WO2002024905A1 - Procede permettant de determiner la mutation genetique - Google Patents

Procede permettant de determiner la mutation genetique Download PDF

Info

Publication number
WO2002024905A1
WO2002024905A1 PCT/JP2001/007942 JP0107942W WO0224905A1 WO 2002024905 A1 WO2002024905 A1 WO 2002024905A1 JP 0107942 W JP0107942 W JP 0107942W WO 0224905 A1 WO0224905 A1 WO 0224905A1
Authority
WO
WIPO (PCT)
Prior art keywords
primer
dna
nucleic acid
size
pcr
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/JP2001/007942
Other languages
English (en)
Japanese (ja)
Inventor
Nobuyuki Hamajima
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Individual
Original Assignee
Individual
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Individual filed Critical Individual
Priority to AU2001286218A priority Critical patent/AU2001286218A1/en
Publication of WO2002024905A1 publication Critical patent/WO2002024905A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • 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

Definitions

  • the present invention provides a method for easily discriminating the presence or absence of a gene mutation on a chromosome assumed in advance and the type on an allele.
  • PCR-RFLP combining the polymerase chain reaction (PCR) method and restriction enzyme cleavage (Erlich et al., Science, 255, 1643, pp. 1991) ) Is a typical example.
  • PCR-RFLP polymerase chain reaction
  • restriction enzyme cleavage Erlich et al., Science, 255, 1643, pp. 1991
  • DNA microarray method has been developed very recently (Brown et al., Nat. Gent. ⁇ 21, p. 33, 1999).
  • the PCR-RFLP method cannot be said to be a rapid method because the testing process involves a restriction enzyme treatment of 3 to 24 hours.
  • the DNA microarray method is extremely expensive to carry out, and is very disadvantageous in terms of processing a large number of samples. Therefore, there has been a demand for the development of a new method for discriminating gene mutations that can rapidly and inexpensively process a large amount of samples.
  • the present inventors can utilize a large number of samples quickly and inexpensively by taking advantage of the PCR method as it is, and further utilizing the restrictions on primer DNA sequence design, which should be noted when performing the PCR method.
  • a new method for identifying gene mutations Confronting two-pair primers PCR) that can be processed in a single step has been developed.
  • the present invention provides a primer DNA having a nucleic acid corresponding to a mutant nucleic acid at the 3 ′ end, a nucleic acid corresponding to a normal nucleic acid at 3 ′, a primer DNA having an end at a 3 ′ end, and two types thereof facing the same.
  • This is a method for determining the presence or absence of a predetermined gene mutation by confirming the size of a DNA fragment generated by a PCR reaction using four types of primer DNAs containing the same primer DNA.
  • the present invention utilizes at least two sets of primer DNA sequences that are designed to have a desired base sequence in advance.
  • the outline of the present invention is based on the target gene for which the presence or absence of a mutation is to be determined.
  • Figure 1 shows an example of SNPs assuming that the base on allele X is X and the nucleic acid corresponding to base X on allele Y is Y.
  • two pairs of primers DNA are designed as four types of primers DNA.
  • One set is a set for which allele X is to be amplified, and another set is a set for which allele Y is to be amplified.
  • one primer DNA (antisense primer 1R) is synthesized as the nucleic acid X 'complementary to the nucleic acid X, and the other end is synthesized as the nucleic acid X'. Synthesize primer 1F and combine them into one.
  • a sense primer DNA (primer 1F) whose 3 end is a nucleic acid Y is synthesized, and further downstream from the primer 2F.
  • the antisense primer DNA (primer 2R), which is located opposite to the primers, is synthesized to form another combination.
  • the size of the specific DNA amplification product observed after each reaction is a base pair (size a ), B is a base pair (size b).
  • size a and size b need to be distinguished in molecular weight by a suitable method such as gel electrophoresis. However, this may be achieved by arbitrarily adjusting the position of the primer DNA on the target gene so as to give a desired size difference.
  • a PCR reaction is carried out on a gene suspected of having a mutation at the same time using the two sets of primer DNAs described above, and the size of the PCR product is confirmed. The presence or absence of mutation and the type of mutation between alleles can be determined.
