JP2002523111A - Method of measuring a patient's ability to metabolize a particular drug - Google Patents

Abstract

  (57) [Summary] It has now been found that it is possible to carry out a simple test to determine a patient's ability to metabolize a particular drug by applying a method comprising the following steps: Isolating and / or preparing a detectable amount of single-stranded DNA from said sample using said method; b) hybridizing said single-stranded DNA obtained in step a) with a detection primer comprising a plurality of nucleotide residues ( However, the primer is a defined point mutation in the single-stranded DNA encoding the cytochrome P450 isoform protein (the point mutation is known to affect the ability of the isoform protein to metabolize the drug. And is complementary to the target nucleotide sequence that is 5 'immediately adjacent to When the outgoing primer hybridizes to the target nucleic acid, a nucleotide residue identical to the first or second nucleotide residue of the point mutation to be detected between the defined point mutation and the 3 'end of the detection primer is detected. Not present); c) extending the primer using a polymerizing agent in a mixture comprising one or more nucleoside triphosphates, provided that the mixture comprises means for detecting incorporation of the nucleoside triphosphate into the nucleic acid polymer; Or at least one nucleoside triphosphate complementary to the second nucleotide residue, and optionally one or more chain-terminated nucleoside triphosphates); d) detecting incorporation of the nucleoside triphosphate using said means; This allows the sample to undergo cytochrome P450 isoform tamper. To determine whether it contains the point mutation in the protein.

Description

DETAILED DESCRIPTION OF THE INVENTION

The present application relates to an assay method for monitoring the metabolism of a specific drug in an individual. More particularly, the present invention relates to the presence of a point mutation in the isoform of cytochrome P450, which point mutation is known to affect the ability of the isoform to metabolize the drug. How to decide. The present invention also relates to primers and diagnostic kits suitable for carrying out the above invention.

[0002] incorporated herein as all documents cited are references in the following description of the background art.

[0003] Single nucleotide variations are estimated to occur at a frequency of about 1 per 1000 nucleotides in the human genome (Cooper et al., J. Hum. Genet. (1985) 69: 201.
). Although many of these mutations do not result in a phenotype, the majority of genetic disorders known to date are caused by single nucleotide polymorphisms. Thus, detecting single nucleotide mutations in particular genes is of increasing interest to understand the pathogenesis of many genetic diseases.

For metabolism of drugs, xenobiotics are oxidized (phase I) or conjugated to xenobiotics (phase II)
Enzymes are involved. The major route of drug metabolism in phase I is maintained by a group of enzymes called cytochrome P450s, which are mainly present in the endoplasmic reticulum of the liver (Lind
er et al., Clinical Chemistry (1997) 43: 254). Cytochrome P450 (CYP) consists of a supergene family of oxidases of various functions that metabolize a number of xenobiotics, including drugs. To date, more than 30 of these enzymes have been characterized in humans, each with different catalytic specificities and specific controls. Because of the diversity of these enzymes, they have been further classified into subpopulations or isoforms based on their sequence homology. Polymorphisms in the catalytic capacity of these enzymes result in the appearance of various phenotypes that differ in their ability to metabolize drugs. Extensive metabolism (EM) of a drug is characteristic of a normal population and represents a wild-type allele. Poor metabolism (PM) is due to poor or inability to catalyze a particular enzyme, which is often caused by mutation or deletion of a gene.
On the other hand, hyperactive metabolism (UEM) is generally caused by gene duplication.

[0005] The most important isoform proteins involved in drug metabolism are CYP2D6, CYP2C
9, CYP2C19 and CYP3A4. Some of these CYP isoform proteins are known to be polymorphic and include omeprazole (a proton pump inhibitor), phenytoin (an anticonvulsant), verapamil (a calcium antagonist)
The metabolic capacity of many drugs such as propanolol (a beta inhibitor) changes.
The CYP2C9 isoform protein is involved in the hydroxylation of tolbutamide, phenytoin and especially S-warfarin. In particular, CYP2C9 converts S-warfarin to the inactive phenolic metabolite S-7-hydroxywarfarin, thereby modulating the pharmaceutical activity of the drug. Again, polymorphisms of these enzymes result in differences in their ability to metabolize drugs. The genetic basis for this polymorphism is a single nucleotide mutation, which represents two allelic variants, CYP2C9 ^* 2 and CYP2C9 ^* 3. The CYP2C9 ^* 2 allele has arginine replaced by cysteine at amino acid 144 of the protein and CYP2C9 ^* 3 has isoleucine replaced by leucine at position 359. The frequency of these alleles has been reported to be between 7 and 19% in the Caucasus population. Although homozygous individuals for these alleles are relatively rare, in vitro studies of warfarin metabolism have shown that these mutant proteins impair the catalytic ability (Steward et al., Pharmacogenetics (19
97) 7: 361). For example, CYP2C9 ^* 3 has only 5% of the catalytic ability of S-warfarin compared to the wild-type enzyme CYP2C9.

