WO1994017204A1 - Process for amplifying and process for detecting polynucleotide sequences in a solid phase - Google Patents

Process for amplifying and process for detecting polynucleotide sequences in a solid phase Download PDF

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Publication number
WO1994017204A1
WO1994017204A1 PCT/EP1994/000143 EP9400143W WO9417204A1 WO 1994017204 A1 WO1994017204 A1 WO 1994017204A1 EP 9400143 W EP9400143 W EP 9400143W WO 9417204 A1 WO9417204 A1 WO 9417204A1
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WO
WIPO (PCT)
Prior art keywords
polynucleotide sequence
amplified
amplification
plastic carrier
activated
Prior art date
Application number
PCT/EP1994/000143
Other languages
German (de)
French (fr)
Inventor
Gustav F. Jirikowski
Original Assignee
Cytech Biomedical, Inc.
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Filing date
Publication date
Application filed by Cytech Biomedical, Inc. filed Critical Cytech Biomedical, Inc.
Publication of WO1994017204A1 publication Critical patent/WO1994017204A1/en

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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
    • C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase

Definitions

  • the invention relates to a method for the amplification of a nucleic acid or polynucleotide sequence and in particular to a method for the detection of a nucleic acid or polynucleotide sequence on a solid phase, in which the method is used for the amplification of the nucleic acid or polynucleotide sequence.
  • PCR The polymerase chain reaction (in the following abbreviated by PCR), which is described in US Pat. No. 4,683,202 and US Pat. No. 4, was developed in vitro 683 195.
  • PCR a defined DNA sequence is created using two flanking polynucleotide primers, the
  • A the repeated reaction cycle, which involves the steps of DNA denaturation,
  • Primer binding ("annealing") and polymerization includes to amplify the defined DNA sequence exponentially.
  • polynucleotide sequences can be (for double stranded present nucleic acids), amplified up to a length of about 6000 bases (when single stranded present nucleic "n acids) or base pairs, it being understood also from a ein ⁇ Zigen or a few copies of the nucleic acid . Since an RNA molecule can be converted into a DNA molecule by the reverse transcriptase reaction, the
  • RNA sample for the PCR was originally also available as RNA.
  • the reactions of these known amplification methods usually take place in a liquid medium.
  • This has the disadvantage that, for the availability of the amplified polynucleotide sequence for further applications, the amplification product is made by complex process steps, e.g. by chromatographic processes or by electrophoretic separation processes, must be obtained and possibly cleaned.
  • REPLACEMENT LEAF The object is achieved in that the polynucleotide sequence is amplified in the presence of a plastic carrier, with at least a part of the surface of the plastic carrier coming into contact with the polynucleotide sequence being activated by increasing the surface content, so that the amplified polynucleotides be bound to the activated surface of the plastic carrier.
  • Another object of the present invention is a method for the detection of a polynucleotide sequence on a solid phase, in which the polynucleotide sequence is amplified according to the above-mentioned method and bound to the plastic support as a solid phase, and the amplified polynucleotide sequence subsequently is determined.
  • FIG. 1 shows a device with which the method according to the invention for amplification and the method for detecting a polynucleotide sequence are preferably carried out.
  • FIG. 1A shows a schematic top view
  • FIG. IB shows a schematic cross section along the line 2-2 from FIG. 1A of the device.
  • any technique can be used to amplify a nucleic acid or polynucleotide sequence in which the polynucleotide sequence is reproduced or reproduced.
  • the amplification systems TAS, 3SR and the Q-beta replicase system are suitable, in particular, however, the PCR, as they are mentioned above and have been cited with source evidence.
  • thermostable reaction components such as, for example, thermostable DNA polymerases for PCR, which are sufficiently known in the prior art.
  • thermostable DNA polymerases from Thermus aquaticus, Th ⁇ rmus flavis or Thermococcus litoralis are suitable.
  • the enzymes required for the amplification reactions do not have to come from enzyme isolations, but can also be of cloned origin.
  • nucleic acid or polynucleotide sequences can be amplified according to the invention, such as double-stranded and single-stranded DNA and RNA.
  • the polynucleotide sequence to be amplified can itself be part of a longer polynucleotide sequence, for example part of a gene, a regulatory sequence, a viral or bacterial DNA or RNA sequence, a messenger RNA (m-RNA) or a plasmid.
  • m-RNA messenger RNA
  • the reaction batch for the amplification of a desired polynucleotide sequence is brought into contact with a plastic support in an aqueous medium which contains the additives required for the corresponding amplification reaction, at least part of the surface coming into contact with the amplification batch of the plastic carrier
  • REPLACEMENT LEAF is in the activated state.
  • Activation means that the surface area in the activated surface area of the plastic carrier has been increased, or in other words that the actual surface or actual surface has been increased compared to the outer surface (the envelope of the surface profile) of the plastic carrier. Activation can be accomplished by mechanical or chemical means. Detailed explanations of the type of plastic carrier to be used and its activation are given in pending PCT applications PCT / EP 92/02256 (filing date: October 8, 1992) and PCT / EP 92/02432 (filing date: 23. October 1992), the descriptions of which are considered part of the disclosure of the present application.
  • a material is used for the plastic carrier which has a matrix or base material or material made of copoly er or polymer blend.
  • This material can be easily activated by chemical or mechanical means to achieve the desired activation.
  • the material can optionally also contain other customary additives, such as dyes, processing aids, stabilizers, crosslinking agents, plasticizers, fillers and the like.
  • Copolymers or polymer blends which can be used are those which are mentioned in the above-mentioned PCT applications No. 92/02256 and No.
  • the plastic carrier that is to be used in the method according to the invention namely at least part of the surface area of the plastic carrier that is to come into contact with the amplification attachment, has been brought into an activated state by treating at least this part of the surface area of the plastic support with a solvent which one polymer component of the copolymer or the polymer blend dissolves more easily than the other or the other polymer multicomponent (s) of the copolymer or polymer blend.
  • solvents such as ethers, for example diethyl ether, esters, alcohols, ketones, aldehydes, acids, bases, unsaturated or aliphatic (linear, branched or cyclic) hydrocarbons, aromatic hydrocarbons, halogenated hydrocarbons and similar materials are suitable, for example Partially dissolve the polymeric material so that the polymer matrix is opened; as a result, the surface of the plastic carrier treated with the solvent is increased in its actual surface or actual surface.
  • ethers for example diethyl ether, esters, alcohols, ketones, aldehydes, acids, bases, unsaturated or aliphatic (linear, branched or cyclic) hydrocarbons, aromatic hydrocarbons, halogenated hydrocarbons and similar materials
  • solvents such as ethers, for example diethyl ether, esters, alcohols, ketones, aldehydes, acids, bases, unsaturated or aliphatic (linear, branched or cyclic) hydrocarbon
  • the polynucleotide sequence to be amplified like the polynucleotides that are synthesized in the course of the amplification process, are bound to the activated surface of the plastic carrier. This happens automatically during the amplification reaction taking place, without an additional step for binding the polynucleotides formed to the plastic carrier being necessary. Binding capacity, affinity and stability of the activated surface of the plastic carrier with respect to the polynucleotides are so high that the polynucleotides continuously formed during the amplification reaction adhere firmly to the activated plastic carrier and no longer diffuse from its surface .
  • the low molecular weight components of the authorization approach e.g. Primers and nucleotides used in the PCR approach are essentially in the liquid phase, while the high-molecular polynucleotides, the
  • REPLACEMENT LEAF are formed during the amplification and are largely bound to the solid phase of the activated carrier surface.
  • the length of the polynucleotide sequence to be amplified should not be too short so that the polynucleotides formed during the amplification are bound efficiently to the activated surface of the plastic support. It suitably has a sequence length of at least 100, preferably at least 200 and more preferably at least 500 nucleotides or base pairs.
  • polynucleotide sequences can be amplified in one approach and bound to the plastic support in the method according to the invention.
  • several amplification stages are carried out, only the last amplification stage being carried out in the presence of a surface-activated plastic carrier, while the remaining preceding amplification stages done in a conventional manner in a purely aqueous medium.
  • Multi-stage amplification methods are known to the person skilled in the art, for example for PCR.
  • the method for the amplification of a polynucleotide sequence with the aid of primers such as, for example, in the PCR reaction, to first immobilize an excess of pri on the support and to hybridize the polynucleotide sequence to be amplified to the primer, and then join the PCR reaction.
  • primers such as, for example, in the PCR reaction
  • Such a previously fixed primer can be referred to as a "capture primer 1 * , and is preferably fixed covalently.
  • the reaction (amplification) product remains anchored to the support.
  • the plastic carrier can be activated on its entire surface or only in surface areas which are provided for contact with the amplification approach. It may even be desirable that only a smaller portion of the plastic carrier that comes into contact with the amplification approach has been activated.
  • the plastic carrier to be used in the method according to the invention will suitably itself be the vessel in which the amplification reaction takes place.
  • a vessel can have the shape of a tube, a microcentrifuge tube or a depression of a microtiter plate, for example.
  • a microtiter plate has the advantage that it has numerous depressions (generally 96) and can therefore accommodate many amplification approaches for simultaneously carrying out the method according to the invention.
  • FIG. 1 shows a device which is particularly suitable for carrying out the amplification method according to the invention.
  • the components of the micro (titer) device include a micro (titer) mask 101, a cover plate 103 and a plate 104 of the plastic carrier, which is used as an activated plastic carrier for binding the amplified polynucleotide sequence.
  • the micro (titer) mask 101 has a multiplicity of cylindrical cavities 102, the cavities 102 each being intended to accommodate individual amplification approaches.
  • a sealing layer 105 made of elastic plastic material is preferably introduced between the plastic carrier plate 104 and the micro (titer) mask 101, which has corresponding recesses (not shown) corresponding to the individual cavities 102, so that the amplification approaches come into contact with the plastic carrier plate 104.
