The disadvantage of this method is the need to put one or two PCR reactions at each analyzed locus.

Analysis of high resolution melting curves (HRM analysis) is a fairly simple method for screening mutations. However, for the analysis of some sequences (including regions of genes with a high degree of homology), this method is not suitable. In addition, HRM analysis can only be performed on a device with a special channel for HRM and special software, which requires equipping the research laboratory with expensive equipment. [Chang YS, Lee SS, Liu TY, Chen YC, Lu HC, Chang JG. Direct assessment of cytochrome P450 2D6 genotypes by high-resolution melting analysis and DNA sequencing. Environ Toxicol Pharmacol. 2014; 38 (3): 821-828. Ghasemi Z, Hashemi M, Ejabati M, et al. Development of a high-resolution melting analysis method for CYP2C19 * 17 genotyping in healthy volunteers. Avicenna J Med Biotechnol. 2016; 8 (4): 193-199.].

All of the above methods involve setting individual reactions for each analyzed locus. To solve a number of problems, it makes sense to form multiplex panels that allow simultaneous analysis of several polymorphic markers. Such test systems include various hybridization options with biochips. For example, the AmpliChip CYP450 test system created by Roche based on Affymetrix microchip technology was approved by the US Food and Drug Administration and is used to individually select psychotropic drugs and their dosages. This test reveals up to 33 polymorphisms and mutations of the CYP2D6 gene and 3 polymorphisms of the CYP2C19 gene, however, this method is expensive and requires complex sample preparation [Jain K.K. Applications of AmpliChip CYP450. Mol Diagn. 2005; 9 (3): 119-27. M.C. Rebsamen, J Desmeules, Y Daali, A Chiappe, A Diemand, C Rey, J Chabert, P Dayer, D Hochstrasser and M F Rossier. The AmpliChip CYP450 test: cytochrome P450 2D6 genotype assessment and phenotype prediction. The Pharmacogenomics Journal (2009) 9, 34-41].

High volumes of data for the analysis of polymorphic markers are provided by high-performance sequencing technologies, which make it possible to obtain genome or exome sequences or panels of specific genes. However, this method is expensive, time-consuming, time-consuming and requires verification of the results. Interpretation of such a volume of data for clinical practice is also difficult. This approach is mainly used in research when searching for new markers [Fabbri C, Tansey KE, Perlis RH, Hauser J, Henigsberg N, Maier W, Mors O, Placentino A, Rietschel M, Souery D, Breen G, Curtis C, Lee SH , Newhouse S, Patel H, O'Donovan M, Lewis G, Jenkins G, Weinshilboum RM, Farmer A, Aitchison KJ, Craig I, McGuffin P, Schruers K, Biernacka JM, Uher R, Lewis CM. Effect of cytochrome CYP2C19 metabolizing activity on antidepressant response and side effects: Meta-analysis of data from genome-wide association studies. Eur Neuropsychopharmacol. 2018 Aug; 28 (8): 945-954.].

A large number of test systems have been patented for the analysis of polymorphic genetic markers to determine individual sensitivity to antidepressants, including those that allow the analysis of genetic variants of the CYP2C19 and CYP2D6 genes.

Many patents provide methods for analyzing the polymorphism of one of the genes (CYP2C19 or CYP2D6). In Chinese patent CN104946759 A from 2015-09-30, a method is described for determining the single nucleotide polymorphism of the CYP2C19 -806 C> T * 17 gene by SNaPshot PCR. In Chinese patent CN 106048076 A from 2016-10-26, the definition of 2 CYP2C19 polymorphisms (* 2 and * 3) is described by real-time allele-specific PCR. Chinese patent CN 101565749 B dated 2012-01-11 describes a method for determining the polymorphisms CYP2C19 681 G> A * 2, CYP2C19 636 G> A * 3 on a liquid microchip. US20040091909A1 2002-07-05 describes a method for determining allelic variants of CYP2D6 (* 2, * 3, * 4, * 5, * b, * 7, * 8, * 9, * 10, * 11, * 12, * 13, * 14, * 15, * 16, * 17, * 1 × 2, * 2 × 2, 4 × 2 and * Nx2). US patent US 20050196771 A1 from 2002-04-11 describes a method for detecting 28 nucleotide substitutions and changing the number of copies of the CYP2D6 gene. French patent application WO 2011092596 A2 of 2011-08-04 describes the definition of allelic variants of CYP2D6 * 3, * 4, * 5, * 6, * 7, * 8, * 11, * 12, * 19, * 40.