[0006] Warfarin is a vitamin K-dependent coagulation factor II, VII, IX and X in the liver.
Is a widely used coumarin-type anticoagulant that acts by inhibiting the synthesis of. S-warfarin intake is indicated in all diseases where prevention of vigorous coagulation is a decisive factor for treating patients effectively. Examples of such diseases are acute emboli of the heart, lungs or brain. In these cases, treatment is often combined with heparin. A more specific application is a disease that requires lifelong treatment with anticoagulants. Examples of such diseases include recurrent venous thrombosis, pulmonary embolism and chronic atrial fibrillation. A major problem in the use of this drug is the widespread interaction with other drugs and nutritional factors. The complex treatment of patients with this drug involves the risk of severe bleeding in as many as 9% of patients per year (Fihn et al., Anne
n. Intern. Med. (1996) 124: 970; Steward et al., Pharmacogenetics (1997) 7: 361).
Therefore, pre-treatment assessment of the CYP2C9 status of potential patients treated with warfarin could significantly reduce the risk of drug side effects.
In addition, CYP2C9 metabolizes the conversion of the anticonvulsant valproic acid (VDA) to its unsaturated metabolite 4-ENE-VPA. 4-ENE-VPA is a drug that acts as a liver toxicant and causes several deaths per year in the United States (Sa
Deque et al., J. Pharmacol. Exp. Ther. (1997) 283: 698).

[0007] The CYP2C19 isoform protein is a procyclic inhibitor such as imipramine,
Antimalarial drug precursors such as guanyl and omeprazole or pentaprazole
(Or 5-hydroxylation) of a proton pump inhibitor such as
(Linder et al., Clinical Chemistry (1997) 43: 254). This sub-
Millie is genetically affected by a single nucleotide mutation (SNP) in the wild-type allele.
Is polymorphic. The M1 allele is G₆₈₆-A₆₈₆Including the substitution
This results in an abnormally spliced CYP2C19 mRNA. As a result,
Sexualized shortened proteins are produced. The M2 allele is G₆₄₁-A ₆₄₁ Including substitutions, which result in premature stop codons. Therefore, these two
Alleles represent a poor metabolic phenotype.

[0008] Detection of single point mutations (SNPs), such as the mutations described above, can be performed using a variety of techniques. In general, such assays can be divided into two approaches. In other words, there are two types of detection: SNP detection by separation of DNA sequences by electrophoresis, and SNP detection by a technique using a solid support. The technique using a solid support has several advantages over the electrophoretic separation technique. First, solid phase assays require relatively few and simple operations and can be easily fully automated. Solid-phase assays are also convenient because non-radioactive methods can be used, and furthermore, these assays give numerical results so that, for example, statistical processing is possible.

As in the case of solid phase assays, different assay types can be distinguished. These techniques include hybridization or sandwich hybridization using sequence-specific oligonucleotide probes, such as "reverse dot blots". These techniques require very careful design of sequence-specific probes and require frequent monitoring of reaction conditions. Therefore, it can only be performed in highly specialized laboratories. Similar problems exist with sequence-specific amplification. This is because this technique requires careful optimization of the PCR conditions. Therefore, this technique can also be performed only in highly specialized laboratories. Finally, sequencing of defined DNA sequences requires expensive equipment and trained personnel, which are currently only available in highly specialized laboratories.

[0010] Simple analytical tests to determine the genetic status of an individual prior to ingestion of the drug, as the CYP2C19 and CYP2C9 enzymes metabolize various drugs that, when overdosed, pose a potential threat to patient health There is a request for Because knowledge of an individual's genetic status prior to ingestion of a drug can substantially reduce the risk of side effects of the drug.