  • the bases that are problematic in the gene are both X alleles. Can be determined.
  • the problematic base on both alleles is YY
  • the size of the band confirmed after the PCR reaction is the size b and size c nodes, which means that the size b and size c
  • the band is specifically amplified, it means that the base in question in the gene can be determined to be Y for both alleles. If, after the PCR reaction, three types of nodes, size a, size b, and size c, are observed, the base on the allele X becomes X based on the same principle as described above.
  • c a + b-(d-1) where the base on the allele Y can be determined to be a heterozygote of Y.
  • d is the sum of the number of bases of both 1R and 2F primers.
  • nucleic acid X ′ is 3 and antisense primer 1R at the end is replaced with nucleic acid X3 and sense primer 2F at the end is replaced with antisense primer 1F.
  • Combination of primer 2R and anti-primer having nucleic acid Y at the 3 'end instead of sense primer 2F having nucleic acid Y at the 3' end for allele Y The present invention can also be carried out by combining 1R and sense primer 1F and performing a PCR reaction in the coexistence of these.
  • the size of the transcript is also size a, size b, and size c. When the size c is YY type, the size a and the size c are respectively confirmed.
  • nucleic acid corresponding to the mutant nucleic acid and the nucleic acid corresponding to the normal nucleic acid according to the present invention mean the mutant nucleic acid and the normal nucleic acid or the nucleic acid complementary thereto as in the above two examples.
  • the PCR reaction and the confirmation of the molecular weight of the PCR product are completed in two steps, and the PCR product is treated with the restriction enzyme over several hours.
  • the time required for discrimination can be significantly reduced.
  • the PCR-RFLP method there is a restriction that a restriction enzyme site must be designed in advance so that a fragment having a molecular weight difference required for fractionation after restriction enzyme treatment is generated. Therefore, it may be difficult to design an appropriate primer DNA depending on the nucleotide sequence of the target gene, but the PCR-CTPP method of the present invention has no such restrictions at all. It also has an advantage in the flexibility of primer-DNA design.
  • the present invention does not require cleavage of a PCR product with a restriction enzyme, it is not affected by various artificial by-products due to the restriction enzyme treatment, and the determination can be performed reliably. Can be done.
  • the PCR-CTPPP method of the present invention has an advantage even compared to the DNA microarray method. In other words, no special equipment is required, and the cost difference when processing a large amount of specimens is significantly lower than that of the DNA microarray method.
  • the present invention is useful for discrimination of so-called polymorphism, but can also be used for discrimination of a causative gene or a cancer gene of a specific disease, and mutation is substitution, addition, or deletion. Any of the above is applicable.
  • DNA containing the target gene to be discriminated can be subjected to a PCR reaction from blood or other tissues collected from a patient or a healthy person by a method commonly used by those skilled in the art in the field to which the present invention belongs.
  • the present invention can be carried out using this as a sample as long as it is prepared as a DNA of the order.
  • the design of the primer DNA used in the method of the present invention is based on three terminal Besides the determination of the nucleic acid in step 1 and that the difference between the molecular weight (base pair number) of the amplified DNA product and the PCR product when using the primer DNA is appropriate. There are no separate restrictions.
  • the size difference between the DNA product and the size of the PCR product can be appropriately determined depending on the sensitivity of the method for detecting the amplified product, but it is sufficient if there are tens to hundreds of base pairs in general. In the case of using a high-sensitivity detection method such as capillary-electrophoresis, the difference may be several base pairs.
  • Such a primer DNA design can be easily performed by a general person skilled in the art to which the present invention belongs based on the nucleotide sequence of the gene to be identified and the position of the mutation. It is something.
