WO2004111231A1

WO2004111231A1 - Genetic polymorphism detection kit

Info

Publication number: WO2004111231A1
Application number: PCT/JP2004/008125
Authority: WO
Inventors: Nobutaka Kusaba
Original assignee: Nipro Corporation
Priority date: 2003-06-13
Filing date: 2004-06-10
Publication date: 2004-12-23

Abstract

A kit capable of readily detecting genetic polymorphism (SNP) with respect to multidrug resistance (MDR1) gene of genomic DNA sampled from human. In particular, a kit capable of readily detecting 1236th genetic polymorphism (SNP) of exon 12, 2677th genetic polymorphism (SNP) of exon 21 or 3435th genetic polymorphism (SNP) of exon 26, preferably simultaneously detecting these genetic polymorphisms (SNP), with respect to MDR1 gene of genomic DNA sampled from human. There is further provided a genetic polymorphism detection kit whereby the future possibility of side effects caused by taking of a steroid drug can be foreseen from detection results.

Description

Specification

Gene polymorphism detection kit

Technical field

[0001] The present invention is directed to the detection of the polymorphism (SNP) of the multidrug resistance (MDR1) gene at the 1236th position of etason 12, the 2677th position of etason 21, or the 3435th position of etason 26 in genomic DNA collected from humans The present invention relates to a gene polymorphism detection kit and a detection method that can be easily performed.

Background art

[0002] Since their discovery, steroids have been used as therapeutic agents in various fields such as collagen disease and dermatitis because of their efficacy. On the other hand, you may be able to avoid taking medications that are more likely to cause side effects such as bone disease, diabetes, cataracts and glaucoma.

[0003] The MDR1 gene is a gene encoding a P-glycoprotein, which is a type of transport protein that transports harmful substances out of cells, and has been actively studied. It is known that the type of the base at a specific position in this MDR1 gene differs depending on the individual, and is called a gene polymorphism (SNP). This SNP is thought to influence the potential side effects of the drug.

[0004] Therefore, a method of predicting the resistance of a patient to a steroid in advance by examining the SNP of the MDR1 gene has been actively studied.

[0005] Candidates for SNP of the MDR1 gene relating to steroid drug resistance include exon 12 at 1236, exon 21 at 2677 or exon 26 at 3435. For example, it is known that the 1236th base of exon 12 is thymine (T) or cytosine (C). Since human chromosomes are dimers, there are three types of SNPs, TT, TC and CC, and it is said that these SNPs are related to steroid side effects (Non-patent Documents 1 and 2). ).

[0006] However, at present, there has been only a very powerful and time-consuming detection method such as directly analyzing a nucleotide sequence using a sequencer. From this, it is considered that detecting the SNP of the MDR1 gene is very useful. [0007] Non-patent document 1: Tanabe et al., The Journal of pharmacology and experimental therapeutics, 297 (3), 1137

Non-Patent Document 2: Hoffmeyer et al., Proceedings of the National Academy of Sciences of the United States of America, Vol. 97 No. 7 3473-3478, March 28 (2000)

Disclosure of the invention

Problems the invention is trying to solve

[0008] Therefore, there is a need for the development of a kit and a detection method that can more easily detect a gene polymorphism in the MDR1 gene.

Means for solving the problem

[0009] The present invention provides

(1) A kit for detecting a genetic polymorphism (SNP) in genomic DNA contained in a biological sample collected from a human, which does not contain at least the following probe group (i), (ii) or (iii): Gene polymorphism detection kit;

(1) A first probe group consisting of the DNA of SEQ ID NO: 1 and the DNA of SEQ ID NO: 2:

(ii) a second probe group consisting of the DNA of SEQ ID NO: 3, the DNA of SEQ ID NO: 4, and the DNA of SEQ ID NO: 5:

(iii) a third probe group consisting of DNA of SEQ ID NO: 6 and DNA of SEQ ID NO: 7, or DNA of SEQ ID NO: 6 and DNA of SEQ ID NO: 8,

(2) The kit for detecting a gene polymorphism according to (1), wherein the probe group comprises a detection strip immobilized on a carrier surface;

(3) The gene polymorphism detection kit according to (2), wherein the carrier is a bullet polymer or a polyester;

(4) The gene polymorphism detection kit according to (2), wherein the carrier is polyethylene, polypropylene, polystyrene, or polyethylene terephthalate.

