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WO1991006675A1 - Nouvelle sonde a base d'oligonucleotides - Google Patents

Nouvelle sonde a base d'oligonucleotides Download PDF

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Publication number
WO1991006675A1
WO1991006675A1 PCT/JP1990/001404 JP9001404W WO9106675A1 WO 1991006675 A1 WO1991006675 A1 WO 1991006675A1 JP 9001404 W JP9001404 W JP 9001404W WO 9106675 A1 WO9106675 A1 WO 9106675A1
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WO
WIPO (PCT)
Prior art keywords
dna
probe
oligonucleotide probe
human
gene
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/JP1990/001404
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English (en)
Japanese (ja)
Inventor
Tamotsu Hashimoto
Koichi Kurose
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.)
Sanofi Aventis KK
Original Assignee
Hoechst Japan Ltd
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 Hoechst Japan Ltd filed Critical Hoechst Japan Ltd
Publication of WO1991006675A1 publication Critical patent/WO1991006675A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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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
    • 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 relates to a novel mini-satellite DNA which can be used as a DNA probe for diagnosis of shrines.
  • Shosho diagnosis is 1) Early diagnosis of Shosho disease, 2) Application to the so-called DNA Bingerprint method for paternity and criminal investigation, 3) causes of viral and bacterial infections Diagnosis of bacteria with high sensitivity.
  • the DNA finger print method can be widely used for individual identification.
  • all or part of the target gene can be used as a DNA probe to directly detect mutations in the gene, and mutations in restriction enzyme sites around the gene are used.
  • Restriction fragment length polymorphism RFLP
  • RFLP Restriction fragment length polymorphism
  • minisatellite DNA can be used for personal identification and family identification (for example, ucleic Acid Research. Vol. IB, 10953-10971. 1988; Science, Vol. 235, 1B1BlG22, 1987).
  • Mini satellite DNA is a so-called repetitive sequence DNA in which one to ten base sequences are repeated in one unit, and these units are repeated in tandem.
  • the repetition rate is as high as 1000 times, and the repetition rate is different for each contraceptive gene, thus obtaining a variety (Review: Ryo Konan, Experimental Medicine, Vol. 7, No. 2,
  • the patterns of mutations in the gene that can be detected by the DNA finger print method are transmitted from the parent to the child by means of center transmission.
  • PCR DNA polymerase chain reaction
  • hair roots, semen, blood From small samples such as skin fragments, it is possible to apply criminal investigations that can identify criminals and victims using patterns that differ for each individual.
  • the minisatellite DNA provided by the present invention is a kind of protease inhibitor involved in the regulation of the fibrinolytic system contained in human plasma.
  • -Plasmid inhibition This is a base sequence found by the present inventors near the gene of Yuichi.
  • Human ⁇ . -Plasmin inhibitors are normally detected in the blood of normal subjects at about 6 mg ZcW and inhibit plasmin, which has a thrombolytic effect, to suppress the progress of thrombolysis, and eventually to cause inflammation and cancer metastasis. It is thought to contribute to the prevention of various abnormal physiological phenomena.
  • the present inventors have isolated the gene for the human “ 2- plasmin inhibitor” and analyzed the upstream region that regulates the expression of the gene. As a result, the D- D sequence of 45 nucleotides was identified.
  • Oligonucleotides having this sequence are labeled and used as probes.
  • Human chromosomal DNA is digested with appropriate restriction enzymes and electrophoresed in agarose gel, and Southern blot hybridisation is performed.
  • restriction it is possible to detect a difference in the pattern of the restriction enzyme digested fragments and to determine the number of copies of the repetitive sequence.
  • the present inventors have determined human ⁇ .
  • the minisatellite array found near the plasmin inhibitor gene consists of 45 nucleotides, and at least seven heterogass sites are found in the sub-branches. This could be used to identify more specific individuals and families by direct sequencing by the polymerase chain reaction method. You.