  • the plastic backing plate is at least in the areas with the
  • REPLACEMENT LEAF cylindrical cavities 102 are connected, activated, but can also be activated on the entire surface side facing the micro (titer) mask 101.
  • the cover plate 103 which can be made of any materials, closes off the individual cavities 102 from one another and from the outside.
  • the seal should be designed in particular with regard to increased vapor pressure phases in the individual vessels (wells) 102, which occur when amplification processes go through high-temperature cycles, such as, for example, in the case of PCR.
  • a further elastomer sealing layer can also be introduced between the micro (titer) mask 101 and the cover plate 103.
  • any fastening devices can be used to assemble the individual components of the device, as can be seen in FIG. 1B.
  • Groove closures 106, 107 are preferably used to assemble the device.
  • other fastening or locking devices known to the person skilled in the art are also suitable, e.g. Clamps, clips, screw closures or the like.
  • a pressure is preferably applied by the fastening or closure device between the cover plate 103 and the micro (titer) mask 101 and / or between the plastic carrier plate 104 and the micro (titer) mask 101 in order to seal the individual To improve void chambers 102.
  • the activated plastic carrier plate 104 can preferably be removed from the device 1. This enables bwz. facilitates a simple further procedure, for example for the qualitative or quantitative detection of the amplified polynucleotide sequence explained below.
  • a preferred embodiment consists in that the activated plastic carrier plate 104 contains "capture primer" at least in the activated area.
  • the surface of the reaction vessel used as the plastic support can be fully or partially activated, provided that at least part of the surface of the individual reaction vessels that comes into contact with the polynucleotide sequence in the amplification mixture is activated. It can be advantageous, for example, that only the bottom area of the depression of the respective reaction vessel receiving the amplification approach has been activated, while the remaining area of the surface which comes into contact with the amplification approach and the remaining surface of the reaction vessel is in an unactivated state. so as to achieve a concentration of binding of the polynucleotides formed during the amplification in a smaller area.
  • the plastic carrier can also be in forms other than the form of the reaction vessel itself.
  • the plastic carrier in the form of beads, granules, fibers, disks, foils, rods and the like can be brought into contact with the amplification batch containing the defined polynucleotide sequence.
  • the shape of the plastic carrier and the extent of activation of the plastic carrier can be selected as desired or depending on the requirements for the respective application of the method according to the invention or the polynucleotides obtained by the method according to the invention become.
  • the method according to the invention can be used in many areas.
  • REPLACEMENT LEAF detectable amount of a polynucleotide sequence a significantly larger amount of the polynucleotide sequence is prepared and obtained in a simple manner.
  • the polynucleotide sequence amplified by the method according to the invention in particular a DNA amplified by the PCR, is furthermore outstandingly suitable for cloning purposes and other genetic engineering applications.
  • a further field of application is offered by the nucleic acid sequence analysis of the polynucleotide sequence amplified by the method according to the invention.
  • the polynucleotides amplified and produced by the method according to the invention can remain on the plastic carrier, or the polynucleotides can be removed from the plastic carrier in a further process step be removed.
  • the bond between the polynucleotides and the plastic carrier based on hydrophobic interaction can be achieved by suitable chemical means, e.g. be broken up by solvents. Possibly. the plastic carrier can also be completely dissolved by suitable solvents. In both cases, the synthesized polynucleotides are then present in a liquid medium, so that they are then isolated by purification methods familiar to the person skilled in the art.
  • Another object of the present invention is a method for the detection of a specific polynucleotide sequence on a solid phase, in which the polynucleotide sequence to be detected is amplified according to the method described above and at the same time bound to the plastic support as a solid phase and subsequently detected or loading is true.
  • the method described above for amplifying a polynucleotide sequence is part of this method for detecting the polynucleotide sequence, so that the explanations given above also apply to this embodiment of the present invention.
  • DNA-specific fluorescent dyes such as Ethidium bromide, DAPI or the bis-benzimidazole dye 33258 Hoechst (see V. W. Bums in Photochem. Photobiol. Rev., pages 87 to 103 (1980)) can be used.
  • the detection method according to the invention is preferably carried out in such a way that the polynucleotide sequence to be detected is amplified by means of the PCR, using either labeled primers and / or labeled nucleotides, with the purpose that the labels are replaced by the respective because of the building blocks (primers and / or nucleotides) that are incorporated into the synthesized polynucleotides by the PCR.
  • the markings thus incorporated can then be easily determined and are a function of the amount of polynucleotides formed. With the aid of suitable standard comparison values, quantitative detection determinations of a polynucleotide sequence to be determined can thus be carried out in a simple manner.
  • amplification approaches other than PCR can also be used in order to mark using appropriate,
  • the separation between the fixed binding of the synthesized polynucleotides and the non-binding of the possibly labeled low molecular weight building blocks can be improved by adding a detergent, the amount of detergent added depending on the particular conditions (type of activated carrier, type and amount of Building blocks, length of the primers and the synthesized polynucleotides, etc.) can be adapted within the scope of the expert knowledge.
  • any type of marking can be used which is well known to the person skilled in the art from the prior art.
  • Suitable for example are radio labels, such as 3 H-, 125 J- and 3 P-labeled nucleotides and primers; Markings with chromophores, fluorophores; time-delayed fluorescence and chemiluminescence-generating marking systems; in particular markings with reporter groups, such as haptens or biotin, which enable immunochemical methods or with the biotin avidin / streptavidin system.
  • bromine and digi-toxigenin-modified nucleoside triphosphates have proven themselves as hapten-labeled nucleotide building blocks.
  • Combinations of different types of labels can of course also be used, in particular if several defined polynucleotide sequences are to be detected in one batch.
  • labeled building blocks primary and / or nucleotides
  • these are preferably used in combination with unlabeled analogues of the building blocks in the amplification reaction.
  • Suitable relationships between the labeled building blocks and the unlabeled analogs are known (see above references by H.A. Er ⁇ lich et al. And T. Maniatis et al.). It is often also desirable to add the marked building blocks only in certain stages of the amplification cycles, in particular in the last amplification cycles or even only in the last amplification cycle.
  • FIA fluorescence immunoassays
  • EIA enzyme immunoassays
  • the detection method according to the invention is particularly simple, but also very sensitive, when hapten or biotin labels are used, the incorporation of which into the synthesized polynucleotides is determined by enzyme immunoassays, in particular in connection with a Antibody detection reagent conjugated to an enzyme, the precipitating
  • REPLACEMENT LEAF Detection dyes form, which are deposited on the plastic support at the location of the polynucleotide bond. Further explanations on suitable detection methods, in particular by immunochemical techniques, are given in the above-mentioned pending PCT applications No. 92/02256 and No. 92/02432, to which reference is expressly made here .
  • Determination methods using automated detection devices e.g. gray-level scanning.
  • a particularly high specificity of the detection of defined polynucleotide sequences is achieved in that the amplified polynucleotide sequence bound to the plastic support is determined by means of the nucleic acid hybridization technique.
  • detection takes place via labeled nucleic acid probes which bind specifically to the polynucleotide sequence to be detected or to partial regions of this polynucleotide sequence.
  • the detection determination is again carried out by means of a suitable label which carries the nucleic acid probe, it also being possible to use the above-mentioned types of label here.
  • Suitable emulsifiers or detergents include, for example, Triton X-100 (octylphenoxypolyethoxyethanol), Tween 20 (polyoxyethylene sorbiton), SDS (sodium dodecyl sulfate), non-ionic, anionic or cationic surfactants, borates and beefs.

Abstract

In a process of amplifying a polynucleotide sequence in the presence of a plastic substrate, at least part of the surface of the plastic substrate in contact with the polynucleotide sequence is activated by increasing its surface content, so that the amplified polynucleotides are bound to the activated surface of the plastic substrate. This amplification process may be used for detecting a polynucleotide sequence in a solid phase, in that the amplified polynucleotide sequence bound to the plastic substrate may be determined on the plastic substrate.

Description

Verfahren zur Amplifikation und Verfahren zum Nachweis von Polynucleotidsequenzen an fester Phase Amplification methods and methods for the detection of solid phase polynucleotide sequences
Die Erfindung betrifft ein Verfahren zur Amplifikation einer Nucleinsäure- bzw. Polynucleotidsequenz und insbesondere ein Verfahren zum Nachweis einer Nucleinsäure- bzw. Polynucleotid¬ sequenz an fester Phase, bei dem das Verfahren zur Amplifika¬ tion der Nucleinsäure- bzw. Polynucleotidsequenz verwendet wird.The invention relates to a method for the amplification of a nucleic acid or polynucleotide sequence and in particular to a method for the detection of a nucleic acid or polynucleotide sequence on a solid phase, in which the method is used for the amplification of the nucleic acid or polynucleotide sequence.