(5) The gene polymorphism detection kit according to (1), comprising at least all of the following probe groups (i), (ii) and (iii):

(i) a first probe group consisting of the DNA of SEQ ID NO: 1 and the DNA of SEQ ID NO: 2:

(ii) a second sequence consisting of the DNA of SEQ ID NO: 3, the DNA of SEQ ID NO: 4 and the DNA of SEQ ID NO: 5 Probe groups:

(6) The polymorphism detection kit according to (1), comprising at least all of the following probe groups (i), (ii) and (iii):

(iii) a third probe group consisting of the DNA of SEQ ID NO: 6 and the DNA of SEQ ID NO: 8,

(7) The gene polymorphism detection kit according to (1), which comprises at least the following (i), (ii) or (iii);

(i) a first probe group consisting of the DNA of SEQ ID NO: 1 and the DNA of SEQ ID NO: 2, and the DNA consisting of the DNA of SEQ ID NO: 9 and the DNA of SEQ ID NO: 10, or the DNA of SEQ ID NO: 9 and the DNA of SEQ ID NO: 11 First primer pair:

(ii) a second probe group consisting of the DNA of SEQ ID NO: 3, the DNA of SEQ ID NO: 4 and the DNA of SEQ ID NO: 5, and a second primer pair consisting of the DNA of SEQ ID NO: 12 and the DNA of SEQ ID NO: 13:

(iii) a third probe group consisting of the DNA of SEQ ID NO: 6 and the DNA of SEQ ID NO: 7, or the DNA of SEQ ID NO: 6 and the DNA of SEQ ID NO: 8, and the third probe group consisting of the DNA of SEQ ID NO: 14 and the DNA of SEQ ID NO: 15 3 primer pairs.

(8) Described in (7) in which a fluorescent substance or a radioisotope is modified on the DNA of SEQ ID NOs: 9 to 15, the DNA of SEQ ID NOs: 9, 12, and 14, or the DNA of SEQ ID NOs: 10, 11, 13, and 15 Gene polymorphism detection kit,

(9) The gene according to (7), wherein a biotin is modified at the 5 ′ end of the DNA of SEQ ID NOS: 9 to 15, the DNA of SEQ ID NOs: 9, 12, and 14, or the DNA of SEQ ID NOs: 10, 11, 13, and 15 Polymorphism detection kit,

(10) In addition to (9), which contains streptavidin, nitroblue tetrazolium (NBT) and 5_bromo-4-chloro-3_indolylphosphatase p-toluidinyl salt (BCIP) Gene polymorphism detection kit, and

(11) The gene polymorphism detection kit according to claim 7, comprising at least all of the following (i), (ii) and (iii):

(i) a first probe group consisting of the DNA of SEQ ID NO: 1 and the DNA of SEQ ID NO: 2, and a first primer pair consisting of the DNA of SEQ ID NO: 9 and the DNA of SEQ ID NO: 11:

(iii) A third probe group consisting of the DNA of SEQ ID NO: 6 and the DNA of SEQ ID NO: 7, and a third primer pair consisting of the DNA of SEQ ID NO: 14 and the DNA of SEQ ID NO: 15.

The invention's effect

[0010] The gene polymorphism detection kit of the present invention easily detects the 1236th SNP of exon 12, 2677th of exon 21, or 3435th of exon 26 of the MDR1 gene in genomic DNA collected from a human. It is preferable that the above three SNPs can be simultaneously detected. From these results, it is possible to predict the possibility of side effects in the future when taking steroids.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the result of detection of the 1236th SNP of exon 12 of the MDR1 gene.