A number of methods have also been patented that allow analyzing the polymorphism of the CYP2C19 and CYP2D6 genes: Japanese patent JP2004537989A from 2001-06-11 involves the analysis of liver cells (hepatocytes), which is associated with technical difficulties, the method described in US patent US 20060229824 A1 from 2006- 03-03 is expensive and requires complex sample preparation.

Recently, low-density gel microchips are widely used for basic scientific research, as well as in medical diagnostics, due to their low cost and the ability to create relevant panels for certain tasks. The method consists in the amplification of DNA fragments and the subsequent hybridization of a fluorescently labeled target DNA with allele-specific oligonucleotides in three-dimensional gel cells of the microchip. [Gel-based microarrays in clinical diagnostics in Russia. Gryadunov D, Dementieva E, Mikhailovich V, Nasedkina T, Rubina A, Sawateeva E, Fesenko E, Chudinov A, Zimenkov D, Kolchinsky A, Zasedatelev A. Expert Rev Mol Diagn. 2011 Nov; 11 (8): 839-53].

Disclosure of the invention

The objective of the present invention is to provide a method for determining genetic markers that cause individual sensitivity to antidepressants (SSRIs and TCAs). The marker panel includes the most informative and frequently encountered genetic markers, the determination of which allows individualizing therapy with SSRI and TCA groups.

The essence of the invention is to provide a method for the analysis of single nucleotide polymorphism (SNP, Single nucleotide polymorphism) of the genes CYP2C19 and CYP2D6, the products of which are involved in the metabolism of SSRIs and TCAs. This method allows you to simultaneously identify the following polymorphic markers in the genes: CYP2C19 * 1 / * 2 / * 3 / * 17 (rs4244285, rs4986893, rs12248560) and CYP2D6 * 1 / * 3 / * 4 / * 10 / * 41 (rs35742686, rs3892097 , rs1065852, rs28371725).

The main features of this invention are multiplex single-stage PCR followed by hybridization of PCR products on a biochip containing a set of immobilized oligonucleotide probes.

The first important feature of the invention is a set of primers for amplification of the analyzed loci, which is used to obtain fluorescently-labeled products in the required amount for hybridization on a biochip. One primer from a pair of primers for each locus consists of a part complementary to the gene region (at the 3'-end) and a universal sequence (at the 5'-end) corresponding to the universal primer. The primer sequences for PCR are given in Sequence Listing 1. (SEQ ID NO: 1-15).

The second important feature of the invention is a biochip containing a set of immobilized oligonucleotide probes, the sequences of which are shown in Sequence Listing 2. (SEQ ID NO: 16-29). Oligonucleotide probes are immobilized in the cells of the hydrogel microchip, as described in patent RU 2206575 (publication date 2003-06-20) at a concentration of 2000 μM. The arrangement of the probes in the cells of the biochip is shown in FIG. 1.

A set of PCR primers is used to carry out one-step PCR to obtain a fluorescently-labeled product in two rounds for hybridization on a biochip. Next, hybridization of fluorescently-labeled DNA fragments obtained after PCR is performed with oligonucleotide probes immobilized in the biochip cells located on a plastic substrate. After hybridization and washing of the biochip, the obtained fluorescence pattern is analyzed, based on which a report is made on the genotype of the test sample. An example of a hybridization pattern is shown in FIG. 2.

Brief description of tables and figures

Figure 1. The layout of the probes in the cells of the biochip for the analysis of polymorphic markers in the genes CYP2C19 and CYP2D6 to determine individual sensitivity to antidepressants, (a) the location of the cells on the biochip; (b) the interpretation of the symbols. (M) - cells containing the fluorescent dye Su5, permanently glowing, for orientation and control of the intensity of the glow.

Figure 2. Hybridization pattern obtained using a biochip for the analysis of polymorphic markers in the CYP2C19 and CYP2D6 genes to determine individual sensitivity to antidepressants (patient No. 1).