[0011] It by applying the method comprising the following steps Gaiyo the invention it is possible to perform a simple test for measuring the ability of a patient to metabolize certain pharmaceutical became now be apparent: a) isolating and / or preparing a detectable amount of single-stranded DNA from said sample using known methods; b) detecting the single-stranded DNA obtained in step a) comprising a plurality of nucleotide residues Hybridize with a primer (provided that the primer is a cytochrome P
A defined point mutation in the single-stranded DNA encoding the 450 isoform protein, wherein the point mutation is known to affect the ability of the isoform protein to metabolize the drug. ) Is complementary to a target nucleotide sequence that is immediately adjacent and 5 'to, and thus, when the detection primer hybridizes to the target nucleic acid, between the defined point mutation and the 3' end of the detection primer. No nucleotide residue identical to the first or second nucleotide residue of the point mutation to be detected at c.); C) extending the primer with a polymerizing agent in a mixture comprising one or more nucleoside triphosphates (However, the mixture may contain a means for detecting the incorporation of nucleoside triphosphate into the nucleic acid polymer. Complementarity of at least one of the nucleoside triphosphates to the nucleotide residues, and one or more chain terminating nucleoside triphosphates optionally); d) the nucleoside triphosphates incorporation detected using said means,
Thereby, it is determined whether the sample contains the point mutation of the cytochrome P450 isoform protein.

[0012] The solid phase minisequencing techniques which are disclosed in the detailed description WO 91/13075 of invention provides an inexpensive and reliable assay can be carried out in any laboratory if laboratory equipped with a thermal cycler. Also, this approach does not require specially trained personnel. Further, the solid phase mini-sequencing technique does not require radiolabeled nucleotides,
It therefore represents a standard that is more secure than the approach using radiolabeled nucleotides. Finally, this approach offers an excellent possibility of detecting homozygous or heterozygous alleles in defined samples.

[0013] Accordingly, the present invention provides a single-chain D encoding a cytochrome P450 isoform protein.
A method for determining the ability of a cell in a sample to metabolize a particular drug, comprising detecting a defined point mutation in NA. The point mutation is one that is known to affect the ability of the isoform protein to metabolize the drug.

[0014] In another embodiment, the invention is directed to a detection primer useful in the above-described method. This primer hybridizes to the target nucleotide sequence located immediately adjacent and 5 'to the point mutation in the DNA encoding the isoform of cytochrome P450.

In another embodiment, the present invention relates to a diagnostic kit for performing the above-described method. The kit includes at least one detection primer described above and at least two amplification primers derived from a sequence encoding a cytochrome P450 isoform protein (provided that the amplification primer has a subsequence of the cytochrome P450 coding sequence to which the detection primer hybridizes). Selected to be amplified), and a DNA polymerizing agent.

As disclosed herein, the term “pharmaceutical” refers to a drug that is metabolized by cytochrome P450 isoform proteins. Examples of such drugs are omeprazole, pentaprazole, phenytoin, verapamil, propanol, tolbutamide, S-
Warfarin, tricyclic antidepressants (eg imipramine) and antimalarial drug precursors (eg proguanil).

As disclosed herein, the term “detection primer” refers to an oligonucleotide that hybridizes to the immediately adjacent 5 ′ site with respect to a defined point mutation. The term "amplification primer" refers to one of two primers that form a primer pair used according to well-known amplification procedures, such as PCR. Both the detection primer and the amplification primer according to the present invention comprise 8-70 nucleotides, preferably 10-30 nucleotides, most preferably 15-25 nucleotides.

As disclosed herein, the term “affinity pair” refers to a pair of chemical, preferably biochemical, compounds that bind specifically and tightly 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, and the like.

As disclosed herein, the term “polymerizing agent” means a DNA polymerizing agent. Examples of such polymerization agents include the Klenow fragment of E. coli DNA polymerase I. However, any other DNA polymerase can be used in the method of the present invention.

According to the present invention, the presence of a point mutation can be detected by adding a labeled nucleotide to a detection primer. Any type of detectable label, such as a member of an affinity pair, a radionucleotide, a fluorescent compound, an enzyme that induces luminescence or a color change, can be combined with a conventional nucleotide to obtain a labeled nucleotide. Alternatively, modified nucleotides such as chain-terminating dideoxynucleotides can be used. Those skilled in the art will know how to select suitable labeled nucleotides when practicing the method according to the invention, as well as how to select suitable detection techniques.