FIG. 2 shows the result of detection of the 2677th SNP of exon 21 of the MDR1 gene.

FIG. 3 shows the result of detection of the SNP at position 3435 of exon 26 of the MDR1 gene.

FIG. 4 shows the results of simultaneous detection of the above three types of SNPs.

FIG. 5 shows the results of single detection of the 1236th SNP of exon 12 of the MDR1 gene in FIG.

FIG. 6 shows the results of single detection of the 2677th SNP of exon 21 of the MDR1 gene in FIG.

FIG. 7 shows the result of single detection of the SNP at position 3435 in exon 26 of the MDR1 gene in FIG. BEST MODE FOR CARRYING OUT THE INVENTION

[0012] The present invention is a kit for detecting SNP in genomic DNA contained in a biological sample collected from a human. The biological sample in the present invention includes blood, saliva, hair, and the like, and is preferably various human cells such as blood cells, epidermal cells, and mucosal cells. Methods for extracting DNA from these biological samples can be performed by known methods, for example, phenol extraction, guanidine thiocinnate extraction, vanadyl ribonucleoside complex extraction, and the like.

[0013] The gene polymorphism detection kit of the present invention comprises a probe group comprising at least the following (i), (ii) or (iii).

(iii) a third probe group consisting of the DNA of SEQ ID NO: 6 and the DNA of SEQ ID NO: 7, or the DNA of SEQ ID NO: 6 and the DNA of SEQ ID NO: 8;

The DNA of SEQ ID NO: 18 in the above (i), (ii) and (iii) is a probe for detecting the SNP of the MDR1 gene. These DNAs can be synthesized by a commercially available DNA / RNA synthesizer or the like, and the synthesis method is not limited.

[0014] (i) The first probe group consisting of the DNA of SEQ ID NO: 1 and the DNA of SEQ ID NO: 2 can detect the 1236th SNP of etason 12. It is known that the 1236th base of exon 12 is thymine (T) or cytosine (C). Since human chromosomes are dimers, there are three types of SNPs: TT, TC and CC. The DNA probe of SEQ ID NO: 1 is capable of detecting when the base at position 1236 of exon 12 is cytosine (C), and the DNA probe of SEQ ID NO: 2 is capable of detecting the base force at position 1236 of exon 12 ( T) can be detected. Therefore, if it is detected only with the DNA probe of SEQ ID NO: 1, it will be CC type, if it is detected only with the DNA probe of SEQ ID NO: 2, it will be TT type, if it is detected with both the DNA probes of SEQ ID NOs: 1 and 2, It can be easily detected as CT type.

(Ii) a second DNA consisting of the DNA of SEQ ID NO: 3, the DNA of SEQ ID NO: 4, and the DNA of SEQ ID NO: 5 Can detect the 2677th SNP of exon 21. It is known that the 2677th base of exon 21 is guanine (G), adenine (A) or thymine (T). Since human chromosomes are dimers, there are six types of SNPs: GG, AA, TT, GA, GT, and AT. The DNA probe of SEQ ID NO: 3 can be detected when the base at position 2677 of exon 21 is guanine (G), and the DNA probe of SEQ ID NO: 4 is that the base of position 2677 of etason 21 is adenine (A) And the DNA probe of SEQ ID NO: 5 can be detected when the 2677th base of exon 21 is thymine (T). Therefore, GG type when detected with only the DNA probe of SEQ ID NO: 3, AA type when detected with only the DNA probe of SEQ ID NO: 4, and TT type when detected with only the DNA probe of SEQ ID NO: 5 GA type when detected with DNA probes of SEQ ID NOS: 3 and 4, GT type when detected with DNA probes of SEQ ID NOs: 3 and 5, detected with DNA probe of SEQ ID NOs: 4 and 5 In this case, it can be easily detected as AT type.