The implementation of the invention

To ensure optimal conditions for multiplex PCR, it is necessary to use primers that provide specific and effective annealing on the target at the same temperature (for one stage), and they should not form secondary structures at this temperature, including under multiplex PCR conditions. For optimal (stable and specific) production of the amount of fluorescently-labeled PCR product necessary for hybridization on a biochip, such parameters as the concentration of reagents in the reaction mixture and the temperature-time mode of reactions should be selected. As a result of the work, primers and conditions for PCR were selected that allow efficient production of the studied DNA loci under multiplex PCR conditions.

For immobilization on a biochip, oligonucleotides are selected that allow identification of all selected gene regions for analysis.

Oligonucleotide probes should be selected in accordance with the following criteria:

1) The oligonucleotide probe must have high specificity for the analyzed locus.

2) It is preferable that the variable nucleotide is located in the middle region of the probe, since such a probe design allows better discrimination between perfect and imperfect duplexes.

3) The oligonucleotide probe should not form stable secondary structures under the conditions under which hybridization is carried out, since the presence of secondary structures can lead to a decrease in the efficiency of hybridization.

On a biochip, for the analysis of polymorphic markers in the CYP2C19 and CYP2D6 genes to determine individual sensitivity to antidepressants, 14 highly specific differentiating oligonucleotide probes were immobilized (Sequence Listing 2. (SEQ ID NO: 16-29)), the structure of which provides highly specific binding to fully complement DNA targets. A bright fluorescent signal is observed only in cells in which, during hybridization, a perfect duplex was formed between the oligonucleotide probe and the fluorescently-labeled PCR product.

Here is the sequence of analysis using this method. Amplification of PCR products for subsequent hybridization on a biochip is carried out by multiplex one-stage PCR, and a DNA sample obtained from a patient is used as a matrix for the reaction.

PCR can be performed using any type of thermostable polymerase (Taq polymerase, Taq polymerase with hot start, Tth polymerase, Tfl polymerase, Pfu polymerase, Vent polymerase, DeepVent polymerase and other commercially available enzymes isolated from thermophiles ) running in the appropriate buffer. To build a new chain, a mixture of dNTPs (dATP, dGTF, dCTP, dTTP) is added to the buffer. For PCR, ready-made commercially available kits containing all the necessary components except primers can also be used.

During multiplex single-stage PCR, mainly single-stranded products are produced (by adding an excess of universal primer), and with its fluorescent labeling using dUTP-Su5, which can be incorporated into a growing DNA chain. Any fluorochrome without restriction (for example, FITC, Texas red, Su-3, etc.), as well as biotin, can also be used as a fluorescent label.

The synthesis of primers and oligonucleotide probes is carried out using such chemical approaches as the phosphodiester method, hydrophosphoryl method, etc. For the synthesis of primers, automatic DNA / RNA synthesizers, for example, manufactured by Applied Biosystems (USA), are used. To immobilize the biochip in the hydrogel cells, an active group is added to the 5'- or 3'-end of the oligonucleotide probes (for example, a free amino group can be introduced at the 3'-end using the 3'-Amino-Modifier C7 CPG 500, Glen Research) ), USA).

For PCR use primers SEQ ID NO: 1-14 at a concentration of 1 picomol / μl, and universal primer SEQ ID NO: 15 at a concentration of 50 picomol / μl. The primer sequences are given in Sequence Listing 1.

Next, hybridization of fluorescently labeled DNA fragments obtained by PCR is performed with oligonucleotide probes immobilized in gel cells, the sequences of which are regions complementary to the sequences of the major or minor allele at the studied loci.

Hybridization of the PCR product with oligonucleotide probes on a biochip can be carried out in any hybridization buffer, for example, in a SSPE buffer with formamide or with guanidine. Before hybridization, the PCR product is heated at 95 ° C for 5 minutes, then cooled on ice for 2 minutes, after which the hybridization mixture is applied to the biochip. Hybridization is carried out for 12-14 hours at 37 ° C. Subsequent washing of the biochip can be carried out in any buffer known in the art with the addition of salt (SSC, SSPE, etc.) or in distilled water.

The analyzed DNA fragment under conditions (reaction composition, temperature and hybridization time) under which hybridization is carried out forms perfect duplexes only with oligonucleotide probes fully complementary to it. The fluorescence signal is detected only in those cells in which a perfect duplex was formed. If the duplex is imperfect (at least one unmatched nucleotide is present), then there is no fluorescence signal. The fluorescence signal is visualized, for example, using a portable biochip analyzer equipped with a CCD camera and special software (BIOCHIP-IMB LLC, Moscow, Russia).

