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WO2004111231A1 - Necessaire de detection du polymorphisme genetique - Google Patents

Necessaire de detection du polymorphisme genetique Download PDF

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Publication number
WO2004111231A1
WO2004111231A1 PCT/JP2004/008125 JP2004008125W WO2004111231A1 WO 2004111231 A1 WO2004111231 A1 WO 2004111231A1 JP 2004008125 W JP2004008125 W JP 2004008125W WO 2004111231 A1 WO2004111231 A1 WO 2004111231A1
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Prior art keywords
dna
seq
probe group
probe
iii
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English (en)
Japanese (ja)
Inventor
Nobutaka Kusaba
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Nipro Corp
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Nipro Corp
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    • 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
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • 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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • 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
  • SNP polymorphism
  • MDR1 multidrug resistance
  • steroids have been used as therapeutic agents in various fields such as collagen disease and dermatitis because of their efficacy.
  • you may be able to avoid taking medications that are more likely to cause side effects such as bone disease, diabetes, cataracts and glaucoma.
  • 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.
  • SNP gene polymorphism
  • 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.
  • 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). ).
  • 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)
  • the present invention provides
  • 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;
  • a first probe group consisting of the DNA of SEQ ID NO: 1 and the DNA of SEQ ID NO: 2:
  • the gene polymorphism detection kit according to (1) which comprises at least the following (i), (ii) or (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 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.
  • 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
  • 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.
  • the gene polymorphism detection kit of the present invention comprises a probe group comprising at least the following (i), (ii) or (iii).
  • 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.
  • 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)
  • 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.
  • 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).
  • 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
  • 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
  • AT type when detected with only the DNA probe of SEQ ID NO: 3 and 4
  • it can be easily detected as AT type.
  • 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.
  • T thymine
  • C cytosine
  • 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.
  • 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.
  • a silane coupling agent having an amino group such as aminoethoxysilane is used.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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).
  • a DN containing a site of a gene polymorphism to be detected in advance In order to use the nucleic acid for detection, a DN containing a site of a gene polymorphism to be detected in advance
  • 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.
  • 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.
  • the number of bases of the nucleic acid to be amplified is 100-400 base pairs, preferably 150-300 base pairs.
  • 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.
  • the kit for detecting a genetic polymorphism according to the present invention includes at least the following (i), (ii) or (m).
  • 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
  • the primers of SEQ ID Nos. 10, 11, 13 and 15 correspond to the reverse primer.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the present invention further includes a kit capable of simultaneously detecting two places, preferably three places, as described above.
  • 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.
  • 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).
  • Exon 21 SNP at position 2677 is more likely to cause side effects of steroids in GT, GA, TT, ⁇ , and ⁇ ⁇ humans GG type humans are more susceptible to side effects than other SNPs ,.
  • 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 ⁇ l of terminal transferase (SOunit / zz1), 10 ⁇ l of thymidine triphosphate (lOpmolZxl), 4 ⁇ l of DNA probe of SEQ ID NO: 1 or 2 (50 mM), Prepare 5 ⁇ l of attached NEBuffer4 and 50 ⁇ l of purified water containing 5 a1 of cacodylate buffer supplied with the product, and react at 37 ° C for 4 hours. By adding 50 ⁇ l, a polythymine-added nucleic acid probe 2 ⁇ mol / ⁇ 1 was obtained.
  • 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 / ⁇ 1 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.
  • 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. .
  • a solution (20 x L) of sodium hydroxide (5 ⁇ ) and ethylenediaminetetraacetic acid (0.05 ⁇ ) was added to 20 ⁇ L of the amplified DNA solution, stirred well, and left to stand for 5 minutes to amplify.
  • the DNA was denatured to single strand.
  • 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.
  • Sample Nos. 1 to 12 each have the genetic polymorphism (SNP) shown in Table 2. It was determined that.
  • 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.
  • Type A detection site 4 T-type detection site
  • 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.
  • 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.
  • 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.

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Abstract

La présente invention concerne un nécessaire permettant de détecter facilement le polymorphisme génétique (SNP) par rapport au gène de multirésistance aux médicaments (MDR1) de l'ADN génomique prélevé chez un humain. L'invention concerne plus particulièrement un nécessaire permettant de détecter le 1236ème polymorphisme génétique (SNP) de l'exon 12, le 1677ème polymorphisme génétique (SNP) de l'exon 21, ou le 3435ème polymorphisme génétique (SNP) de l'exon 26, et de préférence, de détecter simultanément ces polymorphismes génétiques (SNP) par rapport au gène MDR1 de l'ADN génomique prélevé chez un humain. L'invention concerne également un nécessaire de détection du polymorphisme génétique permettant, à partir des résultats de détection, de prévoir les éventuels effets secondaires futurs provoqués par la prise d'un stéroïde.
PCT/JP2004/008125 2003-06-13 2004-06-10 Necessaire de detection du polymorphisme genetique Ceased WO2004111231A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2010263810A (ja) * 2009-05-13 2010-11-25 Kumamoto Univ エルロチニブの副作用又は薬効を判定する方法
CN105525005A (zh) * 2016-01-22 2016-04-27 广州金域检测科技股份有限公司 一种检测mdr1基因多态性的引物及其方法

Non-Patent Citations (7)

* Cited by examiner, † Cited by third party
Title
CHEE M. ET AL.: "Accessing genetic information with high-density DNA arrays", SCIENCE, vol. 274, 1996, pages 610 - 614, XP002914032 *
HOFFMEYER S. ET AL.: "Functional polymorphisms of the human multidrug-resistance gene: multiple sequence variations and correlation of one allele with P-glycoprotein expression and activity in vivo", PNAS, vol. 97, no. 7, 2000, pages 3473 - 3478, XP000996264 *
KIM R.B. ET AL.: "Identification of functionally variant MDR1 alleles among European Americans and Africans Americans", CLINICAL PHARMACOLOGY & THERAPEUTICS, vol. 70, no. 2, 2001, pages 189 - 199, XP002033576 *
MANO H.: "genome kagaku o meguru naigai no doko DNA chip", PROTEIN, NUCLEIC ACID AND ENZYME, vol. 46, no. 16, 2001, pages 2625 - 2629, XP002983543 *
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TANABE M. ET AL.: "Expression of P-glycoprotein in human placenta: relation to genetic polymorphism of the multidrug resistance (MDR)-1 gene", THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS, vol. 297, no. 3, 2001, pages 1137 - 1143, XP001030013 *
TSUKUDA K. ET AL.: "Screening of patients with maternally transmitted diabetes for mitochondrial gene mutations in the tRNA Leu(UUR) region", DIABETIC MEDICINE, vol. 14, no. 12, 1997, pages 1032 - 1037, XP002983542 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2010263810A (ja) * 2009-05-13 2010-11-25 Kumamoto Univ エルロチニブの副作用又は薬効を判定する方法
CN105525005A (zh) * 2016-01-22 2016-04-27 广州金域检测科技股份有限公司 一种检测mdr1基因多态性的引物及其方法

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