[0016] The third probe group consisting of the DNA of SEQ ID NO: 6 and the DNA of SEQ ID NO: 7, or the DNA of SEQ ID NO: 6 and the DNA of SEQ ID NO: 8 is capable of detecting the SNP at position 3435 of exon 26. S can. The 3435th base of exon 26 is known to be thymine (T) or cytosine (C). Since human chromosomes are dimers, there are three types of SNPs: TT type, TC type and CC type. The DNA probe of SEQ ID NO: 6 can be detected when the base at position 3435 of exon 26 is thymine (T), and the DNA probes of SEQ ID NOs: 7 and 8 are those whose base at position 3435 of exon 26 is cytosine (C ) Can be detected. Either of SEQ ID NOS: 7 and 8 can be selected. Therefore, CC type when detected only with the DNA probe of SEQ ID NO: 6, TT type when detected with only the DNA probe of SEQ ID NO: 7 or 8, DNA probes of SEQ ID NO: 6 and 7, Alternatively, when it is detected with the DNA probes of SEQ ID NOS: 6 and 8, it can be easily detected as CT type.

The present invention includes a detection strip obtained by physically or chemically immobilizing the DNA probe group on a carrier surface.

The carrier may be a bull-based polymer or polyester, more specifically, polyethylene or polypropylene. Organic materials such as pyrene, polystyrene, polyethylene terephthalate or nitrocellulose, inorganic materials such as glass or silica, and metal materials such as gold and silver are not particularly limited, but molding processability is easy. Organic materials are preferred, and polyesters such as polyethylene terephthalate are more preferred. It is preferable that these carriers are white in order to make the color development easier to detect in the detection by the color development method.

[0018] As a method of physically immobilizing a DNA probe on the surface of the carrier, for example, a polycationic polymer such as polylysine is coated on the surface of the carrier to obtain a DNA such as polyadione. The efficiency of immobilization can be increased by electrostatic interaction with the probe, and the efficiency of immobilization can be increased by adding an unrelated base sequence (such as polythymine chain) to the probe DNA and increasing the molecular weight of the DNA. Can also be given. Further, when the surface of the carrier is glass, etc., a silane coupling agent having an amino group such as aminoethoxysilane is used. When the surface of the carrier is gold, a thiol or disulfide compound having an amino group such as 2-aminoethanethiol is used. It has been known that, by treating the carrier surface with, for example, the immobilization efficiency is improved due to electrostatic interaction with a DNA probe which is a polyanion.

On the other hand, as a method for chemically immobilizing a probe DNA by introducing a functional group into the probe DNA, for example, when the carrier material is an inorganic material such as glass or silicon, the probe for nucleic acid detection is used. The terminal of the polymer may be modified with a functional group capable of a silane coupling reaction such as trimethoxysilane or triethoxysilane, immersed in the solution for 24-48 hours, taken out, and then washed. Alternatively, a probe in which the surface of an inorganic material such as glass or silicon is treated with a silane coupling agent having an amino group such as aminoethoxysilane to aminify the surface of the carrier and a carboxylic acid is introduced into the terminal. There is a method of immobilization by reacting DNA with an amino coupling reaction. Furthermore, when the carrier material is a metal material such as gold or silver, the terminal of the probe for nucleic acid detection is modified with a functional group capable of binding to a metal such as a thiol group or a disulfide group, and the probe is immersed in the solution for 24-48 hours. After removal, it can be washed and fixed.

[0020] In addition, a method of synthesizing a DNA probe directly on a fixed surface by using a lithography technique for immobilizing a synthesized DNA probe directly on the immobilized surface is also known. Clearly, this also includes this method.

[0021] The gene polymorphism detection kit of the present invention preferably detects any one of the SNPs of exon 12 at position 1236, exon 21 at position 2677 or exon 26 at position 3435, and is preferably selected from the above. This is a kit for simultaneously detecting two SNPs, and more preferably a kit for simultaneously detecting the three SNPs. Therefore, a further preferred embodiment of the gene polymorphism detection kit of the present invention includes at least all of the following probe groups (i), (ii) and (iii).

