WO2000012757A1 - Procede de mesure de l'aptitude d'un patient a metaboliser certains medicaments - Google Patents

Procede de mesure de l'aptitude d'un patient a metaboliser certains medicaments Download PDF

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Publication number
WO2000012757A1
WO2000012757A1 PCT/SE1999/001449 SE9901449W WO0012757A1 WO 2000012757 A1 WO2000012757 A1 WO 2000012757A1 SE 9901449 W SE9901449 W SE 9901449W WO 0012757 A1 WO0012757 A1 WO 0012757A1
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Prior art keywords
sequence
point mutation
detection primer
subsequence
primer
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PCT/SE1999/001449
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English (en)
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Dan Hauzenberger
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Sangtec Molecular Diagnostics Ab
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Application filed by Sangtec Molecular Diagnostics Ab filed Critical Sangtec Molecular Diagnostics Ab
Priority to JP2000567740A priority Critical patent/JP2002523111A/ja
Priority to AU58908/99A priority patent/AU5890899A/en
Priority to EP99946509A priority patent/EP1108065A1/fr
Publication of WO2000012757A1 publication Critical patent/WO2000012757A1/fr

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
    • C12N9/0077—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14) with a reduced iron-sulfur protein as one donor (1.14.15)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6858—Allele-specific amplification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2531/00—Reactions of nucleic acids characterised by
    • C12Q2531/10—Reactions of nucleic acids characterised by the purpose being amplify/increase the copy number of target nucleic acid
    • C12Q2531/113—PCR
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2563/00—Nucleic acid detection characterized by the use of physical, structural and functional properties
    • C12Q2563/131—Nucleic acid detection characterized by the use of physical, structural and functional properties the label being a member of a cognate binding pair, i.e. extends to antibodies, haptens, avidin