Biochips are fabricated by sequentially applying acrylamide gel cells to the glass substrate surface, activating the cells and immobilizing modified oligonucleotides in the cells [Analysis of SNPs and other senomic variations using gel-based chips / A. Kolchinsky, A. Mirzabekov // Hum Mutat. - 2002. - Vol. 19. - P. 343-360. Review]. As the substrate, in addition to glass, metals, flexible membranes and plastic can be used (Patent RU 2309959, published 2007-11-10). Biochips can also be made by any other known methods [Arrays of immobilized oligonucleotides-contributions to nucleic acids technology / H. Seliger, M. Hinz, E. Happ // Curr Pharm Biotechnol. - 2003. - Vol. 4. - P. 379-395].

For the manufacture of the biochip, the present invention uses the set of oligonucleotides SEQ ID NO: 16-29 shown in the sequence List 2. The location of specific oligonucleotide probes on the biochip can vary depending on the ease of interpretation of the results.

The following are examples illustrating the possibilities of using a method for the analysis of polymorphic markers in the CYP2C19 and CYP2D6 genes to determine individual sensitivity to antidepressants. Variants and modifications of the invention, which can be implemented without departing from the general concept of the present invention and without involving one's own inventive activity, will also be included in the scope of the claims of the present invention.

Example 1. Amplification of sections of the CYP2C19 and CYP2D6 genes in order to obtain the fluorescently labeled PCR product in the required amount.

DNA was isolated from the patient’s blood using the QIAamp DNA Blood Mini Kit (Qiagen, USA).

The accumulation of sections of the analyzed genes was carried out by the method of single-stage multiplex PCR. PCR was performed in one reaction for each sample. The PCR mixture with a total volume of 25 μl included: 0.5 units. Act. Taq polymerases (SibEnzyme, Russia), PCR buffer (70 mM Tris-HCl (pH 8.3), 16.6 mM (NH ₄ ) SO ₄ , 2.5 mM MgCl ₂ , 200 μM each of dNTPs (SibEnzyme, Russia ), 10 ng DNA, 1 pmol of each primer SEQ ID NO: 1-14, 50 pmol of the universal primer SEQ ID NO: 15, 8 μM fluorescently labeled DUTP-Su5, Amplification was carried out according to the following scheme: 40 cycles (94 ° C 30 s, 65 ° C 30 s, 67 ° C 30 s, 69 ° C 30 s, 72 ° C 20 s), then 40 cycles (94 ° C 30 s, 56 ° C 10 s, 72 ° C 30 s), for T100 device (Bio-Rad, USA).

Example 2. Oligonucleotide biochip for the analysis of polymorphic markers in the genes CYP2C19 and CYP2D6.

Microarray oligonucleotide probes were synthesized on a 394 DNA / RNA Synthesizer (Applied Biosystems, USA) using a standard phosphoamidite procedure. At the 3'-end of the oligonucleotide probe there is a free amino group spacer, which was introduced into the oligonucleotide using the 3'-Amino-Modifier C7 CPG 500 method (Glen Research), USA).

The biochip was prepared by the copolymerization of an oligonucleotide in an acrylamide gel (patent RU 2309959, published 2007-11-10 and RU 2175972, published 2001-11-20). The biochip contains 14 immobilized oligonucleotide probes (SEQ ID NO: 16-29), the list of which is presented in the List of sequences 2. Cells are applied according to the scheme in FIG. 1. Example 3. Hybridization of a labeled product on a biochip

The reaction mixture obtained after the second PCR step described in Example 1 was used for hybridization on a biochip: 10 μl of formamide (Serva, USA), 10 μl of 20 × SSPE (Promega, USA) and 10 μl of amplification. The hybridization mixture was denatured at 95 ° C for 5 minutes, cooled in ice for 2 minutes and applied to a biochip, after which it was left for 12 hours at 37 ° C. After hybridization, the biochip was washed in 1 × SSPE for 10 minutes at room temperature.

Example 4. Registration and interpretation of hybridization results

The hybridization pattern was recorded using a portable biochip analyzer equipped with a CCD camera manufactured by Biochip-IMB LLC.

The genotype of the test sample was determined by the hybridization pattern shown in FIG. 2.