[0022] In the case of simultaneous detection of two or more sites, the probe that can be detected when the base 3435 of exon 26 is thymine (T) is a non-sense probe among the DNA probes of SEQ ID NOS: 7 and 8. It is preferable to select a DNA probe of SEQ ID NO: 8 that can suppress specific adsorption. Therefore, a particularly preferred embodiment of the gene polymorphism detection kit of the present invention includes at least all of the following probe groups (i), (ii) and (iii).

(iii) a third probe group consisting of the DNA of SEQ ID NO: 6 and the DNA of SEQ ID NO: 8;

[0023] In order to use the nucleic acid for detection, a DN containing a site of a gene polymorphism to be detected in advance

A needs to be amplified. Amplification of a nucleic acid can be performed by a known method, such as a PCR method, a NASBA method, a LAMP method, or an ICAN method, on DNA extracted from a human biological sample, and can be preferably performed by a PCR method.

Therefore, it is preferable that the genetic polymorphism detection kit of the present invention contains primers used for PCR. The primer is particularly limited as long as it can amplify DNA containing the 1236th base of exon 12, the 2677th base of exon 21 or the 3435th base of exon 26. Although not required, the number of bases of the nucleic acid to be amplified is 100-400 base pairs, preferably 150-300 base pairs.

More preferably, the primer containing the 1236th DNA of exon 12 has the DNA of SEQ ID NOS: 9 and 10, or the first primer pair consisting of the DNAs of SEQ ID NOS: 9 and 11; The second primer pair consisting of the DNAs of SEQ ID NOS: 12 and 13 is used for amplification of the DNA containing the 2677th position of SEQ ID NOS: 12 and 13. 3 primer pairs.

Therefore, the kit for detecting a genetic polymorphism according to the present invention includes at least the following (i), (ii) or (m).

Here, the DNAs of SEQ ID Nos. 18 to 18 are DNA probes, the primers of SEQ ID Nos. 9, 12 and 14 correspond to the forward primer, and the primers of SEQ ID Nos. 10, 11, 13 and 15 correspond to the reverse primer.

[0027] The PCR method can amplify a nucleic acid by a known technique. For example, (1) a denaturation step in which double-stranded genomic DNA is heat-treated under a reaction condition of about 92 to 95 ° C for about 30 seconds to 1 minute to form a single strand, and about 50% — Under a reaction condition of 65 ° C for about 20 seconds and 1 minute, (2) an annealing step in which at least two kinds of amplification primers are bound to form a double-stranded portion that serves as a PCR reaction starting point, (3 ) Nucleic acid can be obtained by repeating steps (1) and (3) of the chain elongation step, which is carried out using DNA polymerase under the reaction conditions of about 70 75 ° C for about 20 seconds and 5 minutes, 20 to 40 times by the usual method. Can be amplified. [0028] The nucleic acid amplified by the above method is brought into contact with the surface of the carrier on which the DNA probe is immobilized, and is reacted by DNA hybridization. The target DNA is detected by this hybridization reaction. As a detection method, a radioisotope, a fluorescent substance, a chemiluminescent substance, or the like can be used for detection. In order to easily detect these, a label labeled with a reverse primer is used.

[0029] Among the above detection methods, particularly preferred is nitrobutanoletrazolidium (NBT) / 5-bromo-14-chloro-13-indolyl phosphatase p-toluidinyl, which is relatively inexpensive and easy to carry out. This is the salt (BCIP) coloring method. Specifically, a nucleic acid is amplified using a primer modified with avidin at the 3 ′ or 5 ′ end of the above primer. The amplified nucleic acid having avidin at 5 ′ can be brought into contact with streptavidin after DNA hybridization, and then luminescent by reacting with NBT and BCIP.

