WO1998053098A1 - 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 Download PDF

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Publication number
WO1998053098A1
WO1998053098A1 PCT/EP1997/004955 EP9704955W WO9853098A1 WO 1998053098 A1 WO1998053098 A1 WO 1998053098A1 EP 9704955 W EP9704955 W EP 9704955W WO 9853098 A1 WO9853098 A1 WO 9853098A1
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WO
WIPO (PCT)
Prior art keywords
primers
primer
detection
pcr
mutation
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Application number
PCT/EP1997/004955
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German (de)
English (en)
Inventor
Ursula Bilitewski
Dirk Kuhlmeier
Original Assignee
GESELLSCHAFT FüR BIOTECHNOLOGISCHE FORSCHUNG MBH (GBF)
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Application filed by GESELLSCHAFT FüR BIOTECHNOLOGISCHE FORSCHUNG MBH (GBF) filed Critical GESELLSCHAFT FüR BIOTECHNOLOGISCHE FORSCHUNG MBH (GBF)
Priority to CA002290510A priority Critical patent/CA2290510A1/fr
Priority to AU43022/97A priority patent/AU737771B2/en
Priority to JP54983598A priority patent/JP2001525678A/ja
Priority to EP97919050A priority patent/EP0983379A1/fr
Publication of WO1998053098A1 publication Critical patent/WO1998053098A1/fr