Hereinafter, the present invention will be further described with reference to the accompanying drawings and tables. In the drawings and tables: FIG. 1 is a photograph of an electrophoresis gel. In the figure, lanes AE are the following PCR products
Represents: A: CYP2C9^*2 (simplex PCR), B: C
YP2C9^*3 (simplex PCR), C: CYP2C9^*2^*3 (20μ
l^*3, multiplex PCR), D: CYP2C9^*2^*3
(15 μl^*3, multiplex PCR), E: CYP2C9^*2^*3 (10
μl^*3, multiplex PCR). In multiplex PCR, CY
P2C9^*For the two alleles, a constant primer concentration was used and CYP2C9 ^* For three alleles, decreasing concentrations of primers were used. This is a multiplex
This is to optimize Rex PCR conditions.

Table 1 shows the results obtained when subjecting the PCR product shown in FIG. 1 to a mini-sequencing reaction. Both sequence-specific and non-sequence-specific primers as well as complementary or non-complementary nucleotides were used. The number shown in Table 1 is 4
Represents the optical density (OD) value from the ELISA measured at 05 nm. Which table is P
Shows whether the CR product was coated on a streptavidin-coated ELISA plate (row), which sequence primer was used (column), and which nucleotide was used in the sequencing reaction (row).

Table 2 shows 405 nm from nucleotides incorporated by the mini-sequencing reaction.
2 shows the calculated ratio of OD at. The ratios shown in this table were calculated from the OD values in Table 1. The ratio was calculated as follows: complementary nucleotides (40
OD at 5 nm / complementary nucleotide + non-complementary nucleotide (OD at 405 nm). Ratios greater than 0.85 are significant for incorporation of complementary nucleotides when using homozygous alleles.

Experimental Procedures The mini-sequencing technique is based on the amplification of defined genes by PCR (polymerase chain reaction) using biotinylated or conjugated oligonucleotides (primers). Generally, multiplex amplification procedures are used where possible. After amplification, the biotinylated PCR products are fixed in streptavidin-coated microwell plates, and the PCR products are sequenced using allele-specific oligonucleotides. Possible mutations in the fixed PCR product representing the defined allele are detected by mutation-specific incorporation of labeled nucleotides. Incorporation of complementary nucleotides can be detected directly or indirectly using various established detection methods. Using this technique, it is possible to detect homozygous or heterozygous alleles based on single nucleotide mutations within an individual.

Genomic DNA is described in the literature (PCR Protocols, Innis MA, etc., Academic Press 1990;
CR, a practical approach, McPherson, MJ, etc., Oxford University Press, 199
Using any of the established methods described in 1) or any existing DN
It can also be prepared using an A purification kit. DNA prepared in these experiments was prepared by using the QLAamp Blood Kit (Qiagen Inc, USA) according to the instructions provided by the manufacturer. Genomic DNA can be prepared using any sample, including nucleated cells. Typical yields when using the DNA purification kit described above are 10 ng / μl. 250 ng of purified genomic DNA was used as a template in subsequent PCR. The primers used for the PCR reaction are listed in the sequence listing as SEQ ID NOs: 4, 5, 7, 8, 10, 11, 13, and 14. In the following description of the experimental procedure for the CYP gene PCR and mini-sequencing methods, primers specific for the CYP2C9 allele were used. An experimental procedure similar to that described below was also used when studying the CYP2C19 allele except that a CYP2C19-specific oligonucleotide was used.

A 2 × master mix for multiplex PCR of the CYP2C9 allele was prepared as follows: Tris-HCl (100 mM, pH 8.8) • (NH ₄ ) ₂ SO ₄ (30 mM) Triton X- 100 (0.2% volume / volume) • Gelatin (0.02% weight / volume) • dNTPs (0.4 μM) • SEQ ID NOs: 4, 5, 7, 8 (0.4 mM each) • MgCl ₂ (3. 0 mM) • Make up to 500 μl with ddH ₂ O.

For the PCR reaction, 50 μl of the above-mentioned 2 × master mix was added to a PCR tube (
Thin-walled PCR tubes, Perkin-Elmer Inc. USA). 24.5 μl ddH ₂ O, 0.5 μl Taq polymerase (2.5 units, Perkin-Elmer Inc.
USA) and 25 μl of genomic DNA (250 ng) were added to the tube and the reaction mixture was coated with 50 μl of mineral oil.

The temperature conditions for amplification of the CYP2C9 allele were as follows: 96
Initial denaturation step at 2 ° C. for 2 minutes, followed by 96 ° C. (30 seconds), 60 ° C. (30 seconds) (58 ° C. for CYP2C19 allele) and 72 ° C. (30 seconds).
35 cycles. After PCR amplification, 100 μl of the amplified sample is mixed with 400 μl of binding buffer (buffer 1) containing 20 mM sodium phosphate, pH 7.5, 100 mM NaCl and 0.1% (v / v) Tween-20. did.