[0030] Accordingly, the present invention provides fluorescent or fluorescent primers for all the primers of SEQ ID NOs: 9-15, the forward primers of SEQ ID NOs: 9, 12, and 14, or the reverse primers of SEQ ID NOs: 10, 11, 13 and 15. It is preferable to perform RI labeling or use a primer in which a biotin is modified at the 5 'end of the reverse primer.

[0031] The present invention further includes a kit capable of simultaneously detecting two places, preferably three places, as described above. In the kit capable of simultaneous detection, the reverse primer for amplifying DNA containing the 1236th base of exon 12 can suppress nonspecific adsorption of the amplified DNA of SEQ ID NOS: 10 and 11. From the viewpoint, it is preferable to select the reverse primer of SEQ ID NO: 11. Further, it has been disclosed above that the DNA of SEQ ID NO: 8 is preferable as the probe that can be detected when the 3435th base of exon 26 is thymine (T) in the simultaneous detection.

Therefore, the most preferred form of the present invention is the gene polymorphism detection kit according to claim 1, comprising at least all of the following (i), (ii) and (iii).

(ii) a second probe group consisting of the DNA of SEQ ID NO: 3, the DNA of SEQ ID NO: 4 and the DNA of SEQ ID NO: 5, and the second probe group consisting of the DNA of SEQ ID NO: 12 and the DNA of SEQ ID NO: 13. 2 primer pairs:

[0033] From the gene polymorphism detected using the gene polymorphism kit of the present invention, it is possible to predict the possibility that a side effect of a steroid agent will occur. Typically,

(a) Exon 12 1236th SNP force S, CT and TT type humans are less likely to cause side effects of steroids, and CC type humans are more likely to cause side effects than CT and TT types.

(b) Exon 21 SNP at position 2677 is more likely to cause side effects of steroids in GT, GA, TT, ΤΑ, and ヒト humans GG type humans are more susceptible to side effects than other SNPs ,.

(c) Exon 26 SNP at position 3435 is more likely to cause side effects of steroids in type III humans CC type and CT type humans are more likely to have side effects than type III.

It is said that.

Example

[0034] Hereinafter, the present invention will be described in detail with reference to Reference Examples and Examples, but the present invention is not limited to these Examples.

(Example 1: Detection of the 1236th SNP of exon 12)

Polythymine was added to the 5 ′ end of the DNA probes shown in SEQ ID NOs: 1 and 2 (supplied by Sigma Dienosis Japan) using terminal transferase (New England Biolab). Specifically, 4 μl of terminal transferase (SOunit / zz1), 10 μl of thymidine triphosphate (lOpmolZxl), 4 μl of DNA probe of SEQ ID NO: 1 or 2 (50 mM), Prepare 5 μl of attached NEBuffer4 and 50 μl of purified water containing 5 a1 of cacodylate buffer supplied with the product, and react at 37 ° C for 4 hours. By adding 50 μl, a polythymine-added nucleic acid probe 2ρmol / μ1 was obtained.

Then, the polythymine-added nucleic acid probes of SEQ ID NOs: 1 and 2 were diluted with 10 * SSC buffer supplied with the product to a concentration of 1.Opmol / μ1 and separated on a polyester membrane (4 × 0.4cm). 0.5 / i L each time, and irradiate with 312nm ultraviolet ray for 2 minutes And immobilized to create a detection strip.

Blood was collected from 12 randomly selected healthy Japanese, blood cells were fractionated, and genomic DNA was extracted and purified by phenol extraction. A forward primer having the nucleotide sequence shown in SEQ ID NO: 9; a reverse primer having the nucleotide sequence shown in 10 and having a biotin bound to the 5 'end; and Taq DNA polymerase (Roche's diagnostic status) Amplification of the nucleotide sequence containing the 1236th position of the nucleotide sequence of the MDR1 gene was performed by a PCR method using the same method. The PCR reaction conditions were as follows: denaturation step: 94 ° C, 30 seconds; annealing step: 55 ° C, 20 seconds; chain elongation step: 72 ° C, 20 seconds. .

