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EP0983379A1 - Procede et trousse pour la detection de mutations dans des adn a l'aide d'enzymes de restriction - Google Patents

Procede et trousse pour la detection de mutations dans des adn a l'aide d'enzymes de restriction

Info

Publication number
EP0983379A1
EP0983379A1 EP97919050A EP97919050A EP0983379A1 EP 0983379 A1 EP0983379 A1 EP 0983379A1 EP 97919050 A EP97919050 A EP 97919050A EP 97919050 A EP97919050 A EP 97919050A EP 0983379 A1 EP0983379 A1 EP 0983379A1
Authority
EP
European Patent Office
Prior art keywords
detection
primers
primer
dna
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.)
Withdrawn
Application number
EP97919050A
Other languages
German (de)
English (en)
Inventor
Ursula Bilitewski
Dirk Kuhlmeier
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.)
Helmholtz Zentrum fuer Infektionsforschung HZI GmbH
Original Assignee
Helmholtz Zentrum fuer Infektionsforschung HZI GmbH
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 Helmholtz Zentrum fuer Infektionsforschung HZI GmbH filed Critical Helmholtz Zentrum fuer Infektionsforschung HZI GmbH
Publication of EP0983379A1 publication Critical patent/EP0983379A1/fr
Withdrawn legal-status Critical Current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
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6827—Hybridisation assays for detection of mutation or polymorphism
    • C12Q1/683—Hybridisation assays for detection of mutation or polymorphism involving restriction enzymes, e.g. restriction fragment length polymorphism [RFLP]

Definitions

  • the present invention relates to a method for the detection of mutations of DNAs, a DNA being amplified by means of a PCR and the amplicons being cut and detected with one or more restriction enzymes, characterized in that two labeled primers are used, the labeling of the first Primer for binding the amplicons to one or more carriers and the marking of the second primer is used for detection.
  • the present invention further relates to a kit for carrying out the method described above, which is characterized in that it comprises two labeled primers.
  • PCR polymerase chain reaction
  • primers sequence pieces, which define the beginning and the end of the amplified piece of DNA.
  • a thermal cycler is also required, a device that realizes the required temperature profile for a PCR reaction.
  • the above-mentioned degradation takes place with the aid of the restriction enzyme. Depending on the mutation, this cuts the DNA in a sequence-specific manner under defined conditions or there is no cut. Depending on the concentration of the enzyme, this process can take a few hours.
  • This object is achieved by a method for the detection of mutations of DNAs, wherein a DNA is amplified by means of a PCR and the amplicons are cut and detected (analyzed) with one or more restriction enzymes, which method is characterized in that two marked primers are used and the marking of the first primer is used to bind the amplicons (duplicated DNA segments) to one or more supports and the marking of the second primer is for detection (analysis).
  • Carriers that have an activated surface such as, for example, have proven to be particularly advantageous the appropriately treated surface of the reaction vessel or a microtiter plate.
  • the first primers are partially or completely fixed to the supports before, during or after the PCR, while the second primers are preferably bound free or not to the support and are optionally present in excess in the reaction solution.
  • the primers can be added to the reaction solution simultaneously or in succession.
  • the amplicons After the amplicons have been bound to suitable supports by means of the first primers and before and / or after cutting, preferably after cutting the amplicons with one or more of the restriction enzymes described above, the amplicons can be washed with a suitable dishwashing detergent, such as, for example, tem or distilled water, the optional additives such as
  • the first primers are first bound to a suitable support such as the support surface of the vessel in which the PCR reaction takes place.
  • a coupling method must be selected that meets the constraints of the PCR, such as temperature stability.
  • the reaction then proceeds according to Standard conditions, wherein the second primer is not bound to the support and is preferably present in excess in relation to the first primer.
  • FIG. 2 indicates that after the PCR reaction has ended, the amplicon, which contains the sequence to be examined, is surface-fixed. Subsequently, the same restriction enzyme is added (FIG. 3), which, as described above, cuts the wild type, but not the mutation sequence. The result of the assay carried out is illustrated in FIG. 4. After a washing step (not shown), the detectable label is still present on the fixed segment in the event of a mutation. In the wild type, however, no signal is obtained since the enzyme cuts due to the existing interface and, as a result, the separated, labeled segment can be removed by a washing step.
  • the mutation in protein factor V is held responsible for an increased risk of thrombosis, since it inhibits the breakdown of glycoprotein factor V by the serine protease APC (activated protein C).
  • Human factor V is a 330 kDa glycoprotein that interacts with APC and normally cleaved by APC at a very specific point in the sequence: in the human protein behind Arg 506 (arginine as the 506th amino acid in the protein).
  • Arg 506 arginine as the 506th amino acid in the protein.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Immunology (AREA)
  • Physics & Mathematics (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • Biophysics (AREA)
  • Analytical Chemistry (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 offrant la possibilité de détecter des mutations dans des ADN à l'aide d'enzymes de restriction, l'hypothèse étant qu'à cette occasion, la tenue à la coupure de l'enzyme de restriction choisie se modifie en raison de la mutation. Le segment d'ADN correspondant dans le génome, qui contient la mutation, est dupliqué par amplification en chaîne par polymérase (ACP), les amorces utilisées étant respectivement marquées. La première amorce permet la fixation du segment sur une surface porteuse, le marqueur de la deuxième amorce étant utilisé pour permettre la détection. Une enzyme de restriction est incubée avec l'amplicon obtenu par ACP, le segment muté n'étant pas coupé si le site de coupure est supprimé par la mutation. Une réaction de détection intervient après la fixation des segments sur une surface porteuse, et le marqueur de la deuxième amorce est éliminé dans le type sauvage, tandis qu'on peut l'identifier chez le mutant. On peut donc avoir une pluralité de marqueurs qui permettent une détection directe ou indirecte. On dispose ainsi d'un procédé rapide pour déceler certaines mutations dans le génome sans devoir recourir à des méthodes traditionnelles dispendieuses.
EP97919050A 1997-05-21 1997-09-10 Procede et trousse pour la detection de mutations dans des adn a l'aide d'enzymes de restriction Withdrawn EP0983379A1 (fr)