Subsequently, 50 μl of the solid phase is coated with streptavidin, for example, a commercially available microwell plate (MWP) (Labsystems, Helsinki, Finl.
and). The MWP was then incubated at 22 ° C. for 15 minutes. After incubation, the immobilized PCR sample was denatured with a denaturing solution containing NaOH (50 mM) at 22 ° C. for 1 minute. MWP was added to Tris-HCl (40 mM, pH
8.8), EDTA (1 mM), NaCl (50 mM) and Tween-20 (0.1%
) Was washed using a buffer solution (buffer solution 2).

For the mini-sequencing reaction, all wells of the MWP were incubated with 5 μl of the appropriate mini-sequencing primer (final concentration 0.1 μM) diluted in 10 × DNA polymerase buffer (buffer 3). The buffer 3 is Tris-HCl
(500 mM, pH 8.8), (NH ₄ ) ₂ SO ₄ (150 mM), MgCl ₂ (15 mM), Triton X-100 (1% volume / volume), gelatin (0.1% weight / volume), DNA polymerase (0.1 units), containing fluorescein-12-dNTP complementary to the nucleotide to be detected (final concentration 0.1 μM), dd
The final volume was 50 μl with H ₂ O.

The MWP was incubated at 55 ° C. for 30 minutes. After the mini-sequencing reaction, the MWP
Was washed with buffer 2. Incorporated nucleotides into Hepes (25 mM)
, NaCl (125 mM), MgCl ₂ (2 mM), BSA (1%) and Tween-
Anti-FIT diluted with buffer (buffer 4) containing 20 (0.3% volume / volume)
Alkaline phosphatase (AP) conjugated to monoclonal antibody C (0.75 U
/ Ml). After incubation at 22 ° C. for 15 minutes, the plate was washed with buffer 2. Detection of bound monoclonal antibody was performed with a detection buffer (buffer 5) containing diethanolamine (10.6% w / v), MgCl ₂ (0.05% w / v) and paranitrophenyl phosphate (4 mg / ml). By incubating the MWP at 22 ° C. for 20 minutes. Detection of the incorporated dNTPs was performed using a commercially available spectrophotometer
Performed at 5 nm.

Results PCR amplification and mini-sequencing were performed using CYP2C9 and CYP2C19 specific primers. The results shown below show amplification and mini-sequencing of the CYP2C9 allele. Human genomic DNA was purified as described in the methods section. 250 ng of genomic DNA was subjected to PCR as described above. Representative experimental results are shown in FIG.

Next, a minisequencing reaction of the amplified DNA was performed as described above. By using allele specific sequencing primers, incorporated dNTPs could be detected in a subsequent detection step as described above. Table 1 shows the results.

[0034]

[Table 1]

To obtain a numerical value, the ratio of OD was calculated based on the following equation. Table 2 shows the results.

[0036]

[Table 2]

These results demonstrate that gene-specific primers can be used to amplify and sequence cytochrome P450-specific alleles based on PCR and mini-sequencing techniques as described above. Shown in

[Sequence list]

[Brief description of the drawings]

FIG. 1 is a photograph of an electrophoresis gel.

[Procedural Amendment] Submission of translation of Article 34 Amendment of the Patent Cooperation Treaty

[Submission date] November 22, 2000 (200.11.22)

[Procedure amendment 1]

[Document name to be amended] Statement

[Correction target item name] Claims

[Correction method] Change

[Correction contents]

[Claims]

[Procedure amendment 2]

[Document name to be amended] Statement

[Correction target item name] 0037

[Correction method] Change

[Correction contents]

These results demonstrate that gene-specific primers can be used to amplify and sequence cytochrome P450-specific alleles based on PCR and mini-sequencing techniques as described above. Shown in