[0037] A solution (20 x L) of sodium hydroxide (5Μ) and ethylenediaminetetraacetic acid (0.05Μ) was added to 20 µL of the amplified DNA solution, stirred well, and left to stand for 5 minutes to amplify. The DNA was denatured to single strand. In this sample solution, a solution (1 mL) of sodium dodecinole sulfate (0.01 w / v%), sodium chloride (1.8 w / v%), sodium taenoate (1. Ow / v%) and the above detection strip One piece was dried and shaken at a reaction temperature of 45 ° C. for 30 minutes to react. Then, add alkaline phosphatase-labeled streptavidin, add nitroblue tetrazolium (NBT) and 5_bromo_4_cloth-3-indolinolephosphatase p-toluidinole salt (BCIP), and place on the detection strip. The alkaline phosphatase bound to the sample bound to each probe was colored (FIG. 1). Table 1 shows a schematic diagram that serves as a criterion for judging the results in FIG.

[0038] [Table 1]

1: Color confirmation marker

2: C-type detection site

3: T-type detection site

Calligraphy: coloring

From the results in Fig. 1, Sample Nos. 1 to 12 each have the genetic polymorphism (SNP) shown in Table 2. It was determined that.

[0040] [Table 2]

In addition, the result of measuring the nucleotide polymorphism at position 3435 of exon 26 of the MDR1 gene using a sequencer separately from Sample Nos. 1 to 12 is consistent with the results in Table 2, and thus the gene polymorphism of the present invention is The detection kits and methods have been shown to be very useful.

(Example 2: Detection of the 2677th SNP of exon 21)

Next, a detection strip on which the DNA probes of SEQ ID NOs: 3, 4, and 5 were immobilized was prepared. SNPs were detected in the same manner as in Example 1 from blood of 12 healthy Japanese randomly selected (Fig. 2). SEQ ID NOS: 12 and 13 were used as primers in the PCR method. Table 3 shows a schematic diagram that serves as a criterion for judging the results in FIG.

[Table 3]

1: Color confirmation marker

2: G-type detection site

3: Type A detection site 4: T-type detection site

Hata: Color development

Further, from the results in FIG. 2, it was determined that Sample Nos. 1 to 10 had the SNPs shown in Table 5, respectively.

[Table 4]

On the other hand, the result of separately measuring the 2677th SNP of exon 21 of the MDR1 gene by a sequencer was consistent with the result in Table 4.

(Example 3: Detection of SNP at position 3435 of exon 26)

Next, a detection strip on which the DNA probes of SEQ ID NOS: 6 and 7 were immobilized was prepared. SNP was detected in the same manner as in Example 1 from blood of 10 healthy Japanese randomly selected (Fig. 3). SEQ ID NOS: 14 and 15 were used as primers in the PCR method. Table 5 shows a schematic diagram used as a criterion for judging the results in FIG.

[Table 5]

1: Color confirmation marker

2: C-type detection site

3: T-type detection site Hata: Color development

Further, from the results in FIG. 2, it was determined that Sample Nos. 1 to 10 had the SNPs shown in Table 6, respectively.

[0050] [Table 6]

On the other hand, the result of separately measuring the SNP at position 3435 of exon 26 of the MDR1 gene by a sequencer was consistent with the result in Table 6.

(Example 4: Simultaneous detection of three SNPs)

Next, a detection strip was prepared on which the DNA probes of SEQ ID NOS: 16 and 8 were immobilized. Simultaneous detection of three SNPs was performed from the blood of nine randomly selected healthy Japanese (Figure 4). Using three sets of six primers of SEQ ID NOS: 9 and 11 to 15 in the PCR method, PCR was performed simultaneously at three locations under the same conditions as in Example 1. For detection, the above PCR solution 20 / L was used. Fig. 4 shows the results. Table 7 shows a schematic diagram that is used as a criterion for judging the results in FIG.