Definitions

  • the present application relates to an assay method for monitoring metabolism of certain drugs in an individual. More particularly, the invention relates to a method for determining the presence of point mutations in isoforms of cytochrome P450, which point mutations are known to affect the isoforms' abilities to metabolise said drugs. The invention also relates to primers and diagnostic kits that are suitable for carrying out the invention.
  • Single nucleotide variations have been estimated to occur in a frequency of approximately one out of thousand nucleotides in the human genome (Cooper et al., J. Hum. Genet. (1985) 69:201). Many of these mutations may not give rise to a phenotype but a great number of the genetic diseases known to date are caused by single nucleotide polymorphisms. As a consequence, detection of single nucleotide mutations in specific genes will become of increasing interest in order to understand the ethiology of many genetic diseases.
  • Phase I Drug metabolism involves enzymes that either oxidise (phase I) or conjugate (phase II) xenobiotics.
  • phase II conjugate
  • cytochrome P450 which are located in the endoplasmatic reticulum primarily in the liver (Under et al., Clinical Chemistry (1997) 43:254).
  • Cytochromes P450 are comprised by a super gene family of mixed function oxidases that metabolises a large number of xenobiotics including drugs. Thirty or more of these enzymes have been characterised in the human so far, each with distinct catalytic specificity and unique regulation.
  • the most important isoforms involved in drug metabolism are CYP2D6, CYP2C9, CYP2C19 and CYP3A4.
  • CYP isoforms are known to be polymorphic which results in differential capacities in metabolising drugs such as omeprazole (proton pump inhibitors), phenytoin (anti-convulsant), verapamil (calcium-antagonists), propanolol (beta-blockers) and many others.
  • the C YP2C9 isoform is involved in hydroxylation of tolbutamide, phenytoin and S-warfarin among others.
  • CYP2C9 converts S-warfarin into the inactive phenolic metabolite S-7-hyckoxywarfarin and thereby controls the pharmacological activity of this drug.
  • polymorphisms among these enzymes exist resulting in differential capacities to metabolise drugs.
  • the genetic basis of this polymorphism is single nucleotide mutations resulting in the expression of two allelic variants, CYP2C9*2 and CYP2C9*3.
  • the CYP2C9*2 allele has cysteine substituted for arginine at amino acid 144 in the protein and CYP2C9*3 has leucine substituted for isoleucine at position 359.
  • CYP2C9*3 possesses only 5% of the catalytic capacity for S-warfarin as compared to the CYP2C9 wild type enzyme.
  • Warfarin is a widely used anticoagulant of coumarin type which acts by blocking synthesis of the vitamin K-dependant coagulation factors II, VII, IX and X in the liver.
  • the indications for taking S-warfarin are all diseases where prevention of extensive blood clotting is a crucial factor in the efficient treatment of patients. Examples of such *-»
  • diseases are acute embolic diseases of heart, lung or brain. In these cases the treatment is often combined with heparine. More specific indications are diseases where a lifelong treatment with anti-coagulants is required. Such diseases include recurrent venous thrombosis, pulmonary embolism and chronic atrial fibrillations.
  • diseases include recurrent venous thrombosis, pulmonary embolism and chronic atrial fibrillations.
  • the major difficulties with the use of this drug are a broad range of interactions with other drugs as well as nutritional factors. The complicated treatment of patients with this drug carries the risk of serious hemorrhage in as much as 9 % per patient year (Fihn et al, Ann. Intern. Med. (1996) 124:970; Steward et al., Pharmacogenetics (1997) 7:361).
  • C YP2C9 metabolises the transformation of the anti-convulsant Valproic acid ( VD A) into the unsaturated metabolite 4-ENE-VPA.
  • VD A anti-convulsant Valproic acid
  • 4_ENE-NPA acts hepatotoxic and causes several deaths yearly in the US (Sadeque et al., J. Pharmacol. Exp. Ther. (1997) 283:698).
  • the CYP2C19 isoform is involved in 4-hydroxylation (or 5-hydroxylation) of tricyclic antidepressants such as irnipramin, anti-malarial prodrags as for instance proguanil and proton pump inhibitors such as omeprazole or pentaprazole (Linder et al., Clinical Chemistry (1997) 43:254).
  • This subfamily is polymorphogenic due to single nucleotide mutations (S ⁇ P) of the wild type allele.
  • the Ml allele contains a G 68 6-A 686 substitution which creates a novel aberrantly spliced CYP2C19 mR ⁇ A. This results in the production of an inactivated truncated protein.
  • the M2 allele contains a G 64 ⁇ -A 64I substitution resulting in a premature stop codon. Therefore, these two alleles represent poor metabolise phenotypes.
  • S ⁇ P single-point mutations
  • Detection of single-point mutations can be performed using different techniques.
  • such assays can be subdivided into techniques where detection of S ⁇ P:s involves electrophoretic separation of DNA sequences and techniques using solid supports.
  • Techniques using solid supports have several advantages as compared to electrophoretic separation techniques. Firstly, the solid-phase assays involve relatively few and simple manipulations that are amenable to full automation. Secondly, non-radioactive methods can conveniently be used in the solid phase assays and thirdly, these assays give numerical results allowing e.g. statistical treatment.