  • the preparation of the primer DNA itself can be easily performed using a so-called automatic DNA synthesizer or other general-purpose equipment or a synthesis method.
  • the PCR reaction in the present invention can also be performed under general reaction conditions that do not include special reagents or reaction steps, and commercially available PCR reactions can be used.
  • PCR reaction is performed using the primer D N
  • each primer set It is also possible to carry out the PCR reaction step by step.
  • the PCR product can be detected by any method available to those skilled in the art, such as agarose gel electrophoresis, polyacrylamide gel electrophoresis, or capillary electrophoresis. , High-performance liquid chromatography (HPLC) or gas chromatography, such as chromatography, mass spectrometry (GC-MS), etc. . Gel electrophoresis is preferred as a simpler method.
  • Example 1-Adrenoreceptor-2 (BAR-2) Polymorphism Detection of Adrenoceptor 2 (BAR-2), which has been reported to be involved in obesity and / or metabolic dysfunction Testing for polymorphism was performed.
  • the nucleobase changes from C to G, and the 27th glutamine of the receptor protein is mutated to glutamic acid.
  • a peripheral blood sample of 7 ml was prepared from 20 patients who gave written consent.
  • chromosomal DNA was extracted from the buffy coat fraction and used as the chromosomal DNA in the following tests.
  • the primer DNA of SEQ ID NO: 1 was synthesized as the primer DNA for the allele of the normal nucleic acid C, and the primer DNA of SEQ ID NO: 2 was synthesized as the antisense primer DNA.
  • the primer DNA of SEQ ID NO: 3 was synthesized as the sense primer DNA for the allele of the mutant nucleic acid G, and the primer DNA of SEQ ID NO: 4 was synthesized as the antisense primer: DNA.
  • 30 ng to 100 ng of the chromosomal DNA prepared above was combined with 0.15 mM dNTP, 25 pmol of each primer DNA, and 0.5 unit of Evening Kara Shuzo T aq (R) enzyme, 1 5 m M of M g C l 2 and including 2.
  • CC type normal homozygous in which two kinds of PCR products of 279 base pairs and 204 base pairs are observed, and 279 base pairs, 204 base pairs, 11 Heterotype (CG type) in which three types of PCR products of 0 base pairs are observed, and mutant homo type (GG type) in which two types of PCR products of 279 base pairs and 110 base pairs are observed )
  • CG type Heterotype
  • GG type mutant homo type
  • IL-1B IL-1B
  • the primer DNA of SEQ ID NO: 5 was synthesized as the sense primer DNA for the allele of the normal nucleic acid C, and the primer DNA of SEQ ID NO: 6 was synthesized as the antisense primer DNA.
  • a primer DNA of SEQ ID NO: 7 was synthesized as a primer DNA for an allele of a different nucleic acid T, and a primer DNA of SEQ ID NO: 8 was synthesized as an antisense primer DNA. From 30 ng of chromosomal DNA prepared in Example 1: LOO ng to 0.15 mM dNTP, 25 pmol of each primer DNA, 0.5 unit Evenings Kara Shuzo Ltd.
  • T aq (TM) enzyme was added PCR reactions 1 2 containing 5 mM of M g C l 2.
  • the reaction solution 2 5 ⁇ 1 comprising 1 0 XPCR buffer.
  • the reaction conditions were denaturation at 94 ° C for 5 minutes, followed by 25 cycles of 60 seconds at 94 ° C, 60 seconds at 54 ° C, and 60 seconds at 72 ° C. Finally, a final extension reaction is performed at 72 ° C for 5 minutes.
  • PCR products were visualized by 2% agarose gel and ethidium bromide staining. Figure 3 shows the results.
  • CC type normal homozygous in which two kinds of PCR products, 240 base pairs and 155 base pairs, are observed, 240 base pairs, 155 base pairs, and 125 base pairs Hetero-type (CT type) in which three types of PCR products of 2 base pairs are observed, and mutant homo-type (TT type) in which two types of PCR products of 240 base pairs and 122 base pairs are observed )
  • CT type Tero-type
  • TT type mutant homo-type
  • FIG. 1 is a diagram schematically illustrating the principle and scheme of the method of the present invention.
  • FIG. 2 is an electrophoresis photograph of the PCR reaction product performed in Example 1.
  • Lane M is a 100 base pair size marker
  • lane 2, 4, 6, 7, 9, 11, 11, 12, 15, 17, 20 are normal.
  • lanes 1, 5, 8, 13, 13, 14, 18 , 19 are heterotypes (CG type)
  • lanes 3, 10, and 16 are mutant homotypes (GG type).
  • FIG. 3 is an electrophoresis photograph of the PCR reaction product performed in Example 2.
  • Lane M is a 100 base pair size marker
  • lanes 13, 15, and 18 are normal heterotype (CC type)
  • lanes 1, 2, 4, 5, 7, 9, 9 and 1 0 and 14 are heterozygous (CT type)
  • lanes 3, 6, 8, 11, 12, 16, 17, 19 and 20 are mutant homozygous (TT type) . .