[Sequence List] SEQUENCE LISTING <110> AB Sangtec Medical <120> New method <130> primers <140> SE9802897-0 <141> 1998-08-28 <160> 19 <170> PatentIn Ver. 2.1 <210> 1 <211> 1746 <212> DNA <213> Homo sapiens <400> 1 cttcaatgga tccttttgtg gtccttgtgc tctgtctctc atgtttgctt ctcctttcaa 60
tctggagaca gagctctggg agaggaaaac tccctcctgg ccccactcct ctcccagtga 120
ttggaaatat cctacagata gatattaagg atgtcagcaa atccttaacc aatctctcaa 180
aaatctatgg ccctgtgttc actctgtatt ttggcctgga acgcatggtg gtgctgcatg 240
gatatgaagt ggtgaaggaa gccctgattg atcttggaga ggagttttct ggaagaggcc 300
atttcccact ggctgaaaga gctaacagag gatttggaat cgttttcagc aatggaaaga 360
gatggaagga gattcggcgt ttctccctca tgacgctgcg gaattttggg atggggaaga 420
ggagcattga ggaccgtgtt caagaggaag cccgctgcct tgtggaggag ttgagaaaaa 480
ccaaggcttc accctgtgat cccactttca tcctgggctg tgctccctgc aatgtgatct 540
gctccattat tttccagaaa cgtttcgatt ataaagatca cgaatttctt aacttgatgg 600
aaaaattgaa tgaaaacatc aggattgtaa gcaccccctg gatccagata tgcaataatt 660
ttcccactat cattgattat ttcccgggaa cccataacaa attacttaaa aaccttgctt 720
ttatggaaag tgatattttg gagaaagtaa aagaacacca agaatcgatg gacatcaaca 780
accctcggga ctttattgat tgcttcctga tcaaaatgga gaaggaaaag caaaaccaac 840
agtctgaatt cactattgaa aacttggtaa tcactgcagc tgacttactt ggagctggga 900
cagagacaac aagcacaacc ctgagatatg ctctccttct cctgctgaag cacccagagg 960
tcacagctaa agtccaggaa gagattgaac gtgtcattgg cagaaaccgg agcccctgca 1020
tgcacgacag gggccacatg ccctacacag atgctgtggt gcacgaggtc cagagataca 1080
tcgacctcat ccccaccagc ctgccccatg cagtgacctg tgacgttaaa ttcagaaact 1140
acctcattcc caagggcaca accatattaa cttccctcac ttctgtgcta catgacaaca 1200
aagaatttcc caacccagag atgtttgacc ctcgtcactt tctggatgaa ggtggaaatt 1260
ttaagaaaag taactacttc atgcctttct cagcaggaaa acggatttgt gtgggagagg 1320
gcctggcccg catggagctg tttttattcc tgaccttcat tttacagaac tttaacctga 1380
aatctctgat tgacccaaag gaccttgaca caactcctgt tgtcaatgga tttgcttctg 1440
tcccgccctt ctatcagctg tgcttcattc ctgtctgaag aagcacagat ggtctggctg 1500
ctcctgtgct gtccctgcag ctctctttcc tctggtccaa atttcactat ctgtgatgct 1560
tcttctgacc cgtcatctca cattttccct tcccccaaga tctagtgaac attcagcctc 1620
cattaaaaaa gtttcactgt gcaaatatat ctgctattcc ccatactcta taatagttac 1680
attgagtgcc acataatgct gatacttgtc taatgttgag ttattaacat attattatta 1740
aataga 1746 <210> 2 <211> 743 <212> DNA <213> Homo sapiens <400> 2 tcagaaatat ttgaagcctg tgtggctgaa taaaagcata caaatacaat gaaaatatca 60
tgctaaatca ggcttagcaa atggacaaaa tagtaacttc gtttgctgtt atctctgtct 120