1010
In der Vergangenheit sind zahlreiche Verfahren entwickelt wor¬ den, um eine spezifische Nucleinsäure- bzw. Polynucleotidse¬ quenz künstlich bzw. in-vitro zu amplifizieren. Als erstes und inzwischen weitverbreitetes Verfahren zur Amplifikation einerIn the past, numerous methods have been developed to artificially amplify a specific nucleic acid or polynucleotide sequence or in vitro. As the first and now widely used method for the amplification of a
•_= Polynucleotidsequenz in-vitro wurde die Polymerase-Kettenreak- tion (engl. "polymerase chain reaction", im folgenden abge¬ kürzt durch PCR), entwickelt, die beschrieben ist US-A-4 683 202 und US-A-4 683 195. Bei der PCR wird eine definierte DNA- Sequenz mittels zweier flankierender Polynucleotid-Pri er, dieThe polymerase chain reaction (in the following abbreviated by PCR), which is described in US Pat. No. 4,683,202 and US Pat. No. 4, was developed in vitro 683 195. In PCR, a defined DNA sequence is created using two flanking polynucleotide primers, the
20 jeweils so an einen terminalen Sequenzbereich der komplementä¬ ren DNA-Stränge der definierten DNA-Sequenz binden, daß die 3'-Enden der Primer zueinandergerichtet sind, und auf diese Weise eine gegenläufige DNA-Polymerase-abhängige Synthese neue komplementärer DNA-Stränge ablaufen, um über einen häufig wie- 20 each bind to a terminal sequence region of the complementary DNA strands of the defined DNA sequence in such a way that the 3 'ends of the primers are directed towards one another, and in this way an opposite DNA polymerase-dependent synthesis of new complementary DNA strands takes place to talk about a frequently-
A. A- derholten Reaktionszyklus, der die Schritte DNA-Denaturierung,A. A- the repeated reaction cycle, which involves the steps of DNA denaturation,
Primer-Bindung ("annealing" ) und Polymerisation umfaßt, die definierte DNA-Seguenz expoπentiell zu amplifizieren. Auf diese Weise können Polynucleotidsequenzen bis zu einer Länge von etwa 6000 Basen (bei einzelsträngig vorliegenden Nuclein- „n säuren) oder Basenpaaren (bei doppelsträngig vorliegenden Nucleinsäuren) , amplifiziert werden, wobei auch von einer ein¬ zigen oder wenigen Kopien der Nucleinsäure ausgegangen werden kann. Da ein RNA-Molekül durch die Reverse Transcriptase-Reak- tion in ein DNA-Molekül umgewandelt werden kann, kann diePrimer binding ("annealing") and polymerization includes to amplify the defined DNA sequence exponentially. In this manner, polynucleotide sequences can be (for double stranded present nucleic acids), amplified up to a length of about 6000 bases (when single stranded present nucleic "n acids) or base pairs, it being understood also from a ein¬ Zigen or a few copies of the nucleic acid . Since an RNA molecule can be converted into a DNA molecule by the reverse transcriptase reaction, the
-„_ Nucleinsäureprobe für die PCR ursprünglich auch als RNA vor- 35 liegen.- “_ Nucleic acid sample for the PCR was originally also available as RNA.
ERSATZBLATT Neben der PCR haben sich noch andere Verfahren zur Amplifika- tion von Nucleinsäure- bzw. Polynucleotidsequenzen herausge¬ bildet, wie das auf Transkription basierende Amplifikationssy- stem (abgekürzt TAS, siehe D.Y. Kwo et al., Proceedings of the National Academy of Sciences USA , Seiten 1173 bis 1177 (1989)), das nach retroviralem Replikationsmuεter ablaufende "Sequenz-Replikationssystem" (abgekürzt 3SR, siehe JP.C. Gua- telli et.al., Proceedings of the National Academy of Sciences USA £7, Seiten 1874 bis 1878 (1990)) und das Q-beta Replicase- Systera (siehe F.R. Kramer und P.M. Lizadi, Nature 339, Seiten 401 bis 402 (1989)).REPLACEMENT LEAF In addition to PCR, other methods for amplifying nucleic acid or polynucleotide sequences have emerged, such as the transcription-based amplification system (abbreviated TAS, see DY Kwo et al., Proceedings of the National Academy of Sciences USA, Pages 1173 to 1177 (1989)), the "sequence replication system" (abbreviated 3SR, which follows the retroviral replication pattern), see JP.C. Guatelli et.al., Proceedings of the National Academy of Sciences USA £ 7, pages 1874 to 1878 (1990)) and the Q-beta replicase system (see FR Kramer and PM Lizadi, Nature 339, pages 401 to 402 (1989)).
Die Reaktionen dieser bekannten Amplifikationsverfahren finden üblicherweise in flüssigem Medium statt. Dies hat den Nach¬ teil, daß für die Verfügbarkeit der amplifizierten Polynucleo¬ tidsequenz für weitere Anwendungen das Amplifikationsprodukt durch aufwendige Verfahrenεschritte, z.B. durch chromatogra¬ phische Verfahren oder durch elektrophoretische Trennverfah¬ ren, gewonnen und ggf. gereinigt werden muß.The reactions of these known amplification methods usually take place in a liquid medium. This has the disadvantage that, for the availability of the amplified polynucleotide sequence for further applications, the amplification product is made by complex process steps, e.g. by chromatographic processes or by electrophoretic separation processes, must be obtained and possibly cleaned.
Für viele Anwendungsbereiche, in denen eine Amplifikation einer definierten Polynucleotidsequenz gewünscht oder erfor¬ derlich ist, insbesondere zum Nachweis einer spezifischen Po¬ lynucleotidsequenz, besteht das Bedürfnis, ein Verfahren, wel¬ ches die Amplifikation einer Polynucleotidsequenz umfaßt, we¬ sentlich zu vereinfachen.For many areas of application in which an amplification of a defined polynucleotide sequence is desired or necessary, in particular for the detection of a specific polynucleotide sequence, there is a need to significantly simplify a method which comprises the amplification of a polynucleotide sequence.
Es ist daher Aufgabe vorliegender Erfindung, ein Verfahren zur Amplifikation einer Polynucleotidsequenz zur Verfügung zu stellen, bei dem die amplifizierte Polynucleotidsequenz ohne weitere Schritte aus dem Ansatz zur Amplifikation gewonnen werden kann und die so gewonnene, amplifizierte Polynucleotid¬ sequenz leicht weiteren Anwendungen, insbesondere einer Nach¬ weisbestimmung, zugänglich ist.It is therefore an object of the present invention to provide a method for the amplification of a polynucleotide sequence in which the amplified polynucleotide sequence can be obtained from the amplification approach without further steps and the amplified polynucleotide sequence obtained in this way can easily be used in other applications, in particular in an after ¬ determination of access is accessible.
ERSATZBLATT Die Aufgabe wird dadurch gelöst, daß die Polynucleotidsequenz in Gegenwart eines Kunststoff-Trägers amplifiziert wird, wobei mindestens ein Teil der mit der Polynucleotidsequenz in Kon¬ takt tretenden Oberfläche des Kunststoff-Trägers aktiviert wurde durch Erhöhung des Oberflächeninhalts, so daß die ampli- fizierten Polynucleotide an die aktivierte Oberfläche des Kunststoff-Trägers gebunden werden.REPLACEMENT LEAF The object is achieved in that the polynucleotide sequence is amplified in the presence of a plastic carrier, with at least a part of the surface of the plastic carrier coming into contact with the polynucleotide sequence being activated by increasing the surface content, so that the amplified polynucleotides be bound to the activated surface of the plastic carrier.
Ein weiterer Gegenstand vorliegender Erfindung besteht in einem Verfahren zum Nachweis einer Polynucleotidsequenz an fe¬ ster Phase, bei dem die Polynucleotidsequenz nach dem oben ge¬ nannten Verfahren amplifiziert und an den Kunststoff-Träger als feste Phase gebunden wird, und die amplifizierte Poly¬ nucleotidsequenz anschließend bestimmt wird.Another object of the present invention is a method for the detection of a polynucleotide sequence on a solid phase, in which the polynucleotide sequence is amplified according to the above-mentioned method and bound to the plastic support as a solid phase, and the amplified polynucleotide sequence subsequently is determined.
Vorteilhafte Ausgestaltung der vorliegenden Erfindung sind in den Unteransprüchen gekennzeichnet.Advantageous embodiments of the present invention are characterized in the subclaims.
Die Erfindung wird im folgenden näher erläutert.The invention is explained in more detail below.
Figur 1 zeigt eine Vorrichtung, mit der das erfindungsgemäße Verfahren zur Amplifikation und das Verfahren zum Nachweis einer Polynucleotidsequenz vorzugsweise ausgeführt wird.FIG. 1 shows a device with which the method according to the invention for amplification and the method for detecting a polynucleotide sequence are preferably carried out.
Figur 1A stellt eine schematische Draufsicht, und Figur IB stellt einen schematischen Querschnitt entlang der Linie 2-2 aus Figur 1A der Vorrichtung dar.FIG. 1A shows a schematic top view, and FIG. IB shows a schematic cross section along the line 2-2 from FIG. 1A of the device.
Zur Amplifikation einer Nucleinsäure- bzw. Polynucleotidse¬ quenz kann irgendeine Technik verwendet werden, bei der die Polynucleotidsequenz vervielfältigt bzw. reproduziert wird. Geeignet sind beispielsweise die Amplifikationssysteme TAS, 3SR und das Q-beta Replikase-System, insbesondere jedoch die PCR, wie sie oben genannt sind und mit Quellennachweis angege¬ ben wurden.Any technique can be used to amplify a nucleic acid or polynucleotide sequence in which the polynucleotide sequence is reproduced or reproduced. For example, the amplification systems TAS, 3SR and the Q-beta replicase system are suitable, in particular, however, the PCR, as they are mentioned above and have been cited with source evidence.
ER In den Amplifikationsreaktionen, welche Schritte unter Bedingungen erhöhter Temperatur durchlaufen, werden vorzugs¬ weise thermostabile Reaktionskomponenten verwendet, wie zum Beispiel thermostabile DNA-Polymerasen für die PCR, die im Stand der Technik hinreichend bekannt sind. Geeignet sind bei¬ spielsweise die thermostabilen DNA-Polymerasen aus Thermus aquaticus, Thβrmus flavis oder Thermococcus litoralis . Die für die Amplifikationsreaktionen erforderlichen Enzyme müssen nicht von Enzymisolierungen stammen, sondern können auch klo- nierten Ursprungs sein.HE In the amplification reactions, which steps are carried out under conditions of elevated temperature, preference is given to using thermostable reaction components, such as, for example, thermostable DNA polymerases for PCR, which are sufficiently known in the prior art. For example, the thermostable DNA polymerases from Thermus aquaticus, Thβrmus flavis or Thermococcus litoralis are suitable. The enzymes required for the amplification reactions do not have to come from enzyme isolations, but can also be of cloned origin.