  • Human alpha 2 - JP 64 as a plus Mi N'i down human bitter gene fragments - but 2577 discloses the nucleotide sequence disclosed in this application the first Mechionin residues it is and leader sequence of c D New alpha (mature alpha eta - the Asuparagin residues of a Mi - terminal of plus Mi N'i down arsenide Vita one protein + 1 and the time - 39 th ⁇ Mi (Corresponding to the acid residue).
  • the present inventor has alpha 2 - bra scan Mi Ni Nhi and click port-learning the ⁇ Ko chromosome bitter, the Minisate Lai bets DNA sequences disclosed in the present invention the upstream of these first E click Seo down Found out and completed the present invention.
  • RI radioisotope
  • non-RI methods for labeling with an enzyme or chemiluminescent luminescent material are used. Have been used because of their high operational safety and because they do not require special facilities.
  • RI labeling method and the 32 [rho using T 4 port I J click Reochi Dokinaze is 5 '- how to label the end or D New Alpha polymerase I and D Nase l by combining 32 P, - labeling Dokishi DOO
  • nickel transduction method for labeling oligonucleotides there is a nickel transduction method for labeling oligonucleotides, and these labeling methods are commercially available using dedicated kits (eg, Bethesda Research Laboratories of Ameri Power Co., Ltd.).
  • Non-RI labeling and detection methods include horseradish pero idase (HRP) tDNA.
  • HRP horseradish pero idase
  • these probe DNAs are chemically synthesized using a commercially available DNA synthesizer (for example, ABI 381A of Applied Biosystems, USA). Can also be synthesized. When the base chain is long, an appropriate gene fragment can be cloned and separated and purified. In order for the labeled DNA probe to form a stable hybridisation and generate a specific signal, more than 12 oligonucleotides are detectable, but more non-specific. 12 to 17 or more, preferably 20 or more oligonucleotides to control the background of the reaction and to detect sharp and accurate patterns Can be used.
  • the starting point may be anywhere in the base sequence of GCAGCG 'or its complementary strand, but it is sufficient if it contains at least 12 base sequences. Examples are shown below to explain the present invention more specifically. However, the present embodiment does not limit the present invention. ⁇ Example 1
  • FIG. 1 shows a restriction enzyme map of the 2-plasmin inhibitor gene, exon positions, 45 bp repeat sequences, and their nucleotide sequences.
  • DNA prepared from human liver was partially digested with restriction enzyme Sau3AI, and fractionated by sucrose density gradient centrifugation. A DNA fragment corresponding to 10 to 20 kb was incorporated into IL-47.1 vector (purchased from Amersham) and described in Maniatis. T. et al., Molecular Cloning. Cold Spring Harbor Lab., (1982). In vitro packaging according to went. As a result of the titration, there were approximately 1 ⁇ 10 6 independent phage mosquitoes.
  • the human gene library was screened by the plaque hybridization method. Basically, the method described in Mani at is, T. et al., Molecular Cloning, Cold Spring Harbor Lab., (1982) was followed. Probe is, Tone et al, J. Biochem, 102, 1033- 1041 (1987), human alpha 2 - Using flops la scan Mi emissions Lee down heat Vita over c DNA. Labeling of probes, - it was carried out by Stevenage click tiger Nsuresho emissions with [alpha 32 [rho] d CTP. Approximately 1 ⁇ 10 6 plaques were transferred to a nitrocellulose filter, hybridized with the probe, washed, and autoradiographed to detect positive clones.
  • DNA is prepared from the above positive clones, digested with appropriate restriction enzymes, and subjected to restriction enzyme mapping. Fragments of each restriction enzyme are then converted to PUC118, pUC119, pHSG398, or pHSG. Cloned to a part of polylinker of 399 (all manufactured by Takara Shuzo). Using a delay kit for kilo-sequence (manufactured by Takara Shuzo Co., Ltd.), a delay mutant of the above-mentioned recombinant plasmid is prepared and cloned to pUC118 or pUC119. See Vieira, J. et al., Methods in Enzymolgy, 152.
  • Leukocytes obtained from 3 Oml of blood by dextran specific gravity centrifugation were subjected to lOOm MEDTA, 50mM Tris ⁇
  • 45-mer oligonucleotide having the following base sequence CAGGACAGTGAGGGTGGGAAGGAGCCTGATGCAGGGAGTGAGGCG
  • Table 1 shows the chain length and number of copies of each sample (a to i).
  • FIG. 2 is an autoradiogram obtained by performing Southern blot hybridization using a 45nier nucleic acid probe after chromosomal DNA of 9 people was converted with Bst. Arrows indicate band positions and numbers indicate their length.
  • the oligonucleotide probe of the present invention can be used as a DNA probe for gene diagnosis, and as a diagnostic agent for personal identification, family identification, or shoal disease. Used.