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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
    • C12Q1/6813—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.
  • Restriction enzymes are used here, which recognize double-stranded DNA in a sequence-specific manner and can cut at the appropriate place. Approximately 500 different restriction enzymes with over 100 different interfaces are currently characterized. This high specificity is used to detect the defects. Because a mutation that triggers a pathological change often lies in the region of such an enzyme interface. The mutation changes the interface so that the strand to be examined is not cut. In other cases, a mutation first creates a possible interface for an enzyme that was not previously available.
  • 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.
  • agarose gel electrophoresis has been used to analyze the product: an agarose polymer is dissolved in a buffer solution by heating and after a cooling phase, the gelling polysaccharide is poured into a device in which the electrophoresis is to be carried out. When the gel has cooled completely, the samples are applied and the size-dependent migration of the DNA fragments in the agarose gel occurs with the application of a potential difference.
  • the fragments can be visualized e.g. with the addition of fluorescent intercalates, substances that are embedded in the DNA and are visible under UV light.
  • a mutation can be diagnosed if a specific change in the restriction pattern becomes apparent in the gel.
  • the invention is therefore based on the object of developing a method and a kit with which the mutation forms described above can be detected simply, reproducibly and with a high sample throughput, the disadvantages of gel electrophoresis being avoided.
  • 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).
  • the PCR used in this method can be carried out as usual, the primers described above being used as primers.
  • 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 second primers should preferably have labels that can be easily detected by conventional methods when they are detected.
  • one or more restriction enzymes can be added before, during or after the binding of the DNA segments (amplicons from the PCR) to the carrier, the addition after the binding being preferred.
  • the restriction enzymes can be incubated with the amplicons.
  • restriction enzymes can be used as restriction enzymes, it of course depending on the sequence to be cut which enzyme (s) are used in the specific case.
  • 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 detection methods are selected so that the respectively selected marking (the label) of the second primer can be detected.
  • the detection can be carried out directly or indirectly according to the marking of the second primer.
  • the DNA is first amplified and the amplicon is then bound to a suitable support, the label on the first primer serving as the coupling mediator. If necessary, the first primers can also have several corresponding labels.
  • 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.
  • the desired DNA segment is formed in the solution, but hybridization and amplification also take place on the surface of the support, initiated by the bound primer.
  • the desired products are obtained which are bound directly to the support. Excess PCR starting materials (e.g. free nucleotides etc.) and unbound products are removed in a rinsing step.
  • the segment is then cleaved by the enzyme, optionally a further rinsing step and the detection as described above.
  • step 3 of this scheme the coupling to the support, for example a microtiter plate, could then be carried out via a biotin-binding protein, for example via streptavidin or avidin, which would be adsorbed or covalently bound, for example on the surface of the wells the microtiter plate.
  • a biotin-binding protein for example via streptavidin or avidin, which would be adsorbed or covalently bound, for example on the surface of the wells the microtiter plate.
  • streptavidin or avidin which would be adsorbed or covalently bound, for example on the surface of the wells the microtiter plate.
  • Microtiter plates coated with streptavidin are commercially available (Boehringer Mannheim).
  • a covalent coupling to the support could be provided for primer 1, for example on a microtiter plate.
  • phosphorylated primers are suitable, for example, which could react in a carbodiimide reaction with appropriately activated carriers.
  • An example of suitable functional groups of the support, for example the microtiter plates, would be amino groups. Such microtiter plates are also commercially available.
  • primer 2 can be used in scheme 1 and in scheme 2.
  • examples of these are fluorescein, fluorescein derivatives, rhodamine, rhodamine derivatives, Cy5 (fluorescent dye), Cy3 (fluorescent dye) or compounds which can be detected indirectly.
  • Fluorescent dyes These primers can then be directly detected using a suitable fluorimeter.
  • Digoxin indirect detection can be performed by adding an (enzyme-labeled) antibody.
  • Biotin Indirect detection can be performed by adding an avidin / enzyme conjugate, for example by adding avidin or streptavidin.
  • the binding of the respective recognition protein can either be detected, as is customary in other microtiter plate assays, by adding suitable enzyme substrates, with peroxidase or alkaline phosphatase being suitable enzymes (enzyme reaction provides colored products), or else it can then be followed directly if the coupling is not made on microtiter plates, but on suitable sensors.
  • suitable sensors can be, for example, planar waveguides, as are known from biosensor technology, and on which the same coupling reactions can be carried out with the aid of primer 1 as have been described above for microtitre plates.
  • the DNA to be examined can have an interface as a wild type or as a mutant.
  • the interface is removed in the selected example.
  • a point mutation is sufficient to prevent the enzyme from specifically recognizing and cutting the segment.
  • the restriction enzyme, PCR starting materials and, if appropriate, separated PCR products which contain the detectable label can be removed by a rinsing step before the actual detection.
  • the detection step analyzes whether the label is present or not. If there is a mutation, the detectable label is present and a reaction is obtained depending on the selected label. In the case of enzyme labeling one would e.g. observe a catalyzed color reaction. If there is no mutation, the enzyme can cut specifically and the label is removed. There is no detectable signal.
  • the interface is created in the selected example.
  • a point mutation can be sufficient for the enzyme to specifically recognize and cut the segment.
  • PCR reactants and any separated PCR products that contain the detectable La ⁇ bel be removed.
  • the detection step analyzes whether the label is present or not. If there is a mutation, the detectable label is not present and no reaction is obtained. If there is no mutation, the enzyme cannot cut and the label is not removed. A detectable signal is obtained.
  • Scheme 1 illustrates the sequence of the first assay setup according to the invention.
  • Fig. 1 shows the amplification of the target using the labeled primers.
  • the PCR is carried out in suitable containers.
  • Figure 2 shows the PCR reaction products labeled with labein.
  • One end of the reaction products has a label that is used for later binding to a support surface and the label at the other end is used for detection.
  • the searched sequence which can contain a potential mutation, is within the amplified segments.
  • FIG. 3 shows the binding of the segments made possible by the first marking; in Fig. 4 it is examined by the action of a restriction enzyme whether the interface of this enzyme has been removed by a mutation or not. If a mutation is present, as shown in FIG. 5, the interface is omitted and the detectable labels are not removed. If there is no mutation, the enzyme can cut. The separated segment is removed by a simple washing step and consequently no signal is obtained at the bound part of the segment.
  • Scheme 2 demonstrates the second structure of the assay according to the invention. 1 shows that part of the first primer, which is used for binding to the support surface, is already bound before the start of the PCR. The second primer, which can be detected, is in excess in the solution.
  • 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.
  • a kit for carrying out the methods disclosed above is provided, which is characterized in that it comprises two labeled primers, it preferably additionally containing free nucleotides and a polymerase.
  • the first primer is suitable for binding PCR reaction products to one or more supports and the second primer is used for detection, the support preferably comprising an activated surface.
  • the method according to the invention and the kit according to the invention have the following advantages over the prior art: -
  • the described embodiments enable time savings compared to gel electrophoretic detection.
  • the second embodiment in particular has advantages since the PCR and the analysis of the products take place in one container. Repipetting is no longer necessary; the handling of the samples is simplified and the associated sources of error are eliminated.
  • the mutation is detected immediately after cleavage by the enzyme and not only after gel electrophoresis.
  • 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.
  • This mutation means that the Arg 506 (product of the nucleotide sequence CGA) in the protein is replaced by glutamine (gin as the product of the nucleotide sequence CAA) and the cleavage by the protease described above therefore no longer takes place.
  • primers are used which are used to amplify a region of the gene which comprises the corresponding nucleotide sequence by means of a PCR reaction. For example, fragments of 267 base pairs in length were obtained.