Einen Überblick über neuere Entwicklungen und die Reaktionsbe¬ dingungen für die PCR werden z.B. gegeben in: H.A. Erlich, R. Gibbs und H.H. Kazazian: "Polymerase Chain Reaction", in "Current Communications in Molecular Biology", Cold Spring Harbor Laboratory Press (1988), sowie in: T. Maniatis et al., "Molecular cloning. A Laboratory Manual", Cold Spring Harbor Laboratory Press (1988).An overview of recent developments and the reaction conditions for PCR are e.g. given in: H.A. Erlich, R. Gibbs and H.H. Kazazian: "Polymerase Chain Reaction", in "Current Communications in Molecular Biology", Cold Spring Harbor Laboratory Press (1988), and in: T. Maniatis et al., "Molecular cloning. A Laboratory Manual", Cold Spring Harbor Laboratory Press (1988).
Es können jegliche Arten von Nucleinsäure- bzw. Polynucleotid¬ sequenzen gemäß Erfindung amplifiziert werden, wie doppel- strängige und einzelsträngige DNA und RNA. Die zu amplifizie- rende Polynucleotidsequenz kann selbst Teil einer längeren Po¬ lynucleotidsequenz sein, beispielsweise Teil eines Gens, einer regulatorischen Sequenz, einer viralen oder bakteriellen DNA- oder RNA-Sequenz, einer Boten-RNA (m-RNA) oder eines Plasmids.Any kind of nucleic acid or polynucleotide sequences can be amplified according to the invention, such as double-stranded and single-stranded DNA and RNA. The polynucleotide sequence to be amplified can itself be part of a longer polynucleotide sequence, for example part of a gene, a regulatory sequence, a viral or bacterial DNA or RNA sequence, a messenger RNA (m-RNA) or a plasmid.
Der Reaktionsansatz zur Amplifikation einer gewünschten Poly¬ nucleotidsequenz wird in einem wäßrigen Medium, welches die für die entsprechende Amplifikationsreaktion erforderlichen Zusätze enthält, mit einem Kunststoff-Träger in Kontakt ge¬ bracht, wobei mindestens ein Teil der mit dem Amplifikations- ansatz in Kontakt tretenden Oberfläche des Kunststoff-TrägersThe reaction batch for the amplification of a desired polynucleotide sequence is brought into contact with a plastic support in an aqueous medium which contains the additives required for the corresponding amplification reaction, at least part of the surface coming into contact with the amplification batch of the plastic carrier
ERSATZBLATT in aktiviertem Zustand vorliegt.REPLACEMENT LEAF is in the activated state.
Aktivierung bedeutet, daß der Oberflächeninhalt im aktivierten Oberflächenbereich des Kunststo f-Trägers erhöht worden ist, oder mit anderen Worten, daß die tatsächliche Oberfläche bzw. Ist-Oberfläche gegenüber der die Mantelfläche (die Einhüllende des Oberflächenprofils) des Kunststoff-Trägers vergrößert wurde. Die Aktivierung kann durch mechanische oder chemische Mittel bewirkt werden. Ausführliche Erläuterungen zu der Art des zu verwendenden Kunststoff-Trägers sowie zu dessen Akti¬ vierung werden in den anhängigen PCT-Anmeldungen PCT/EP 92/02256 (Anmeldetag: 8. Oktober 1992) und PCT/EP 92/02432 (Anmeldetag: 23. Oktober 1992) gegeben, deren Beschreibungen als Teil der Offenbarung vorliegender Anmeldung gelten.Activation means that the surface area in the activated surface area of the plastic carrier has been increased, or in other words that the actual surface or actual surface has been increased compared to the outer surface (the envelope of the surface profile) of the plastic carrier. Activation can be accomplished by mechanical or chemical means. Detailed explanations of the type of plastic carrier to be used and its activation are given in pending PCT applications PCT / EP 92/02256 (filing date: October 8, 1992) and PCT / EP 92/02432 (filing date: 23. October 1992), the descriptions of which are considered part of the disclosure of the present application.
Vorzugsweise wird in dem erfindungsgemäßen Verfahren für den Kunststoff-Träger ein Material verwendet, das eine Matrix bzw. Grundmaterial oder -stoff aus Copoly er oder Polymerblend aufweist. Dieses Material kann leicht durch chemische oder mechanische Mittel aktiviert werden, um die gewünschte Aktivierung zu erhalten. Das Material kann ggf. noch weitere übliche Zusätze enthalten, wie Farbstoffe, Pro¬ zessierhilfsmittel, Stabilisatoren, Vernetzungsmittel, Weich¬ macher, Füllstoffe und dergleichen. Als Copolymere oder Poly- merblends können solche verwendet werden, die in den oben ge¬ nannten PCT-Anmeldungen Nr. 92/02256 und Nr. 92/02432 genannt sind, insbesondere Copolymere von Styrol und Butadien oder Styrol und Chloropren oder Polymerblends von Polystyrol und Polybutadien oder Polystyrol und Polychloropren. Bei Aktivie¬ rung durch chemische Mittel wurde der Kunststoff-Träger, der in dem erfindungsgemäßen Verfahren verwendet werden soll, und zwar mindestens ein Teil des Oberflächenbereiches des Kunst¬ stoff-Trägers, der mit dem Amplifikationsansätz in Kontakt kommen soll, in einen aktivierten Zustand versetzt, indem mindestens dieser Teil des Oberflächenbereiches des Kunst¬ sto f-Trägers mit einem Lösungsmittel behandelt wurde, welches eine Polymerkomponente des Copolymers bzw. des Polymerblends leichter löst als die andere bzw. die anderen Polymehrkompo- nente(n) des Copolymers bzw. Polymerblends. Zu diesem Zweck eignen sich beispielsweise Lösungsmittel wie Ether, z.B. Diethylether, Ester, Alkohole, Ketone, Aldehyde, Säuren, Ba¬ sen, ungesättigte oder aliphatische (lineare, verzweigte oder zyklische) Kohlenwasserstoffe, aromatische Kohlenwasserstoffe, halogenierte Kohlenwasserstoffe und ähnliche Materialien, wel¬ che das polymere Material teilweise lösen, so daß die Polymer¬ matrix geöffnet wird, im Resultat ist die mit dem Lösungsmit¬ tel behandelte Oberfläche des Kunststoff-Trägers in ihrer tatsächlichen Oberflächen bzw. Ist-Oberfläche erhöht.Preferably, in the method according to the invention, a material is used for the plastic carrier which has a matrix or base material or material made of copoly er or polymer blend. This material can be easily activated by chemical or mechanical means to achieve the desired activation. The material can optionally also contain other customary additives, such as dyes, processing aids, stabilizers, crosslinking agents, plasticizers, fillers and the like. Copolymers or polymer blends which can be used are those which are mentioned in the above-mentioned PCT applications No. 92/02256 and No. 92/02432, in particular copolymers of styrene and butadiene or styrene and chloroprene or polymer blends of polystyrene and Polybutadiene or polystyrene and polychloroprene. When activated by chemical means, the plastic carrier that is to be used in the method according to the invention, namely at least part of the surface area of the plastic carrier that is to come into contact with the amplification attachment, has been brought into an activated state by treating at least this part of the surface area of the plastic support with a solvent which one polymer component of the copolymer or the polymer blend dissolves more easily than the other or the other polymer multicomponent (s) of the copolymer or polymer blend. For this purpose, solvents such as ethers, for example diethyl ether, esters, alcohols, ketones, aldehydes, acids, bases, unsaturated or aliphatic (linear, branched or cyclic) hydrocarbons, aromatic hydrocarbons, halogenated hydrocarbons and similar materials are suitable, for example Partially dissolve the polymeric material so that the polymer matrix is opened; as a result, the surface of the plastic carrier treated with the solvent is increased in its actual surface or actual surface.
Im Rahmen der Erfindung wurde nun überraschenderweise festge¬ stellt, daß die zu amplifizierende Polynucleotidsequenz ebenso wie die Polynucleotide, die im Laufe des Amplifika- tionsprozesses synthetisiert werden, an die aktivierte Ober¬ fläche des Kunststoff-Trägers gebunden werden. Dies geschieht automatisch während der ablaufenden Amplifikationsreaktion, ohne daß ein zusätzlicher Schritt zur Bindung der gebildeten Polynucleotide an den Kunststoff-Träger erforderlich ist. Bin¬ dungskapazität, -affinität und -Stabilität der aktivierten Oberfläche des Kunststoff-Trägers gegenüber den Polynucleoti- den sind so hoch, daß die während der Amplifikationsreaktion fortlaufend gebildeten Polynucleotide fest an dem aktivierten Kunststoff-Träger anhaften und nicht mehr von dessen Oberflä¬ che abdiffundieren. Es wird angenommen, daß sich ein Gleichge¬ wicht zwischen in der flüssigen Phase des wäßrigen Milieus be¬ findlichen, niedermolekularen Komponenten des Amplifikations- ansatzes und an der festen Phase des Kunststoff-Trägers gebun¬ denen höhermolekularen Komponenten des Amplifikationsanεatzes einstellt. D.h. , die niedermolekularen Komponenten des A pli- fikationsansatzes, wie z.B. bei dem PCR-Ansatz verwendete Pri¬ mer und Nucleotide, befinden sich im wesentlichen in der flüs¬ sigen Phase, während die hochmolekularen Polynucleotide, dieIn the context of the invention, it has now surprisingly been found that the polynucleotide sequence to be amplified, like the polynucleotides that are synthesized in the course of the amplification process, are bound to the activated surface of the plastic carrier. This happens automatically during the amplification reaction taking place, without an additional step for binding the polynucleotides formed to the plastic carrier being necessary. Binding capacity, affinity and stability of the activated surface of the plastic carrier with respect to the polynucleotides are so high that the polynucleotides continuously formed during the amplification reaction adhere firmly to the activated plastic carrier and no longer diffuse from its surface . It is assumed that there is an equilibrium between the low-molecular components of the amplification batch in the liquid phase of the aqueous medium and the higher-molecular components of the amplification batch bound to the solid phase of the plastic carrier. That , the low molecular weight components of the authorization approach, e.g. Primers and nucleotides used in the PCR approach are essentially in the liquid phase, while the high-molecular polynucleotides, the
ERSATZBLATT bei der Amplifikation gebildet werden, weitgehend an der fe¬ sten Phase der aktivierten Trägeroberfläche gebunden vorlie¬ gen.REPLACEMENT LEAF are formed during the amplification and are largely bound to the solid phase of the activated carrier surface.