Sample genotype:

CYP2C19_ * 2_G / A

CYP2C19_ * 3_G / G

CYP2C19_ * 17_C / C

CYP2D6 * 3_A / A

CYP2D6 * 4_G / G

CYP2D6 * 10_G / G

CYP2D6 * 41_C / T

List of sequences 1. The sequence of the primers for PCR

SEQ ID NO: Title Sequence 5 ’-> 3’ 1 CYP2C19_ * 2_F CCAGAGCTTGGCATATTGTATCTATACC 2 CYP2C19_ * 2_R TCATTGGATCTCATTACCGAGGGTTGTTGATGTCCATCG 3 CYP2C19_ * 3_F CATTTAGCTTCACCCTGTCATCCC 4 CYP2C19_ * 3_R TCATTGGATCTCATTAGCAAAAAACTTGGCCTTACCTCGAT 5 CYP2C19_ * 17_F TGCCCATCCTGGCGCATTA 6 CYP2C19_ * 17_R TCATTGGATCTCATTAGCATCTCTGGGGGTGTTTTCC 7 CYP2D6 * 3_F TCCCCGTCCTCCTGCATATC 8 CYP2D6 * 3_R TCATTGGATCTCATTAACCTTCTCCATCTCTGCCAGG 9 CYP2D6 * 4_F CCGCCTTCGCCAACCACT 10 CYP2D6 * 4_R TCATTGGATCTCATTAAAGAGACCGTTGGGGCGAAA eleven CYP2D6 * 10_F TCTGGTAGGGGAGAGTCTCAGC 12 CYP2D6 * 10_R TCATTGGATCTCATTAAGTGGCCATCTTCCTGCTCC thirteen CYP2D6 * 41_F AAAGTTCATGGGCCCCCGC 14 CYP2D6 * 41_R TCATTGGATCTCATTACATAGTGGTGGCTGACCTGTTCTC fifteen Uni TCATTGGATCTCATTA

List of sequences 2. The sequences of oligonucleotide probes

SEQ ID NO: Title Sequence 5 ’-> 3’ 16 CYP2C19_ * 2_G ATTATTTCCCGGGAACCCA 17 CYP2C19_ * 2_A ATTATTTCCCAGGAACCCAT 18 CYP2C19_ * 3_G Driptggatcgaggt 19 CYP2C19_ * 3_A 132CTGAATCGAGG 20 CYP2C19_ * 17_C TTCTCAAAGCATCTCTGATG 21 CYP2C19_ * 17_T TTCTCAAAGTATCTCTGATG 22 CYP2D6 * 3_A ACTGAGCACAGGATGACCT 23 CYP2D6 * 3_delA1 ACTGAGCACGGATGACCT 24 CYP2D6 * 4_G ACCCCCAGGACGCCCC 25 CYP2D6 * 4_A ACCCCCAAGACGCCCC 26 CYP2D6 * 10_G GCCTGGTGGGTAGCGTG 27 CYP2D6 * 10_A GCCTGGTGAGTAGCGTG 28 CYP2D6 * 41_C GTACCCTTCCTCCCTCG 29th CYP2D6 * 41_T TGTACCCTTTCTCCCTCG

Claims (7)

1. The method of analysis of polymorphic markers in the genes CYP2C19 and CYP2D6 to determine individual sensitivity to antidepressants, comprising the following stages: (a) - amplification of fragments of the CYP2C19 and CYP2D6 genes using multiplex one-stage PCR, in which DNA isolated from blood or other biomaterial obtained from the patient and primers represented by the sequences SEQ ID NO: 1-14 are used as amplification matrix; (b) - providing the biochip with sets of oligonucleotide probes with sequences SEQ ID NO: 16-29 for analysis and identification of polymorphic markers in the CYP2C19 and CYP2D6 genes to determine individual sensitivity to antidepressants; (c) hybridization of a fluorescently labeled PCR product obtained in step (a) on a biochip obtained in step (b); (d) - registration and interpretation of hybridization results on a biochip carried out at stage (c); (d) - determination of the genotype based on the fluorescence pattern obtained in stage (g). 2. The method according to p. 1, in which to identify polymorphic markers in the genes CYP2C19 and CYP2D6 (in children with acute leukemia), amplified using a set of primers, characterized in p. 1, a biochip is used, which is a substrate with a set of immobilized on it oligonucleotide probes with the sequences shown in SEQ ID NO: 16-29.

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