Applications Claiming Priority (5)

Application Number Priority Date Filing Date Title
DE19721327 1997-05-21
DE19721327 1997-05-21
DE19739612 1997-09-09
DE19739612 1997-09-09
PCT/EP1997/004955 WO1998053098A1 (fr) 1997-05-21 1997-09-10 Procede et trousse pour la detection de mutations dans des adn a l'aide d'enzymes de restriction

Publications (1)

Publication Number Publication Date
EP0983379A1 true EP0983379A1 (fr) 2000-03-08

Family

ID=26036720

Family Applications (1)

Application Number Title Priority Date Filing Date
EP97919050A Withdrawn EP0983379A1 (fr) 1997-05-21 1997-09-10 Procede et trousse pour la detection de mutations dans des adn a l'aide d'enzymes de restriction

Country Status (6)

Country Link
EP (1) EP0983379A1 (fr)
JP (1) JP2001525678A (fr)
AU (1) AU737771B2 (fr)
CA (1) CA2290510A1 (fr)
RU (1) RU2193069C2 (fr)
WO (1) WO1998053098A1 (fr)

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AUPR221400A0 (en) * 2000-12-20 2001-01-25 Murdoch Childrens Research Institute, The Diagnostic assay
JP2006520199A (ja) * 2003-03-19 2006-09-07 ザ ユニバーシティ オブ ブリティッシュ コロンビア 患者結果の指標として有用なプラスミノーゲンアクチベーターインヒビター‐１（ｐａｉ−１）ハプロタイプ

Family Cites Families (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5118605A (en) * 1984-10-16 1992-06-02 Chiron Corporation Polynucleotide determination with selectable cleavage sites
CA2062943A1 (fr) * 1990-04-30 1991-10-31 Lori Lan Cho Cheung Methode d'identification
CA2143428A1 (fr) * 1993-07-09 1995-01-19 Takanori Oka Methode de differenciation de l'acide nucleique et trousse de dosage pour cette differenciation
EP0812211A4 (fr) * 1994-03-18 1998-12-16 Gen Hospital Corp Methodes de detection de polymorphismes amplifies et clives des sites de restriction
FR2718461B1 (fr) * 1994-04-07 1996-05-15 Cis Bio Int Procédé de détection d'un site de restriction dans une séquence d'ADN.
US5851770A (en) * 1994-04-25 1998-12-22 Variagenics, Inc. Detection of mismatches by resolvase cleavage using a magnetic bead support
CA2220467A1 (fr) * 1995-05-11 1996-11-14 Arild Lagerkvist Detection d'erreurs d'appariement par clivage de resolvase sur un support solide
US5753439A (en) * 1995-05-19 1998-05-19 Trustees Of Boston University Nucleic acid detection methods

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO9853098A1 *

Also Published As

Publication number Publication date
CA2290510A1 (fr) 1998-11-26
WO1998053098A1 (fr) 1998-11-26
AU737771B2 (en) 2001-08-30
AU4302297A (en) 1998-12-11
JP2001525678A (ja) 2001-12-11
RU2193069C2 (ru) 2002-11-20

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