Damit eine effiziente Bindung der während der Amplifikation gebildeten Polynucleotide an die aktivierte Oberfläche des Kunststoff-Trägers stattfindet, sollte die Länge der zu ampli- fizierenden Polynucleotidsequenz nicht zu kurz sein. Geeigneterweise weist sie eine Sequenzlänge von mindestens 100, bevorzugt mindestens 200 und weiter bevorzugt mindestens 500 Nucleotide bzw. Basenpaare aufweisen.The length of the polynucleotide sequence to be amplified should not be too short so that the polynucleotides formed during the amplification are bound efficiently to the activated surface of the plastic support. It suitably has a sequence length of at least 100, preferably at least 200 and more preferably at least 500 nucleotides or base pairs.
Selbstverständlich können in dem erfindungsgemäßen Verfahren nicht nur eine, sondern auch mehrere verschiedene, spezifische Polynucleotidsequenzen in einem Ansatz amplifiziert und an den Kunststoff-Träger gebunden werden. Ferner ist es auch im Rah¬ men der Erfindung möglich, daß in dem Verfahren zur Amplifika¬ tion einer Polynucleotidsequenz mehrere Amplifikationsstufen durchgeführt werden, wobei nur die letzte Amplifikationsstufe in Gegenwart eines oberflächenaktivierten Kunststoff-Trägers durchgeführt wird, während die übrigen, vorangehenden Amplifi¬ kationsstufen auf herkömmliche Weise in rein wäßrigem Medium erfolgen. Mehrstufige Amplifikationsverfahren sind dem Fach¬ mann beispielsweise für die PCR bekannt.Of course, not only one, but also several different, specific polynucleotide sequences can be amplified in one approach and bound to the plastic support in the method according to the invention. Furthermore, it is also possible within the scope of the invention that in the method for amplifying a polynucleotide sequence, several amplification stages are carried out, only the last amplification stage being carried out in the presence of a surface-activated plastic carrier, while the remaining preceding amplification stages done in a conventional manner in a purely aqueous medium. Multi-stage amplification methods are known to the person skilled in the art, for example for PCR.
Im Rahmen der Erfindung ist es besonders bevorzugt, bei dem Verfahren zur Amplifikation einer Polynucleotidseguenz mit Hilfe von Primern, wie z.B. bei der PCR-Reaktion, zuerst einen Pri erüberschuß auf dem Träger zu immobilisieren und die zu amplifizierende Polynucleotidsequenz an den Primer zu hybridisieren, und dann die PCR-Reaktion anzuschließen. Ein solcher zuvor fixierter Primer kann als "capture-primer1* bezeichnet werden, und ist vorzugsweise kovalent fixiert. In der nachfolgenden PCR-Reaktion bleibt das Reaktion- (Amplifikations-)Produkt am Träger verankert.In the context of the invention, it is particularly preferred in the method for the amplification of a polynucleotide sequence with the aid of primers, such as, for example, in the PCR reaction, to first immobilize an excess of pri on the support and to hybridize the polynucleotide sequence to be amplified to the primer, and then join the PCR reaction. Such a previously fixed primer can be referred to as a "capture primer 1 * , and is preferably fixed covalently. In the subsequent PCR reaction, the reaction (amplification) product remains anchored to the support.
ERSATZBLATT Je nach Wunsch kann der Kunststoff-Träger auf seiner gesamten Oberfläche oder nur in Oberflächenbereichen, die für den Kon¬ takt mit dem Amplifikationsansatz vorgesehen sind, aktiviert worden sein. Es kann sogar erwünscht sein, daß lediglich ein kleinerer Teilbereich des Kunststoff-Trägers, der mit dem Amplifikationsansatz in Kontakt kommt, aktiviert wurde.REPLACEMENT LEAF Depending on requirements, the plastic carrier can be activated on its entire surface or only in surface areas which are provided for contact with the amplification approach. It may even be desirable that only a smaller portion of the plastic carrier that comes into contact with the amplification approach has been activated.
Der in dem erfindungsgemäßen Verfahren zu verwendende Kunst¬ stoff-Träger wird geeigneterweise selbst das Gefäß sein, in dem die Amplifikationsreaktion abläuft. Ein solches Gefäß kann beispielsweise die Form eines Röhrchens, eines Mikrozentrifu- genröhrchens oder einer Vertiefung einer Mikrotiterplatte ha¬ ben. Eine Mikrotiterplatte hat den Vorteil, daß sie zahlreiche Vertiefungen aufweist (in der Regel 96) und somit viele Ampli- fikationsansätze zur gleichzeitigen Durchführung des erfin¬ dungsgemäßen Verfahrens aufnehmen kann.The plastic carrier to be used in the method according to the invention will suitably itself be the vessel in which the amplification reaction takes place. Such a vessel can have the shape of a tube, a microcentrifuge tube or a depression of a microtiter plate, for example. A microtiter plate has the advantage that it has numerous depressions (generally 96) and can therefore accommodate many amplification approaches for simultaneously carrying out the method according to the invention.
Die Figur 1 zeigt eine Vorrichtung, die zur Durchführung des erfindungsgemäßen Amplifikationsverfahrens besonders gut ge¬ eignet ist. Die Mikro(-titer-)Vorrichtung umfaßt als Bestand¬ teile eine Mikro(-titer-)maske 101, eine Deckplatte 103 sowie eine Platte 104 des Kunststoff-Trägers, der als aktivierter Kunststoff-Träger zur Bindung der amplifizierten Polynucleotidsequenz eingesetzt wird. Die Mikro(-titer-)maske 101 weist eine Vielzahl zylindrischer Hohlräume 102 auf, wobei die Hohlräume 102 jeweils einzelne Amplifikationsansätze aufnehmen sollen. Damit die einzelnen Hohlräume besser gegeneinander abgedichtet sind, wird vorzugsweise zwischen der Kunststoff-Trägerplatte 104 und der Mikro(-titer-)maske 101 eine Dichtungsschicht 105 aus elastischem Kunststoffmaterial eingebracht, welche korrespondierend zu den einzelnen Hohlräumen 102 entsprechende Ausnehmungen (nicht dargestellt) aufweist, damit die Amplifikationsansätze mit der Kunststoff- Trägerplatte 104 in Kontakt treten. Die Kunststoff- Trägerplatte ist also mindestens in den Bereichen, die mit denFIG. 1 shows a device which is particularly suitable for carrying out the amplification method according to the invention. The components of the micro (titer) device include a micro (titer) mask 101, a cover plate 103 and a plate 104 of the plastic carrier, which is used as an activated plastic carrier for binding the amplified polynucleotide sequence. The micro (titer) mask 101 has a multiplicity of cylindrical cavities 102, the cavities 102 each being intended to accommodate individual amplification approaches. So that the individual cavities are better sealed against one another, a sealing layer 105 made of elastic plastic material is preferably introduced between the plastic carrier plate 104 and the micro (titer) mask 101, which has corresponding recesses (not shown) corresponding to the individual cavities 102, so that the amplification approaches come into contact with the plastic carrier plate 104. The plastic backing plate is at least in the areas with the
ERSATZBLATT zylindrischen Hohlräumen 102 in Verbindung stehen, aktiviert, kann jedoch auch auf der ganzen, der Mikro(-titer-)maske 101 zugewandten Oberflächenseite aktiviert sein. Die Deckplatte 103, die aus beliebigen Materialien bestehen kann, schließt die einzelnen Hohlräume 102 gegeneinander und nach außen hin ab. Die Abdichtung sollte insbesondere im Hinblick auf erhöhte Dampfdruckphasen in den einzelnen Gefäßen (Wells) 102 ausgestaltet werden, die auftreten, wenn Am¬ plifikationsverfahren Hochtemperaturzyklen durchlaufen, wie z.B. bei der PCR. Zu diesem Zweck kann auch noch eine weitere Elastomer-Dichtungsschicht zwischen Mikro(-titer-)maske 101 und Deckplatte 103 eingebracht werden.REPLACEMENT LEAF cylindrical cavities 102 are connected, activated, but can also be activated on the entire surface side facing the micro (titer) mask 101. The cover plate 103, which can be made of any materials, closes off the individual cavities 102 from one another and from the outside. The seal should be designed in particular with regard to increased vapor pressure phases in the individual vessels (wells) 102, which occur when amplification processes go through high-temperature cycles, such as, for example, in the case of PCR. For this purpose, a further elastomer sealing layer can also be introduced between the micro (titer) mask 101 and the cover plate 103.
Zum Zusammenbau der einzelnen Bestandteile der Vorrichtung, wie sie in Fig. IB zu sehen ist, können beliebige Befestigungseinrichtungen verwendet werden. Vorzugsweise dienen Nutverschlüsse 106, 107 zum Zusammenbau der Vorrichtung. Geeignet sind jedoch auch andere Befestigungs¬ oder Verschlußeinrichtungen, die dem Fachmann geläufig sind, z.B. Klammern, Spangen, Schraubverschlüsse oder dergleichen. Vorzugsweise wird durch die Befestigungs- oder Verschlußeinrichtung ein Druck zwischen der Deckplatte 103 und der Mikro(-titer-)maske 101 und/oder zwischen der Kunststoff- Trägerplatte 104 und der Mikro(-titer-)maske 101 angelegt, um die Abdichtung der einzelnen Hohlraum-Gefäßkammern 102 zu verbessern.Any fastening devices can be used to assemble the individual components of the device, as can be seen in FIG. 1B. Groove closures 106, 107 are preferably used to assemble the device. However, other fastening or locking devices known to the person skilled in the art are also suitable, e.g. Clamps, clips, screw closures or the like. A pressure is preferably applied by the fastening or closure device between the cover plate 103 and the micro (titer) mask 101 and / or between the plastic carrier plate 104 and the micro (titer) mask 101 in order to seal the individual To improve void chambers 102.