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

Abstract

Sonde à base d'oligonucléotides destinée à être utilisée en tant que sonde d'ADN pour effectuer des diagnostics génétiques, comportant une nouvelle séquence d'ADN minisatellite ainsi que des structures répétitives relatives. Cet ADN minisatellite comprend des séquences de 45 nucléotides répétées dans la même direction, que l'on trouve dans la région amont des gènes de l'inhibiteur d'α2-plasmine, l'un des inhibiteurs de protéase contenus dans le plasma humain et jouant un rôle dans la régulation d'un système fibrinolytique. Le nombre de séquences répétées dépend de l'individu et du degré de consanguinité et peut être transféré de manière génétiquement stable. Il est ainsi possible de procéder à des différenciations en fonction des individus ou des degrés de consanguinité par l'analyse 'Southern blot', en utilisant comme sonde le fragment d'ADN comprenant 45 nucléotides.
PCT/JP1990/001404 1989-11-02 1990-10-31 Nouvelle sonde a base d'oligonucleotides Ceased WO1991006675A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP1284909A JPH03147799A (ja) 1989-11-02 1989-11-02 新規なオリゴヌクレオチドプローブ
JP1/284909 1989-11-02

Publications (1)

Publication Number Publication Date
WO1991006675A1 true WO1991006675A1 (fr) 1991-05-16

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Family Applications (1)

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PCT/JP1990/001404 Ceased WO1991006675A1 (fr) 1989-11-02 1990-10-31 Nouvelle sonde a base d'oligonucleotides

Country Status (3)

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JP (1) JPH03147799A (fr)
AU (1) AU6631090A (fr)
WO (1) WO1991006675A1 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022047038A1 (fr) 2020-08-28 2022-03-03 Illumina, Inc. Détection et filtrage de groupes sur la base d'appels de base prédits par intelligence artificielle
WO2022212180A1 (fr) 2021-03-31 2022-10-06 Illumina, Inc. Appelant de base à base d'intelligence artificielle avec reconnaissance contextuelle

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB2373500B (en) * 2000-02-04 2004-12-15 Aeomica Inc Methods and apparatus for predicting, confirming, and displaying functional information derived from genomic sequence

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS642577A (en) * 1987-06-23 1989-01-06 Teijin Ltd Amino acid sequence of human alpha2-plasmin inhibitor and gene fragment

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS642577A (en) * 1987-06-23 1989-01-06 Teijin Ltd Amino acid sequence of human alpha2-plasmin inhibitor and gene fragment

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
BIO. IND., Vol. 6, No. 9, September 1989, TOMIO YOSHII, IKUO ISHIYAMA, "The Actual State and Future of DNA Fingerprint Technique", p. 692-699. *
NIPPON HOIGAKU ZASSHI, Vol. 41, No. 3, June 1987, MASAMITSU HONMA, IKUO ISHIYAMA, "Medicolegal Use 1 of DNA Fingerprint, Application of Minisatellite DNA for Judgement of Parent and Child", p. 236-241. *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022047038A1 (fr) 2020-08-28 2022-03-03 Illumina, Inc. Détection et filtrage de groupes sur la base d'appels de base prédits par intelligence artificielle
EP4517761A2 (fr) 2020-08-28 2025-03-05 Illumina, Inc. Détection et filtrage de grappes sur la base de valeurs de filtre
WO2022212180A1 (fr) 2021-03-31 2022-10-06 Illumina, Inc. Appelant de base à base d'intelligence artificielle avec reconnaissance contextuelle

Also Published As

Publication number Publication date
AU6631090A (en) 1991-05-31
JPH03147799A (ja) 1991-06-24

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