actttcctag ctctcaaagg tctatggccc tgtgttcact ctgtattttg gcctgaaacc 180
catagtggtg ctgcatggat atgaagcagt gaaggaagcc ctgattgatc ttggaggaga 240
gttttctgga agaggcattt tcccactggc tgaaagagct aacagaggat ttggtaggtg 300
tgcatgtgcc tgtttcagca tctgtcttgg ggatggggag gatggaaaac agagacttac 360
agagctcctc gggcagagct tggcccatcc acatggctgc ccagtgtcag cttcctcttt 420
cttgcctggg atctccctcc tagtttcgtt tctcttcctg ttaggaattg ttttcagcaa 480
tggaaagaaa tggaaggaga tccggcgttt ctccctcatg acgctgcgga attttgggat 540
ggggaagagg agcattgagg accgtgttca agaggaagcc cgctgccttg tggaggagtt 600
gagaaaaacc aagggtgggt gaccctactc catatcactg accttactgg actactatct 660
tctctactga cattcttgga aacatttcag gggtggccat atctttcatt atgagtctgg 720
ttgttagctc atgtgaagcg ggg 743 <210> 3 <211> 323 <212> DNA <213> Homo sapiens <400> 3 cccctgaatt gctacaacaa atgtgccatt tttctccttt tccatcagtt tttacttgtg 60
tcttatcagc taaagtccag gaagagattg aacgtgtgat tggcagaaac cggagcccct 120
gcatgcaaga caggagccac atgccctaca cagatgctgt ggtgcacgag gtccagagat 180
accttgacct tctccccacc agcctgcccc atgcagtgac ctgtgacatt aaattcagaa 240
actatctcat tcccaaggta agtttgtttc tcctacactg caactccatg ttttcgaagt 300
cccaaattca tagtatcatt ttt 323 <210> 4 <211> 20 <212> DNA <213> Homo sapiens <400> 4 ttacaaccag agcttggcat 20 <210> 5 <211> 20 <212> DNA <213> Homo sapiens <400> 5 atcaataaag tcccgagggt 20 <210> 6 <211> 21 <212> DNA <213> Homo sapiens <400> 6 aagtaatttg ttatgggttc c 21 <210> 7 <211> 20 <212> DNA <213> Homo sapiens <400> 7 tgcaatgtga tctgctccat 20 <210> 8 <211> 20 <212> DNA <213> Homo sapiens <400> 8 gttcagggct tggtcaatat 20 <210> 9 <211> 18 <212> DNA <213> Homo sapiens <400> 9 gattgtaagc accccctg 18 <210> 10 <211> 21 <212> DNA <213> Homo sapiens <400> 10 cctagtttcg tttctcttcc t 21 <210> 11 <211> 21 <212> DNA <213> Homo sapiens <400> 11 cagtgatatg gagtagggtc a 21 <210> 12 <211> 18 <212> DNA <213> Homo sapiens <400> 12 aagaggagca ttgaggac 18 <210> 13 <211> 20 <212> DNA <213> Homo sapiens <400> 13 gtccaggaag agattgaacg 20 <210> 14 <211> 20 <212> DNA <213> Homo sapiens <400> 14 catggagttg cagtgtagga 20 <210> 15 <211> 18 <212> DNA <213> Homo sapiens <400> 15 tggtggggag aaggtcaa 18 <210> 16 <211> 18 <212> DNA <213> Homo sapiens <400> 16 gggcttcctc ttgaacac 18 <210> 17 <211> 18 <212> DNA <213> Homo sapiens <400> 17 cacgaggtcc agagatac 18 <210> 18 <211> 19 <212> DNA <213> Homo sapiens <400> 18 ctatcattga ttatttccc 19 <210> 19 <211> 22 <212> DNA <213> Homo sapiens <400> 19 gaaaattatt gcatatctgg at 22