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.
PCT/EP1997/004955 1997-05-21 1997-09-10 Procede et trousse pour la detection de mutations dans des adn a l'aide d'enzymes de restriction WO1998053098A1 (fr)

Priority Applications (4)

Application Number Priority Date Filing Date Title
CA002290510A CA2290510A1 (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
AU43022/97A AU737771B2 (en) 1997-05-21 1997-09-10 Method and kit for the detection of mutations in DNA's using restriction enzymes
JP54983598A JP2001525678A (ja) 1997-05-21 1997-09-10 Ｄｎａの中の突然変異を制限酵素により検出するための方法とキット
EP97919050A 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

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
DE19721327 1997-05-21
DE19721327.8 1997-05-21
DE19739612.7 1997-09-09
DE19739612 1997-09-09

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

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1354059A1 (fr) * 2000-12-20 2003-10-22 Murdoch Childrens Research Institute Procede permettant de detecter si un organisme est homozygote ou heterozygote a l'aide d'amorces marquees et du polymorphisme de longueur des fragments de restriction (rflp)
WO2004083457A1 (fr) * 2003-03-19 2004-09-30 The University Of British Columbia Haplotypes de l'inhibiteur 1 d'activateur de plasminogene (pai-1) utiles comme indicateurs d'evolution chez des patients

Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0360940A2 (fr) * 1988-09-29 1990-04-04 Chiron Corporation Détermination de polynucléotides aux sites de scission sélectables
WO1991017262A1 (fr) * 1990-04-30 1991-11-14 The University Of British Columbia Procede d'etablissement d'identite
EP0664339A1 (fr) * 1993-07-09 1995-07-26 Wakunaga Seiyaku Kabushiki Kaisha Methode de discrimination des acides nucleiques et necessaire d'essai a cette fin
WO1995025538A1 (fr) * 1994-03-18 1995-09-28 The General Hospital Corporation Methodes de detection de polymorphismes amplifies et clives des sites de restriction
FR2718461A1 (fr) * 1994-04-07 1995-10-13 Cis Bio Int Procédé de détection d'un site de restriction dans une séquence d'ADN.
WO1996035809A1 (fr) * 1995-05-11 1996-11-14 Avitech Diagnostics Inc Detection d'erreurs d'appariement par clivage de resolvase sur un support solide
WO1996036731A2 (fr) * 1995-05-19 1996-11-21 Trustees Of Boston University Procedes de detection d'acides nucleiques
WO1997009444A1 (fr) * 1995-09-01 1997-03-13 Variagenics, Inc. Detection de mesappariements par clivage par une resolvase a l'aide d'un support magnetique

Patent Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0360940A2 (fr) * 1988-09-29 1990-04-04 Chiron Corporation Détermination de polynucléotides aux sites de scission sélectables
WO1991017262A1 (fr) * 1990-04-30 1991-11-14 The University Of British Columbia Procede d'etablissement d'identite
EP0664339A1 (fr) * 1993-07-09 1995-07-26 Wakunaga Seiyaku Kabushiki Kaisha Methode de discrimination des acides nucleiques et necessaire d'essai a cette fin
WO1995025538A1 (fr) * 1994-03-18 1995-09-28 The General Hospital Corporation Methodes de detection de polymorphismes amplifies et clives des sites de restriction
FR2718461A1 (fr) * 1994-04-07 1995-10-13 Cis Bio Int Procédé de détection d'un site de restriction dans une séquence d'ADN.
WO1996035809A1 (fr) * 1995-05-11 1996-11-14 Avitech Diagnostics Inc Detection d'erreurs d'appariement par clivage de resolvase sur un support solide
WO1996036731A2 (fr) * 1995-05-19 1996-11-21 Trustees Of Boston University Procedes de detection d'acides nucleiques
WO1997009444A1 (fr) * 1995-09-01 1997-03-13 Variagenics, Inc. Detection de mesappariements par clivage par une resolvase a l'aide d'un support magnetique

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1354059A1 (fr) * 2000-12-20 2003-10-22 Murdoch Childrens Research Institute Procede permettant de detecter si un organisme est homozygote ou heterozygote a l'aide d'amorces marquees et du polymorphisme de longueur des fragments de restriction (rflp)
EP1354059A4 (fr) * 2000-12-20 2004-07-07 Murdoch Childrens Res Inst Procede permettant de detecter si un organisme est homozygote ou heterozygote a l'aide d'amorces marquees et du polymorphisme de longueur des fragments de restriction (rflp)
US7459271B2 (en) 2000-12-20 2008-12-02 Murdoch Childrens Research Institute Method for detecting whether an organism is homozygous or heterozygous using labelled primers and RFLP
WO2004083457A1 (fr) * 2003-03-19 2004-09-30 The University Of British Columbia Haplotypes de l'inhibiteur 1 d'activateur de plasminogene (pai-1) utiles comme indicateurs d'evolution chez des patients
EP2199410A1 (fr) * 2003-03-19 2010-06-23 The University Of British Columbia Polymorphisme du plasminogen activator inhibitor-1 (PAI-1) pour le pronostic de maladie

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CA2290510A1 (fr) 1998-11-26
EP0983379A1 (fr) 2000-03-08
AU737771B2 (en) 2001-08-30
JP2001525678A (ja) 2001-12-11
RU2193069C2 (ru) 2002-11-20
AU4302297A (en) 1998-12-11

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