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Health & Medical Sciences (AREA)
  • Biophysics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Immunology (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Analytical Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Biotechnology (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L'invention concerne un procédé permettant de déterminer de manière économique et rapide une mutation dans un acide nucléique sur un gène. L'invention concerne notamment un procédé permettant de déterminer la présence ou l'absence d'une mutation génétique spécifique, qui consiste à confirmer la taille de fragments d'ADN formés par une réaction PCR en utilisant quatre amorces d'ADN dont une amorce d'ADN dotée d'un acide nucléique correspondant à l'acide nucléique muté à l'extrémité 3', une amorce d'ADN dotée d'un acide nucléique correspondant à l'acide nucléique normal à l'extrémité 3', et deux amorces d'ADN correspondant respectivement à ces amorces d'ADN.
PCT/JP2001/007942 2000-09-19 2001-09-13 Procede permettant de determiner la mutation genetique Ceased WO2002024905A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU2001286218A AU2001286218A1 (en) 2000-09-19 2001-09-13 Method of judging gene mutation

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2000-283996 2000-09-19
JP2000283996A JP2001137000A (ja) 2000-09-19 2000-09-19 遺伝子変異の判別方法

Publications (1)

Publication Number Publication Date
WO2002024905A1 true WO2002024905A1 (fr) 2002-03-28

Family

ID=18768281

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2001/007942 Ceased WO2002024905A1 (fr) 2000-09-19 2001-09-13 Procede permettant de determiner la mutation genetique

Country Status (3)

Country Link
JP (1) JP2001137000A (fr)
AU (1) AU2001286218A1 (fr)
WO (1) WO2002024905A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008066164A1 (fr) * 2006-11-30 2008-06-05 Arkray, Inc. Ensemble d'amorces utilisé dans l'amplification du gène de l'obésité, réactif utilisé dans l'amplification du gène de l'obésité comprenant ledit ensemble d'amorce et son utilisation

Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2001137000A (ja) * 2000-09-19 2001-05-22 Nobuyuki Hamashima 遺伝子変異の判別方法
JP4491276B2 (ja) * 2004-05-17 2010-06-30 日本製粉株式会社 標的ｄｎａ配列において一塩基変異多型の存在を検出する方法及びキット
WO2006030872A1 (fr) * 2004-09-15 2006-03-23 Celltec Projet Management Co., Ltd. Procédé et appareil de mesure de taux de mutation
JP4650420B2 (ja) * 2005-03-30 2011-03-16 東洋紡績株式会社 塩基判定方法及び塩基判定用キット

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0530794A1 (fr) * 1991-09-06 1993-03-10 Roche Diagnostics GmbH Méthode pour la détection d'acides nucléiques ayant une séquence similaire
JPH06167492A (ja) * 1992-11-30 1994-06-14 Otsuka Pharmaceut Co Ltd 変異癌遺伝子の検出測定方法
JPH0889297A (ja) * 1994-09-26 1996-04-09 S R L:Kk Ｄｎａ点突然変異の検出方法及び試薬
JPH10276779A (ja) * 1997-04-01 1998-10-20 Sumitomo Metal Ind Ltd 遺伝子プローブとｄｎａポリメラーゼを用いた変異検出方法
JP2001137000A (ja) * 2000-09-19 2001-05-22 Nobuyuki Hamashima 遺伝子変異の判別方法

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0530794A1 (fr) * 1991-09-06 1993-03-10 Roche Diagnostics GmbH Méthode pour la détection d'acides nucléiques ayant une séquence similaire
JPH06167492A (ja) * 1992-11-30 1994-06-14 Otsuka Pharmaceut Co Ltd 変異癌遺伝子の検出測定方法
JPH0889297A (ja) * 1994-09-26 1996-04-09 S R L:Kk Ｄｎａ点突然変異の検出方法及び試薬
JPH10276779A (ja) * 1997-04-01 1998-10-20 Sumitomo Metal Ind Ltd 遺伝子プローブとｄｎａポリメラーゼを用いた変異検出方法
JP2001137000A (ja) * 2000-09-19 2001-05-22 Nobuyuki Hamashima 遺伝子変異の判別方法

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
BROWN P.O. ET AL.: "Exploring the new world of the genome with DNA microarrays", NAT. GENET., vol. 21, no. 1 (SUPPL.), 1999, pages 33 - 37, XP002907408 *
ERLICH H.A. ET AL.: "Recent advances in the polymerase chain reaction", SCIENCE, vol. 252, 1991, pages 1643 - 1651, XP002907409 *
HAMAJIMA N. ET AL.: "Polymerase chain reaction with confronting two-pair primers for polymorphism genotyping", JPN. J. CANCER RES., vol. 91, no. 9, 25 September 2000 (2000-09-25), pages 865 - 868, XP002907406 *
KENSHI HAYASHI: "Jikken igaku bessatsu; Bio-manual UP series PCR-hou no saishin gijutsu", 15 October 1996, KABUSHIKI KAISHA YOUDOSHA, XP002907407 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008066164A1 (fr) * 2006-11-30 2008-06-05 Arkray, Inc. Ensemble d'amorces utilisé dans l'amplification du gène de l'obésité, réactif utilisé dans l'amplification du gène de l'obésité comprenant ledit ensemble d'amorce et son utilisation
US8021845B2 (en) 2006-11-30 2011-09-20 Arkray, Inc. Probes for detecting obesity gene
JP5279492B2 (ja) * 2006-11-30 2013-09-04 アークレイ株式会社 肥満遺伝子増幅用プライマーセット、それを含む肥満遺伝子増幅用試薬およびその用途