  • a method comprising the steps of a) isolating and/or providing detectable amounts of single-stranded DNA from said sample by using known methods; b) hybridising the single-stranded DNA obtained in step a) with a detection primer comprising a plurality of nucleotide residues, said primer being complementary to a target nucleotide sequence immediately adjacent and 5' in relation to a defined point mutation of a single-stranded DNA encoding a cytochrome P450 isoform, where said point mutation is known to affect said isoform' s ability to metabolise said drug, such that there are no nucleotide residues between the defined point mutation and the 3 ' end of the detection primer that are identical to the first or second nucleotide residues of the point mutation to be detected, when the detection primer is hybridised to the target nucleic acid; c) extending the
  • the solid phase mini-sequencing technique disclosed in WO91/13075 provides a cheap and robust assay which can be performed by any laboratory equipped with a thermal cycler. Moreover, this technique does not require any specially trained personnel. Furthermore, the solid phase mini-sequencing technique does not require radioactively labeled nucleotides. Therefor it exhibits higher safety standards than such techniques.
  • this technique provides an excellent possibility of detecting either homozygote or heterozygote alleles within a defined sample.
  • the present invention relates to a method for determining the ability of cells in a sample to metabolise a certain drug comprising detecting a defined point mutation of a single-stranded DNA encoding a cytochrome P450 isoform, where said point mutation is known to affect said isoform' s ability to metabolise said drug.
  • the present invention relates to detection primers useful in the above mentioned method, which primers hybridise to target nucleotide sequences immediately adjacent and 5' in relation to a point mutation of a DNA, said DNA encoding an isoform of cytochrome P450.
  • the present invention relates to a diagnostic kit for carrying out said method, said kit comprising at least one detection primer as defined above, at least two amplification primers derived from a sequence encoding a cytochrome P450 isoform , said amplification primers being chosen in such a way that a subsequence of said cytochrome P450-encoding sequence to which said detection primer hybridises is amplified, and a DNA-polymerising agent.
  • drug relates to drugs that are metabolised by cytochrome P450 isoforms.
  • examples of such drugs are omeprazole , pentaprazole, phenytoin, verapamil, propanolol, tolbutamide, S-warfarin, tricyclic antidepressants such as imipramin and anti-malarial prodrags such as proguanil.
  • detection primer relates to an oligonucleotide which hybridises to a site immediately adjacent 5' in relation to a defined point mutation.
  • amplification primer relates to one of two primers forming a primer pair that is used according to well-known amplification procedures such as PCR. Both detection primers and amplification primers according to the invention comprises 8 - 70 nucleotides, preferably 10-30 nucleotides, and most preferably 15 - 25 nucleotides.
  • affinity pair relates to a pair of chemical, preferably biochemical, compounds that binds specifically and strongly to each other.
  • examples of such pairs include, but are not limited to antibody-antigen, biotin-avidin/streptavidin, enzyme-substrate, a pair of complementary oligonucleotides, protein A-IgG, etc.
  • the term "polymerising agent” relates to a DNA polymerising agent.
  • An example of such an gent is the Klenow fragment of Escherichia coli DNA polymerase I, but any DNA polymerase can be used in the method of this invention.
  • the presence of point mutations can be detected by adding labelled nucleotides to the detection primer.
  • Any kind of detectable labels such as one member of an affinity pair, radioactive nuclides, fluorescent compounds, enzymes inducing light emissions or colour changes etc. can be bound to an ordinary nuclotide in order to obtain a labelled nucleotide.
  • it is possible to use modified nucleotides such as chain-terminating dideoxynucleotides.
  • the skilled person is well aware of how to choose suitable labelled nucleotides as well as how to choose suitable detection procedures when carrying out the method according to the present invention.
  • fig. 1 discloses a photo of an electrophoresis gel where lanes A-E represent the following PCR products: A: CYP2C9*2 (simplex PCR), B: CYP2C9*3 (simplex PCR), C: CYP2C9*2*3 (20 ⁇ l*3, multiplex PCR), D: CYP2C9*2*3 (15 ⁇ l * 3, multiplex PCR), E: CYP2C9*2*3 (10 ⁇ * 3, multiplex PCR).
  • A-E represent the following PCR products: A: CYP2C9*2 (simplex PCR), B: CYP2C9*3 (simplex PCR), C: CYP2C9*2*3 (20 ⁇ l*3, multiplex PCR), D: CYP2C9*2*3 (15 ⁇ l * 3, multiplex PCR), E: CYP2C9*2*3 (10 ⁇ * 3, multiplex PCR).
  • A-E represent the following
  • table 1 discloses results obtained when the PCR products shown in fig. 1 have been subjected to minisequencing reactions. Both specific and non-specific sequence primers as well as complimentary or non-complimentary nucleotides have been used.
  • the figures shown in table 1 represent optical density (OD) values from an ELISA determined at 405 nm.
  • the table shows which PCR products were coated onto the streptavidin-coated ELISA plate (columns), which sequence primers were used (rows) and which nucleotides were used in the sequencing reaction (columns);
  • table 2 shows the calculated ratio of the OD at 405 nm from nucleotides incorporated by the mini-sequencing reaction.