[Table 7]

1: Color confirmation marker

2: 3435th C-type detection site

3: 3435th T-type detection site

4: G-type detection site at position 2677

5: 2677th T-type detection site

6: 2677th type A detection site

7: 1236th C-type detection site

8: 1236th T-type detection site

Calligraphy: coloring

Also, from the results in FIG. 4, it was determined that Sample No. 19 had the SNPs shown in Table 8, respectively.

[0055] [Table 8]

No.SNP (3435nt) SNP (2677nt) SNP (I236nt)

1 C T G T C T

2 C C G G C C

3 C C G G C T

4 C T G G C C

5 T T T T T T

6 T T G T T T

7 C T T A C T

8 C T G G C C

9 CTGTCT On the other hand, using the detection strip for detecting each SNP, each SNP of the same nine samples was detected according to Examples 13 to 13, respectively. The results agreed with the results in Table 8, confirming that simultaneous detection was possible (Fig. 5-7).

Industrial applicability

[0057] The gene polymorphism detection kit of the present invention easily detects the 1236th SNP of exon 12, the 2677th of etason 21 or the 3435th of exon 26 of the MDR1 gene in genomic DNA collected from humans. Can predict the potential for side effects in the future when taking steroids.

Claims

The scope of the claims

[1] A kit for detecting a gene polymorphism (SNP) in genomic DNA contained in a biological sample collected from a human, comprising at least the following probe group (i), (ii) or (m): Genetic polymorphism detection kit;

[2] The gene polymorphism detection kit according to claim 1, wherein the probe group comprises a detection strip immobilized on a carrier surface.

[3] The gene polymorphism detection kit according to claim 2, wherein the carrier is a Bier polymer or polyester.

[4] The gene polymorphism detection kit according to claim 2, wherein the carrier is polyethylene, polypropylene, polystyrene or polyethylene terephthalate.

[5] The gene polymorphism detection kit according to claim 1, comprising at least all of the following probe groups (i), (ii) and (iii):

[6] The gene polymorphism detection kit according to claim 1, comprising at least all of the following probe groups (i), (ii) and (iii):

[7] The kit for detecting a genetic polymorphism according to claim 1, which comprises at least the following (i), (¾ or (m);

(iii) a third probe group consisting of the DNA of SEQ ID NO: 6 and the DNA of SEQ ID NO: 7, or the DNA of SEQ ID NO: 6 and the DNA of SEQ ID NO: 8, and a third probe group consisting of the DNA of SEQ ID NO: 14 and the DNA of SEQ ID NO: 15 3 primer pairs.

[8] SEQ ID NO: 9-15 DNA, SEQ ID NO: 9, 12 and 14 DNA, or SEQ ID NO: 10,

The genetic polymorphism detection kit according to claim 7, wherein a fluorescent substance or a radioisotope is modified on the DNAs of 11, 13, and 15.

[9] SEQ ID NO: 9-15 DNA, SEQ ID NO: 9, 12 and 14 DNA, or SEQ ID NO: 10,

8. The gene polymorphism detection kit according to claim 7, wherein the 5 'end of the DNA of 11, 13, and 15 is modified with biotin.

[10] The gene polymorphism detection according to claim 9, further comprising streptavidin, nitroblue tetrazolium (NBT), and 5_bromo_4_chloro-3_indolylphosphatase p-toluidinyl salt (BCIP). kit.

[11] The gene polymorphism detection kit according to claim 7, which comprises at least all of the following (i), (ii) and (iii);

(iii) a third probe group consisting of the DNA of SEQ ID NO: 6 and the DNA of SEQ ID NO: 7, and And a third primer pair consisting of the DNA of SEQ ID NO: 14 and the DNA of SEQ ID NO: 15.