Die aktivierte Kunststoff-Trägerplatte 104 ist vorzugsweise von der Vorrichtung 1 abnehmbar. Dies ermöglicht bwz. erleichtert eine einfache weitere Verfahrensweise, beispielsweise zum nachstehend erläuterten qualitativen oder quantitativen Nachweis der amplifizierten Polynucleotid¬ sequenz. Eine bevorzugte Ausführungsform besteht darin, daß die aktivierte Kunststoff-Trägerplatte 104 zumindest in dem aktivierten Bereich "capture-primer" enthält.The activated plastic carrier plate 104 can preferably be removed from the device 1. This enables bwz. facilitates a simple further procedure, for example for the qualitative or quantitative detection of the amplified polynucleotide sequence explained below. A preferred embodiment consists in that the activated plastic carrier plate 104 contains "capture primer" at least in the activated area.
Wie oben bereits erwähnt, kann dabei die Oberfläche des als Kunststoff-Träger verwendeten Reaktioπsgefäßes vollständig oder teilweise aktiviert vorliegen, vorausgesetzt, daß minde¬ stens ein Teil der mit der Polynucleotidseguenz im Amplifika¬ tionsansatz in Kontakt tretenden Oberfläche der einzelnen Re¬ aktionsgefäße aktiviert vorliegt. Es kann beispielsweise vor¬ teilhaft sein, daß nur der Bodenbereich der den Amplifikati¬ onsansatz aufnehmenden Vertiefung des jeweiligen Reaktionsge¬ fäßes aktiviert wurde, während der übrige Bereich der mit dem Amplifikationsansatz in Kontakt tretenden Oberfläche sowie die restliche Oberfläche des Reaktionsgefäßes in nichtaktiviertem Zustand vorliegt, um so eine Konzentration der Bindung der während der Amplifikation gebildeten Polynucleotide auf einer kleineren Fläche zu erreichen.As already mentioned above, the surface of the reaction vessel used as the plastic support can be fully or partially activated, provided that at least part of the surface of the individual reaction vessels that comes into contact with the polynucleotide sequence in the amplification mixture is activated. It can be advantageous, for example, that only the bottom area of the depression of the respective reaction vessel receiving the amplification approach has been activated, while the remaining area of the surface which comes into contact with the amplification approach and the remaining surface of the reaction vessel is in an unactivated state. so as to achieve a concentration of binding of the polynucleotides formed during the amplification in a smaller area.
Der Kunststoff-Träger kann jedoch auch in anderen Formen als in Form des Reaktionsgefäßes selbst vorliegen. Insbesondere kann der Kunststoff-Träger in Form von Kügelchen ("beads"), Granulaten, Fasern, Scheibchen, Folien, Stäbchen und derglei¬ chen mit dem die definierte Polynucleotidsequenz enthaltenden Amplifikationsansatz in Kontakt gebracht werden. Die Form des Kunststoff-Trägers und das Ausmaß der Aktivierung des Kunst¬ stoff-Trägers (ganz oder nur bestimmte Teilbereiche davon) kann wie gewünscht oder je nach Anforderungen für den jeweili¬ gen Anwendungszweck des erfindungsgemäßen Verfahrens oder der durch das erfindungsgemäße Verfahren gewonnenen Polynucleotide gewählt werden.However, the plastic carrier can also be in forms other than the form of the reaction vessel itself. In particular, the plastic carrier in the form of beads, granules, fibers, disks, foils, rods and the like can be brought into contact with the amplification batch containing the defined polynucleotide sequence. The shape of the plastic carrier and the extent of activation of the plastic carrier (entirely or only certain subareas thereof) can be selected as desired or depending on the requirements for the respective application of the method according to the invention or the polynucleotides obtained by the method according to the invention become.
Das erfindungsgemäße Verfahren kann in vielen Bereichen ange¬ wandt werden. So kann beispielsweise mit dem erfindungsgemäßen Verfahren aus einer sehr geringen, d.h. nicht konventionellThe method according to the invention can be used in many areas. For example, with the method according to the invention, a very small, i.e. not conventional
ERSATZBLATT nachweisbaren Menge einer Polynucleotidsequenz eine merklich größere Menge der Polynucleotidsequenz hergestellt und auf einfache Weise gewonnen werden. Die durch das erfindungsgemäße Verfahren amplifizierte Polynucleotidsequenz, insbesondere eine durch die PCR amplifizierte DNA, ist weiterhin hervorra¬ gend für Klonierungszwecke und andere gentechnologische Anwen¬ dungen geeignet. Ein weiteres Anwendungsgebiet wird durch die Nucleinsäure-Sequenzanalyse der durch das erfindungsgemäße Verfahren amplifizierten Polynucleotidsequenz geboten.REPLACEMENT LEAF detectable amount of a polynucleotide sequence a significantly larger amount of the polynucleotide sequence is prepared and obtained in a simple manner. The polynucleotide sequence amplified by the method according to the invention, in particular a DNA amplified by the PCR, is furthermore outstandingly suitable for cloning purposes and other genetic engineering applications. A further field of application is offered by the nucleic acid sequence analysis of the polynucleotide sequence amplified by the method according to the invention.
Falls gewünscht und je nach Anforderungen an die Reinheit der mittels des erfindungsgemäßen Verfahrens amplifizierten Poly¬ nucleotidsequenz im Hinblick auf den jeweiligen Anwendungs¬ zweck, d.h. je nach dem, ob die amplifizierte Polynucleotidse¬ quenz im isolierten Zustand vorliegen muß oder nicht, können die durch das erfindungsgemäße Verfahren amplifizierten und hergestellten Polynucleotide auf dem Kunststoff-Träger ver¬ bleiben, oder die Polynucleotide können von dem Kunststoff- Träger in einem weiteren Verfahrensschritt entfernt werden. Zum Entfernen der Polynucleotide von dem Kunststoff-Träger kann die auf hydrophobe Wechselwirkung beruhende Bindung zwi¬ schen den Polynucleotiden und dem Kunststoff-Träger durch ge¬ eignete chemische Mittel, z.B. durch Lösungsmittel, aufgebro¬ chen werden. Ggf. kann auch der Kunststoff-Träger durch pas¬ sende Lösungsmittel vollständig gelöst werden. In beiden ge¬ nannten Fällen liegen dann die synthetisierten Polynucleotide in flüssigem Medium vor, so daß diese dann durch dem Fachmann geläufige Reinigungsmethoden isoliert werden.If desired and depending on the requirements for the purity of the polynucleotide sequence amplified by means of the method according to the invention with regard to the respective application, i.e. Depending on whether the amplified polynucleotide sequence must be present in the isolated state or not, the polynucleotides amplified and produced by the method according to the invention can remain on the plastic carrier, or the polynucleotides can be removed from the plastic carrier in a further process step be removed. To remove the polynucleotides from the plastic carrier, the bond between the polynucleotides and the plastic carrier based on hydrophobic interaction can be achieved by suitable chemical means, e.g. be broken up by solvents. Possibly. the plastic carrier can also be completely dissolved by suitable solvents. In both cases, the synthesized polynucleotides are then present in a liquid medium, so that they are then isolated by purification methods familiar to the person skilled in the art.
Ein weiterer Gegenstand vorliegender Erfindung besteht in einem Verfahren zum Nachweis einer spezifischen Polynucleotid¬ sequenz an fester Phase, bei dem die nachzuweisende Poly¬ nucleotidsequenz nach dem oben beschriebenen Verfahren ampli¬ fiziert und gleichzeitig an den Kunststoff-Träger als feste Phase gebunden wird und anschließend nachgewiesen bzw. be- stimmt wird. Somit ist das oben beschriebene Verfahren zur Am¬ plifikation einer Polynucleotidεeguenz Bestandteil dieses Ver¬ fahrens zum Nachweis der Polynucleotidsequenz, so daß die oben ausgeführten Erläuterungen auch für diese Ausführungsfor der vorliegenden Erfindung gelten.Another object of the present invention is a method for the detection of a specific polynucleotide sequence on a solid phase, in which the polynucleotide sequence to be detected is amplified according to the method described above and at the same time bound to the plastic support as a solid phase and subsequently detected or loading is true. Thus, the method described above for amplifying a polynucleotide sequence is part of this method for detecting the polynucleotide sequence, so that the explanations given above also apply to this embodiment of the present invention.
Für die Bestimmung der amplifizierten und an der festen Phase gebundenen Polynucleotidsequenz bieten sich zahlreiche Metho¬ den an. Bei der Bestimmung erfolgt ein qualitativer oder quan¬ titativer Nachweis der amplifizierten Polynucleotide.Numerous methods are available for determining the amplified polynucleotide sequence bound to the solid phase. A qualitative or quantitative detection of the amplified polynucleotides takes place during the determination.
Für die grob-quantitative Bestimmung von amplifizierter DNA kann beispielsweise ein Fluoreεzenznachweis mit DNA-spezifi¬ schen Fluoreszenzfarbstoffen, wie z.B. Ethidiumbromid, DAPI oder den bis-Benzimidazol-Farbstoff 33258 Hoechst (s. V.W. Bums in Photochem. Photobiol. Rev. , Seiten 87 bis 103 (1980)) , angewandt werden.For the coarse-quantitative determination of amplified DNA, for example, fluorescence detection with DNA-specific fluorescent dyes, such as Ethidium bromide, DAPI or the bis-benzimidazole dye 33258 Hoechst (see V. W. Bums in Photochem. Photobiol. Rev., pages 87 to 103 (1980)) can be used.