  • the ratio values presented in this table have been calculated from the OD values in table 1.
  • the ratio was calculated as follows: complementary nucleotide (OD at 405 nm/ complementary nucleotide + non- complementary nucleotide (OD at 405 nm).
  • a ratio of > 0.85 is significant for an incorporation of complementary nucleotides when using homozygous alleles.
  • the mini-sequencing technique is based on amplification of defined genes with PCR (Polymerase Chain Reaction) using biotinylated or otherwise conjugated oligonucleotides (primers). In general, where possible a multiplex amplification procedure is utilised.
  • the biotinylated PCR products are immobilised on streptavidin- coated microwell plates and the PCR products are sequenced using an allele-specific oligonucleotide.
  • Possible mutations within the immobilised PCR product representing a defined allele are detected by incorporation of a mutation-specific labelled nucleotide.
  • Incorporatio of a complementary nucleotide can be detected either directly or indirectly utilising various established detection methods. Using this technique, it is possible to detect homozygote or heterozygote alleles based on single nucleotide mutations within an individual.
  • Genomic DNA can be prepared using any established method described in the literature (PCR Protocols, Innis MA et al., Academic Press 1990; PCR, a practical approach, McPherson, MJ et al., Oxford University Press, 1991) or using any DNA purification kit present.
  • the DNA prepared in these experiments has been prepared using the QIAamp Blood Kit (Qiagen Inc, USA) according to the description provided by the manufacturer.
  • Genomic DNA can be prepared using any sample-containing nucleated cells.
  • the typical yield using the above mentioned DNA purification kit is 10 ng/ ⁇ l. 250 ng of purified genomic DNA was used as a template in the subsequent PCR.
  • the primers used for the PCR reactions are described in the sequence listing as SEQ.ID.Nos 4, 5, 7, 8, 10, 11, 13 and 14.
  • a 2x mastemix for the multiplex PCR of CYP2C9 alleles was prepared as follows: • Tris-HCl (100 mM, pH8.8)
  • PCR reaction 50 ⁇ l of the above described 2x mastermix was subject to a PCR tube (thin wall PCR tubes, Perkin-Ehner Inc. USA). 24,5 ⁇ l of ddH20, 0,5 ⁇ l Taq- polymerase (2,5 Units, Perkin-Elmer, Inc, USA) and 25 ⁇ l geonomic DNA (250 ng) was added to the tube and the reaction mix was overlaid with 50 ⁇ l mineral oil.
  • the thermal conditions for amplification of the CYP2C9 alleles were as follows: An initial denaturation step at 96°C for 2 minutes thereafter 96°C (30 sec), 60°C (30 sec) (58°C for the CYP2C19 alleles) and 72°C (30 sec), 35 cycles. Following the PCR amplification, 100 ⁇ l of the amplified sample was mixed with 400 ⁇ l of Binding buffer (buffer 1) containing 20 mM sodium phosphate, pH 7.5, 100 mM NaCl and 0.1 % (v/v) Tween-20.
  • Buffer 1 Binding buffer
  • MWP microwell plates
  • the MWP were then incubated at 22°C for 15 minutes.
  • the immobilized PCR samples were denatured using a denaturing solution containing NaOH (50 mM) for 1 minute at 22°C.
  • the MWP were washed using a buffer (buffer 2) containing Tris-HCl (40 mM, pH 8.8), EDTA (lmM), NaCl (50 mM) and Tween-20 (0.1%).
  • minisequencing reaction every well of the MWP was incubated with an appropriate minisequencing primer (final concentration 0.1 ⁇ M) diluted in 5 ⁇ l of 10 x DNA polymerase buffer (buffer 3) containing Tris-HCl (500 mM, pH 8.8), (NH 4 ) 2 S0 4 (150 mM), MgCl 2 (15mM), Triton X-100 (1% V/V), Gelatin (0.1%W/V), DNA polymerase (0.1 units), flourescein-12-dNTP complementary to the nucleotide to be detected (final concentration of 0.1 ⁇ M) and ddH 2 0 to a final volume of 50 ⁇ l.
  • buffer 3 DNA polymerase buffer
  • Tris-HCl 500 mM, pH 8.8
  • NH 4 ) 2 S0 4 150 mM
  • MgCl 2 15mM
  • Triton X-100 1% V/V
  • Gelatin 0.1%W/V
  • DNA polymerase
  • the MWP were incubated at 55° C for 30 minutes. Following the minisequencing reaction the MWP were washed using buffer 2. Incorporated nucleotides were detected using alkaline phosphatase (AP) conjugated anti-FITC monoclonal antibodies (0.75 U/ml) diluted in a buffer (buffer 4) containing : Hepes (25 mM), NaCl (125 mM), MgCl 2 (2 mM), BSA (1%) and Tween-20 (0.3 % N/V). Incubation was done at 22° C for 15 minutes and the plates were subsequently washed using buffer 2.
  • AP alkaline phosphatase
  • Buffer 4 buffer containing : Hepes (25 mM), NaCl (125 mM), MgCl 2 (2 mM), BSA (1%) and Tween-20 (0.3 % N/V). Incubation was done at 22° C for 15 minutes and the plates were subsequently washed using buffer 2.
  • Detection of bound monoclonal antibodies was performed by incubation the MWP using a detection buffer (buffer 5) containing diethanolamine (10.6 % W/V), MgCl 2 (0.05 % W/V) and para- nitro-phenyl phosphate (4 mg/ml) for 20 minutes at 22° C. Detection of incorporated d ⁇ TP's was done at 405 nm using a commercially available spectrophotometer.
  • PCR amplification and minisequencing has been performed using CYP2C9 and CYP2C19 specific primers. The results shown below demonstiate amplification and minisequencing of the CYP2C9 alleles.
  • Human genomic D ⁇ A was purified as described in the method section. 250 ng of genomic D ⁇ A was subjected to PCR as described above. The results of a representative experiment are demonstrated in figure 1. Next a minsequencing reaction of the amplified DNA was done as decribed above. By using alleles specific sequencing primers incorporated dNTP's could be detected in subsequent detection steps as described above. These results are shown in table 1.