Also Published As

Publication number Publication date
AU2001286218A1 (en) 2002-04-02
JP2001137000A (ja) 2001-05-22

Similar Documents

Publication Publication Date Title
US10329605B2 (en) Method to increase sensitivity of detection of low-occurrence mutations
CN111032881B (zh) 核酸的精确和大规模平行定量
US9920370B2 (en) Haplotying of HLA loci with ultra-deep shotgun sequencing
JP5926183B2 (ja) 疾患関連ｋｉｒハプロタイプの決定
Elmore et al. Identification of a genetic variant associated with abdominal aortic aneurysms on chromosome 3p12. 3 by genome wide association
JP2014506459A (ja) 薬理ゲノミクスバイオマーカーを発見するための方法
JP2023109998A (ja) マイクロサテライト不安定性の検出
WO2017112738A1 (fr) Procédés pour mesurer l'instabilité microsatellitaire
KR20190059018A (ko) Cyp2e1 유전자의 메틸화 수준을 이용한 비만의 예측 또는 진단을 위한 정보제공방법 및 이를 위한 조성물
Steffensen et al. Determination of gene frequencies for two common haemochromatosis mutations in the Danish population by a novel polymerase chain reaction with sequence‐specific primers
WO2002024905A1 (fr) Procede permettant de determiner la mutation genetique
TW202146660A (zh) 用於評估神經傳導物轉運子抑制劑之功效之組合物及方法
JP2023515482A (ja) 核酸のメチル化の差を利用したマーカーの選別方法、メチルまたは脱メチルマーカー及びそのマーカーを利用した診断方法
CN103003428B (zh) 一种用于预测对于抗癌靶向治疗制剂的敏感性的snp
KR101100437B1 (ko) 단일염기 다형을 포함하는 대장암과 관련된 폴리뉴클레오티드, 그를 포함하는 마이크로어레이 및 진단 키트 및 그를 이용한 대장암의 진단방법
JPH11510059A (ja) 遺伝子診断の共優性テスト
KR101167934B1 (ko) Ｔｉｃａｍ1 유전자로부터 유래된 단일염기다형을 포함하는 폴리뉴클레오티드, 이를 포함하는 마이크로어레이 및 진단키트, 및 이를 이용한 자폐 스펙트럼 장애 분석방법
RU2600874C2 (ru) Набор олигонуклеотидных праймеров и зондов для генотипирования полиморфных локусов днк, ассоциированных с риском развития спорадической формы болезни альцгеймера в российских популяциях
Zhou et al. The HumD21S11 system of short tandem repeat DNA polymorphisms in Japanese and Chinese
KR20060131532A (ko) 특정 질병 또는 약물 감수성 관련 다중 단일염기다형의스크리닝 방법
RU2837876C1 (ru) Способ генотипирования полиморфного локуса rs139794067 (G>T) гена MYL3
Ai-Ghalayini et al. Identification of genetic variants associated with myocardial infarction in Saudi Arabia
JP6245796B2 (ja) 原発性胆汁性肝硬変の発症リスク予測マーカー、プローブ、プライマー及びキット並びに原発性胆汁性肝硬変の発症リスク予測方法
KR101167945B1 (ko) Ａｔｇ16ｌ1 유전자로부터 유래된 단일염기다형을 포함하는 폴리뉴클레오티드, 이를 포함하는 마이크로어레이 및 진단키트, 및 이를 이용한 자폐 스펙트럼 장애 분석방법
Hwang et al. Effective screening of informative single nucleotide polymorphisms using the novel method of restriction fragment mass polymorphism

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE ES FI GB GD GE GH GM HR HU ID IL IN IS KE KG KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NO NZ PH PL PT RO RU SD SE SG SI SK SL TJ TM TR TT TZ UA UG US UZ VN YU ZA ZW

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): GH GM KE LS MW MZ SD SL SZ TZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

32PN Ep: public notification in the ep bulletin as address of the adressee cannot be established

Free format text: RULE 69 (1) EPC

122 Ep: pct application non-entry in european phase