Vorzugsweise wird jedoch das erfindungsgemäße Nachweisverfah¬ ren so durchgeführt, daß die nachzuweisende Polynucleotidse¬ quenz mittels der PCR amplifiziert wird, und zwar unter Ver¬ wendung von entweder markierten Primern und/oder markierten Nucleotiden, mit dem Zweck, daß die Markierungen durch die je¬ weiligen Bausteine (Primer und/oder Nucleotide) durch die PCR in die synthetisierten Polynucleotide eingebaut werden. Die somit eingebauten Markierungen können anschließend leicht be¬ stimmt werden und sind eine Funktion für die Menge der gebil¬ deten Polynucleotide. Mit Hilfe passender Standard-Vergleichs¬ werten können somit quantitative Nachweisbestimmungen einer zu bestimmenden Polynucleotidsequenz auf einfache Weise durchge¬ führt werden.However, the detection method according to the invention is preferably carried out in such a way that the polynucleotide sequence to be detected is amplified by means of the PCR, using either labeled primers and / or labeled nucleotides, with the purpose that the labels are replaced by the respective because of the building blocks (primers and / or nucleotides) that are incorporated into the synthesized polynucleotides by the PCR. The markings thus incorporated can then be easily determined and are a function of the amount of polynucleotides formed. With the aid of suitable standard comparison values, quantitative detection determinations of a polynucleotide sequence to be determined can thus be carried out in a simple manner.
Entsprechend können auch andere Amplifikationsansätze als die PCR angewandt werden, um Markierungen über entsprechende, mar-Correspondingly, amplification approaches other than PCR can also be used in order to mark using appropriate,
ERSATZBLATT kierte Bausteine in die gebildeten Polynucleotide, einzubauen und somit einen einfachen Nachweis zu ermöglichen.REPLACEMENT LEAF cated building blocks in the polynucleotides formed, thus allowing easy detection.
Durch die Verwendung des speziell aktivierten Kunststof -Trä¬ gers wird eine bisher nicht erreichte, hervorragende Trennung zwischen -einerseits in die Polynucleotidsequenz eingebauten Markierungen und andererseits in den eingesetzten, niedermole¬ kularen Bausteinen vorliegenden Markierungen bewirkt, indem die in die Polynucleotide eingebauten Markierungen fest an die aktivierte Oberfläche des Kunststoff-Trägers gebunden werden, während die niedermolekularen, markierten Bausteine im wesent¬ lichen in der flüssigen Phase verbleiben. Eventuell schwach mit der festen Phase assoziierte, markierte niedermolekulare Bausteine können leicht im Anschluß an die Amplifikationsreak- tion durch geeignete Waschschritte vollständig entfernt wer¬ den, so daß sich ein spezifischer Nachweis der Polynucleotid¬ sequenz mit hohem Signal : Rausch-Verhältnis ergibt.Through the use of the specially activated plastic carrier, an unprecedented excellent separation between markings built into the polynucleotide sequence on the one hand and markings present in the used, low molecular weight building blocks on the other hand is brought about by the markings built into the polynucleotides being firmly attached the activated surface of the plastic carrier is bound, while the low molecular weight, marked building blocks remain essentially in the liquid phase. Marked low molecular weight building blocks which are possibly weakly associated with the solid phase can easily be completely removed by suitable washing steps after the amplification reaction, so that specific detection of the polynucleotide sequence with high signal: noise ratio results.
Die Trennung zwischen der festen Bindung der synthetisierten Polynucleotide und der Nicht-Bindung der ggf. markierten nie¬ dermolekularen Bausteine kann durch die Zugabe eines Detergens verbessert werden, wobei die Menge des zugesetzten Detergens den jeweiligen Bedingungen (Art des aktivierten Trägers, Art und Menge der Bausteine, Länge der Primer und der syntheti¬ sierten Polynucleotide usw. ) im Rahmen des fachmännischen Kön¬ nens angepaßt werden können.The separation between the fixed binding of the synthesized polynucleotides and the non-binding of the possibly labeled low molecular weight building blocks can be improved by adding a detergent, the amount of detergent added depending on the particular conditions (type of activated carrier, type and amount of Building blocks, length of the primers and the synthesized polynucleotides, etc.) can be adapted within the scope of the expert knowledge.
Zum Zweck der Markierung können jegliche Arten von Markierun¬ gen verwendet werden, die dem Fachmann aus dem Stand der Tech¬ nik hinreichend bekannt sind. Geeignet sind beispielsweise Ra¬ diomarkierungen, wie 3H-, 125J- und 3 P-markierte Nucleotide und Primer; Markierungen mit Chro ophoren, Fluorophoren; zeit¬ verzögerte Fluoreszenz- und Chemilumineszenz-erzeugende Mar¬ kierungssysteme; insbesondere Markierungen mit Reporter¬ gruppen, wie Haptenen bzw. Biotin, welche Nachweisbestimmungs- methoden immunchemischer Art oder mit dem Biotin-Avi- din/Streptavidin-System ermöglichen. Als Hapten-markierte Nucleotid-Bausteine haben sich beispielsweise Brom- und Digi- toxigenin-modifizierte Nucleosidtriphosphate (insbesondere BrdU und Dig-dUTP) in der Praxis bewährt. Selbstverständlich können auch Kombinationen verschiedener Markierungsarten ange¬ wandt werden, insbesondere dann, wenn mehrere definierte Polynucleotidsequenzen in einem Ansatz nachgewiesen werden sollen.For the purpose of marking, any type of marking can be used which is well known to the person skilled in the art from the prior art. Suitable for example are radio labels, such as 3 H-, 125 J- and 3 P-labeled nucleotides and primers; Markings with chromophores, fluorophores; time-delayed fluorescence and chemiluminescence-generating marking systems; in particular markings with reporter groups, such as haptens or biotin, which enable immunochemical methods or with the biotin avidin / streptavidin system. For example, bromine and digi-toxigenin-modified nucleoside triphosphates (especially BrdU and Dig-dUTP) have proven themselves as hapten-labeled nucleotide building blocks. Combinations of different types of labels can of course also be used, in particular if several defined polynucleotide sequences are to be detected in one batch.
Bei Verwendung markierter Bausteine (Primer und/oder Nucleotide) zur Amplifikation und zum späteren Nachweis der amplifizierten Polynucleotidsequenz werden diese vorzugsweise in Kombination mit nicht-markierten Analoga der Bausteine in die Amplifikationsreaktion eingesetzt. Geeignete Verhältnisse zwischen den markierten Bausteinen und den nicht-markierten Analoga sind bekannt (s. obige Literaturstellen von H.A. Er¬ lich et al. und T. Maniatis et al.). Häufig ist es auch er¬ wünscht, die markierten Bausteine nur in bestimmten Stufen der Amplifikationszyklen, insbesondere in den letzten Amplifika- tionszyklen oder sogar nur im letzten Amplifikationszyklus zu¬ zusetzen.When using labeled building blocks (primers and / or nucleotides) for the amplification and later detection of the amplified polynucleotide sequence, these are preferably used in combination with unlabeled analogues of the building blocks in the amplification reaction. Suitable relationships between the labeled building blocks and the unlabeled analogs are known (see above references by H.A. Er¬lich et al. And T. Maniatis et al.). It is often also desirable to add the marked building blocks only in certain stages of the amplification cycles, in particular in the last amplification cycles or even only in the last amplification cycle.
Unter den immunchemischen Bestimmungsmethoden, zu denen auch das Biotin-Avidin/Streptavidin-System gezählt werden kann, stehen dem Fachmann eine große Auswahl aus dem Stand der Tech¬ nik zur Verfügung und es bestehen in dieser Hinsicht keine Li¬ mitierungen. Beispielsweise sind Fluoreszenz-Immunoassays (FIA) und Enzym-Immunoassays (EIA) geeignet. Das erfindungsge¬ mäße Nachweisver ahren ist insbesondere dann sehr einfach, aber auch sehr empfindlich, wenn Hapten- bzw. Biotin-Markie- rungen angewandt werden, deren Einbau in die synthetisierten Polynucleotide durch Enzym-Immunoassays bestimmt werden, ins¬ besondere in Verbindung mit einem Antikörper-Nachweisreagenz, welches mit einem Enzym konjugiert ist, das präzipitierendeAmong the immunochemical methods of determination, to which the biotin-avidin / streptavidin system can also be counted, a large selection from the prior art is available to the person skilled in the art and there are no limitations in this regard. For example, fluorescence immunoassays (FIA) and enzyme immunoassays (EIA) are suitable. The detection method according to the invention is particularly simple, but also very sensitive, when hapten or biotin labels are used, the incorporation of which into the synthesized polynucleotides is determined by enzyme immunoassays, in particular in connection with a Antibody detection reagent conjugated to an enzyme, the precipitating
ERSATZBLATT Nachweisfarbstoffe bildet, die sich auf dem Kunststoff-Träger am Ort der Polynucleotidbindung niederschlagen. Nähere Erläu¬ terungen zu geeigneten Nachweisbestimmungsmethoden, insbeson¬ dere durch immunchemische Techniken, werden in den oben ge¬ nannten, anhängigen PCT-Anmeldungen Nr. 92/02256 und Nr. 92/02432, auf die hier ausdrücklich Bezug genommen wird, gege¬ ben.REPLACEMENT LEAF Detection dyes form, which are deposited on the plastic support at the location of the polynucleotide bond. Further explanations on suitable detection methods, in particular by immunochemical techniques, are given in the above-mentioned pending PCT applications No. 92/02256 and No. 92/02432, to which reference is expressly made here .
Besonders gut geeignet sind Bestimmungsmethoden unter Verwendung automatisierbarer Detektionsvorrichtungen, z.B. dem Graustufen-Scannen ("gray-level scanning") .Determination methods using automated detection devices, e.g. gray-level scanning.