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Abstract

Selon la présente invention, il est désormais possible de mesurer, par un essai simple, l'aptitude d'un patient à métaboliser un médicament donné. Le procédé appliqué comprend les étapes suivantes: a) isoler et/ou fournir une quantité détectable d'ADN monocaténaire provenant de l'échantillon par l'utilisation de procédés connus; b) hybrider l'ADN monocaténaire obtenu à l'étape a) avec une amorce de détection composée d'une pluralité de résidus nucléotidiques, ladite amorce étant complémentaire d'une séquence nucléotidique cible et de 5' par rapport à une mutation ponctuelle définie d'un ADN monocaténaire codant pour un isoforme du cytochrome P450, la mutation ponctuelle affectant l'aptitude de l'isoforme à métaboliser le médicament, de façon telle qu'aucun résidu nucléotidique entre la mutation ponctuelle définie et l'extrémité 3' de l'amorce de détection n'est identique au premier ou au second résidus nucléotidiques de la mutation ponctuelle à détecter, lorsque l'amorce de détection est hybridée à l'acide nucléique cible; c) développer l'amorce par l'adjonction d'un agent polymérisant dans un mélange comprenant un ou plusieurs triphosphates nucléosidiques, le mélange comportant au moins un triphosphate nucléosidique complémentaire soit du premier, soit du second résidu nucléique avec des moyens de détecter l'incorporation du triphosphate nucléosidique dans un polymère d'acide nucléique et, éventuellement, un ou plusieurs triphosphates de terminaison; d) détecter l'incorporation du triphosphate nucléosidique à l'aide desdits moyens, ce qui permet de déterminer si l'échantillon contient la mutation poncutelle de l'isoforme du cytochrome P450.
PCT/SE1999/001449 1998-08-28 1999-08-25 Procede de mesure de l'aptitude d'un patient a metaboliser certains medicaments WO2000012757A1 (fr)

Priority Applications (3)

Application Number Priority Date Filing Date Title
JP2000567740A JP2002523111A (ja) 1998-08-28 1999-08-25 特定医薬を代謝することに対する患者の能力を測定する方法
AU58908/99A AU5890899A (en) 1998-08-28 1999-08-25 A method for measuring a patient's ability to metabolise certain drugs
EP99946509A EP1108065A1 (fr) 1998-08-28 1999-08-25 Procede de mesure de l'aptitude d'un patient a metaboliser certains medicaments

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SE9802897-0 1998-08-28
SE9802897 1998-08-28

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Cited By (8)

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WO2002018639A2 (fr) * 2000-08-30 2002-03-07 Sequenom-Gemini Limited Detection de polymorphismes cyp2c19
WO2004018707A2 (fr) * 2002-08-15 2004-03-04 Biotez Berlin-Buch Gmbh Procede pour identifier des polymorphismes d'un seul nucleotide (snp) dans des genes du metabolisme de medicaments, et kit de test pour la mise en oeuvre dudit procede
EP1561823A1 (fr) * 2004-02-04 2005-08-10 Biotez Berlin-Buch GmbH Procédé pour la détection des polymorphismes de nucléotide simple (SNP) des gênes du métabolisme des médicaments et dispositif pour l'utilisation correspondante
WO2006010266A1 (fr) * 2004-07-30 2006-02-02 Tm Bioscience Pgx, Inc. Et Al Methode de detection de mutations dans le gene codant pour le cytochrome p450-2c19
WO2007034423A2 (fr) * 2005-09-20 2007-03-29 Medicon Hellas S.A. Methode de detection de variations nucleotidiques uniques dans une sequence de nucleotides au moyen de reactifs secs/lyophilises.
EP1781812A1 (fr) * 2004-06-30 2007-05-09 TM Bioscience PGX, Inc. Methode de detection de mutations dans le gene codant pour le cytochrome p450-2c9
WO2008091839A2 (fr) * 2007-01-22 2008-07-31 Siemens Healthcare Diagnostics Inc. Réactifs et procédés pour détecter des polymorphismes dans cyp2c9
EP2055789A1 (fr) * 2006-11-30 2009-05-06 Arkray, Inc. Jeu d'amorces pour l'amplification du gène cyp2c19, réactif pour l'amplification du gène cyp2c19 comprenant ledit jeu d'amorces et utilisation du réactif

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Cited By (15)

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