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Claims (18)

[Claims] 1. A method for determining the ability of a cell in a sample to metabolize a particular drug, comprising the steps of: a) removing a detectable amount of single-stranded DNA from said sample using known methods; B) hybridizing the single-stranded DNA obtained in step a) with a detection primer containing a plurality of nucleotide residues (provided that the primer is a cytochrome P
A defined point mutation in the single-stranded DNA encoding the 450 isoform protein, wherein the point mutation is known to affect the ability of the isoform protein to metabolize the drug. ) Is complementary to a target nucleotide sequence that is immediately adjacent and 5 'to, and thus, when the detection primer hybridizes to the target nucleic acid, between the defined point mutation and the 3' end of the detection primer. No nucleotide residue identical to the first or second nucleotide residue of the point mutation to be detected at c.); C) extending the primer with a polymerizing agent in a mixture comprising one or more nucleoside triphosphates (However, the mixture may contain a means for detecting the incorporation of nucleoside triphosphate into the nucleic acid polymer. Complementarity of at least one of the nucleoside triphosphates to the nucleotide residues, and one or more chain terminating nucleoside triphosphates optionally); d) the nucleoside triphosphates incorporation detected using said means,
Thereby, it is determined whether the sample contains the point mutation of the cytochrome P450 isoform protein. 2. The single-stranded DNA isolated and / or prepared in step a) is
One of the two amplification primers is obtained by performing a modified amplification reaction comprising a first attachment moiety attached to the primer, whereby a double-stranded amplification product wherein only one of the strands comprises the first attachment moiety. (Wherein the first attachment moiety is affinity versus DEF)
INIERA) and then simultaneously or sequentially out of sequence to single stranded amplification product, and convert the strand containing the first attachment moiety to a solid support with the help of the other component of the affinity pair. 2. The method according to claim 1, further comprising the step of removing all unbound substances. 3. The method according to claim 1, wherein the point mutation to be detected contains only one changed nucleotide. 4. A detection primer which hybridizes to a target nucleotide sequence located immediately adjacent and 5 'to a point mutation of a single-stranded DNA encoding a cytochrome P450 isoform protein CYP2C19, comprising the following sequence:
A detection primer consisting of a subsequence of -70 nucleotides, provided that the subsequence always includes the nucleotide located at the 3 'end of the following sequence: 5. The detection primer according to claim 4, comprising a subsequence of 10-30 nucleotides. 6. The detection primer according to claim 5, which is: 7. A detection primer which hybridizes to a target nucleotide sequence located immediately adjacent to and 5 ′ to a point mutation of a single-stranded DNA encoding a cytochrome P450 isoform protein CYP2C19, comprising the following sequence:
A detection primer consisting of a subsequence of -70 nucleotides, provided that the subsequence always includes the nucleotide located at the 3 'end of the following sequence: 8. A detection primer which hybridizes to a target nucleotide sequence located immediately adjacent to and 5 ′ to a point mutation of a single-stranded DNA encoding the cytochrome P450 isoform protein CYP2C19, comprising the following sequence: −
A detection primer consisting of a 50 nucleotide subsequence (provided that the subsequence always includes the nucleotide located at the 3 'end of the following sequence): 9. The detection primer according to claim 8, which is: 10. A detection primer which hybridizes to a target nucleotide sequence located immediately adjacent and 5 ′ to a point mutation of a single-stranded DNA encoding the cytochrome P450 isoform protein CYP2C9, wherein the detection primer has the following sequence: −
A detection primer consisting of a 50 nucleotide subsequence (provided that the subsequence always includes the nucleotide located at the 3 'end of the following sequence): 11. The detection primer according to claim 10, which is: 12. A detection primer which hybridizes to a target nucleotide sequence located immediately adjacent and 5 ′ to a point mutation of a single-stranded DNA encoding a cytochrome P450 isoform protein CYP2C9, comprising the following sequence: −
A detection primer consisting of a 50 nucleotide subsequence (provided that the subsequence always includes the nucleotide located at the 3 'end of the following sequence): 13. A detection primer which hybridizes to a target nucleotide sequence located immediately adjacent to and 5 ′ to a point mutation of a single-stranded DNA encoding a cytochrome P450 isoform protein CYP2C9, comprising the following sequence: −
A detection primer consisting of a 50 nucleotide subsequence (provided that the subsequence always includes the nucleotide located at the 3 'end of the following sequence): 14. The detection primer according to claim 13, which is: 15. A detection primer which hybridizes to a target nucleotide sequence located immediately adjacent and 5 ′ to a point mutation of a single-stranded DNA encoding a cytochrome P450 isoform protein CYP2C9, comprising the following sequence: −
A detection primer consisting of a 50 nucleotide subsequence (provided that the subsequence always includes the nucleotide located at the 3 'end of the following sequence): 16. A cytochrome P450 isoform protein C comprising:
A kit for detecting possible point mutations in YP2C19: a) a detection primer according to any one of claims 4-9; b) a sequence according to SEQ ID NO: 1 and complementary to SEQ ID NO: 1. Amplification primers derived from different sequences (provided that the primers are capable of amplifying a subsequence of the sequence of SEQ ID NO: 1 containing the possible point mutation and a sequence complementary to the detection primer). C) at least one labeled nucleoside triphosphate; and d) a DNA polymerizing agent. 17. A cytochrome P450 isoform C comprising:
Kit for detecting possible point mutations in YP2C9: a) Detection primer according to any one of claims 10-12; b) Complementary to SEQ ID NO: 2 and SEQ ID NO: 2 Amplification primers derived from different sequences (provided that the primers are capable of amplifying a subsequence of the sequence of SEQ ID NO: 2 containing the possible point mutation and a sequence complementary to the detection primer). C) at least one labeled nucleoside triphosphate; and d) a DNA polymerizing agent. 18. A cytochrome P450 isoform C comprising:
Kit for detecting possible point mutations in YP2C9: a) Detection primer according to any one of claims 13-15; b) Complementary to SEQ ID NO: 3 and SEQ ID NO: 3 Amplification primers derived from different sequences (provided that the primer is capable of amplifying a subsequence of the sequence of SEQ ID NO: 3 containing the possible point mutation and a sequence complementary to the detection primer). C) at least one labeled nucleoside triphosphate; and d) a DNA polymerizing agent.