Wenn dies gewünscht wird oder falls dies erforderlich ist,, können, um die Spezifität des erfindungsgemäßen Nachweisver¬ fahrens zu erhöhen, weitere Schritte an die Amplifikation der definierten Polynucleotidsequenz angeschlossen werden. In der Regel sollten Waschschritte zum Abwaschen unspezifisch gebun¬ dener, leicht mit der Oberfläche des Kunststoff-Trägers asso¬ ziierter Markierungssubstanzen durchgeführt werden. Eine be¬ sonders hohe Spezifität des Nachweises definierter Polynucleo¬ tidsequenzen wird dadurch erreicht, daß die amplifizierte und an den Kunststoff-Träger gebundene Polynucleotidsequenz mit¬ tels der Nucleinsäure-Hybridisierungstechnik bestimmt wird. Bei dieser Technik erfolgt die Detektion über markierte Nucleinsäure-Sonden, welche spezifisch an die nachzuweisende Polynucleotidsequenz oder an Teilbereiche dieser Polynucleo¬ tidsequenz binden. Die Nachweisbestimmung erfolgt in diesem Fall wiederum durch eine geeignete Markierung, welche die Nucleinsäure-Sonde trägt, wobei die oben genannten Markie¬ rungsarten auch hier angewandt werden können.If this is desired or if this is necessary, further steps can be connected to the amplification of the defined polynucleotide sequence in order to increase the specificity of the detection method according to the invention. As a rule, washing steps for washing off unspecifically bound marking substances which are easily associated with the surface of the plastic carrier should be carried out. A particularly high specificity of the detection of defined polynucleotide sequences is achieved in that the amplified polynucleotide sequence bound to the plastic support is determined by means of the nucleic acid hybridization technique. In this technique, detection takes place via labeled nucleic acid probes which bind specifically to the polynucleotide sequence to be detected or to partial regions of this polynucleotide sequence. In this case, the detection determination is again carried out by means of a suitable label which carries the nucleic acid probe, it also being possible to use the above-mentioned types of label here.
Einen Oberblick über die Nucleinsäure-Hybridisierungstechnik geben U. Landegren et al., Science 242, Seiten 229 bis 237 (1988), J. Meinkoth und G. Wahl in Analytical Biochemistry 138, Seiten 267 bis 284 (1984) und B.D. Harnes und S.J. HigginsAn overview of the nucleic acid hybridization technique is given by U. Landegren et al., Science 242, pages 229 to 237 (1988), J. Meinkoth and G. Wahl in Analytical Biochemistry 138, pages 267 to 284 (1984) and B.D. Harnes and S.J. Higgins
ERSATZBLATT in "Nucleic Acid Hybridization. A Practical Approach", IRL Press (1985).REPLACEMENT LEAF in "Nucleic Acid Hybridization. A Practical Approach", IRL Press (1985).
Bei dem erfindungsgemäßen Nachweisverfahren sollte darauf ge¬ achtet werden, daß in allen Schritten, die nach dem Amplifi- zierungsschritt in dem erfindungsgemäßen Nachweisverfahren durchgeführt werden, wenn dies erforderlich ist oder gewünscht wird, Emulgatoren bzw. Detergentien in den Inkubationsansätzen vorhanden sind. Dies gilt beispielsweise für die Wasch¬ schritte, die immunchemischen Inkubationsschritte, die Nucleinsäure-Hybridisierungsreaktionen und die Enzym-Substrat- Detektionsreaktionen. Dies unterbindet wirksam die unspezifi¬ sche Bindung markierter Nachweisreagenzien an die aktivierte Oberfläche des Kunststoff-Trägers verursacht jedoch nicht die Abdissoziation der Polynucleotide vom aktivierten Träger. Ge¬ eignete Emulgatoren bzw. Detergentien umfassen beispielsweise Triton X-100 (Octylphenoxypolyethoxyethanol) , Tween 20 (Polyoxyethylensorbiton) , SDS (Natriumdodecylεulfat) , nicht¬ ionische, anionische oder kationische Tenside, Borate und Sei¬ fen.In the detection method according to the invention, care should be taken to ensure that emulsifiers or detergents are present in the incubation batches in all steps which are carried out in the detection method according to the invention after the amplification step. This applies, for example, to the washing steps, the immunochemical incubation steps, the nucleic acid hybridization reactions and the enzyme-substrate detection reactions. This effectively prevents the unspecific binding of labeled detection reagents to the activated surface of the plastic carrier, but does not cause the polynucleotides to dissociate from the activated carrier. Suitable emulsifiers or detergents include, for example, Triton X-100 (octylphenoxypolyethoxyethanol), Tween 20 (polyoxyethylene sorbiton), SDS (sodium dodecyl sulfate), non-ionic, anionic or cationic surfactants, borates and beefs.
ERSATZBLATT REPLACEMENT LEAF

Claims

PATENTANSPRUCHE PATENT CLAIMS
1. Verfahren zur Amplifikation einer Polynucleotidseguenz, dadurch gekennzechnet, daß die Polynucleotidsequenz in Gegenwart eines Kunststoff-Trägers amplifiziert wird, wobei mindestens ein Teil der mit der Polynucleotidsequenz in Kontakt I tretenden Oberfläche des Kunststoff-Trägers aktiviert wurde durch Erhöhung des Oberflächeninhalts, so daß die amplifizierte Polynucleotidsequenz an die aktivierte Oberfläche des Kunststoff-Trägers gebunden wird.1. A method for the amplification of a polynucleotide sequence, characterized in that the polynucleotide sequence is amplified in the presence of a plastic carrier, wherein at least a part of the surface of the plastic carrier coming into contact with the polynucleotide sequence has been activated by increasing the surface content, so that the amplified polynucleotide sequence is bound to the activated surface of the plastic carrier.
2. Verfahren nach Anspruch 1, dadurch gekennzeichnet, daß für2. The method according to claim 1, characterized in that for
15 den Kunststoff-Träger ein Material verwendet wird, das eine Matrix aus Copolymer oder Polymerblend aufweist, und der Kunststoff-Träger durch ein Lösungsmittel aktiviert wurde.15 the plastic carrier is used a material that has a matrix of copolymer or polymer blend, and the plastic carrier has been activated by a solvent.
3. Verfahren nach Anspruch 1 oder 2, dadurch gekennzeichnet, 20 daß die Polynucleotidseguenz durch die Polymerase-3. The method according to claim 1 or 2, characterized in 20 that the polynucleotide sequence by the polymerase
Kettenreaktion amplifiziert wird.Chain reaction is amplified.
4. Verfahren nach einem der Ansprüche 1 bis 3, dadurch gekennzeichnet, daß vor der Amplifikation ein zur Amplifikation4. The method according to any one of claims 1 to 3, characterized in that one before amplification for amplification
?5 der Polynucleotidsequenz erforderlicher Primer an den Kunststoff-Träger gebunden wird und die zu amplifizierende Polynucleotidsequenz an den Primer anhybridisiert wird.5 of the polynucleotide sequence required primer is bound to the plastic support and the polynucleotide sequence to be amplified is hybridized to the primer.
5. Verwendung eines Verfahrens nach einem der Ansprüche 1 bis 4 zur Herstellung einer amplifizierten Menge einer 0 Polynucleotidseguenz.5. Use of a method according to any one of claims 1 to 4 for the preparation of an amplified amount of a 0 polynucleotide sequence.
6. Verfahren zum Nachweis einer Polynucleotidsequenz an fester Phase, dadurch gekennzeichnet, daß die Polynucleotidsequenz nach einem der Verfahren nach Ansprüchen 1 bis 4 amplifiziert 5 und an den Kunststoff-Träger als feste Phase gebunden wird und die amplifizierte Polynucleotidseguenz anschließend bestimmt wird.6. A method for the detection of a polynucleotide sequence on a solid phase, characterized in that the polynucleotide sequence is amplified by one of the methods according to claims 1 to 4 and is bound to the plastic support as a solid phase and the amplified polynucleotide sequence is then determined.
LATT LATT
7. Verfahren nach Anspruch 6, dadurch gekennzechnet, daß die Polynucleotidsequenz mittels der Polymerase-Kettenraktion unter Verwendung von markierten Primern amplifiziert wird und daß die Polynucleotidsequenz durch den Einbau des markierten Primers bestimmt wird.7. The method according to claim 6, characterized in that the polynucleotide sequence is amplified by means of the polymerase chain reaction using labeled primers and that the polynucleotide sequence is determined by the incorporation of the labeled primer.
8. Verfahren nach Anspruch 6, dadurch gekennzechnet, daß die Polynucleotidseguenz mittels der Polymerase-Kettenraktion unter Verwendung von markierten Nucleotiden amplifiziert wird und daß die Polynucleotidsequenz durch den Einbau der markierten Nucleotide bestimmt wird.8. The method according to claim 6, characterized in that the polynucleotide sequence is amplified by means of the polymerase chain reaction using labeled nucleotides and that the polynucleotide sequence is determined by the incorporation of the labeled nucleotides.
9. Verfahren nach einem der Ansprüche 6 bis 8, dadurch gekennzeichnet, daß die amplifizierte Polynucleotidsequenz mittels immunoenzymatischer Detektion bestimmt wird.9. The method according to any one of claims 6 to 8, characterized in that the amplified polynucleotide sequence is determined by means of immunoenzymatic detection.
10. Verfahren nach Anspruch 6, dadurch gekennzeichnet, daß die amplifizierte und an den Kunststoff-Träger gebundene Polynucleotidsequenz mittels der Nucleinsäure-Hybridisierungs- technik bestimmt wird. 10. The method according to claim 6, characterized in that the amplified and bound to the plastic carrier polynucleotide sequence is determined by means of the nucleic acid hybridization technique.
PCT/EP1994/000143 1993-01-22 1994-01-20 Process for amplifying and process for detecting polynucleotide sequences in a solid phase WO1994017204A1 (en)

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