EP0751827B1 - Method of processing nucleic acids - Google Patents

Abstract

PCT No. PCT/EP95/00975 Sec. 371 Date Sep. 19, 1996 Sec. 102(e) Date Sep. 19, 1996 PCT Filed Mar. 16, 1995 PCT Pub. No. WO95/25592 PCT Pub. Date Sep. 28, 1995Method and device for processing nucleic acids in a reaction mixture on a surface that can be temperature-controlled and its immediate vicinity wherein the main space of the reaction mixture remains essentially isothermal. The method has the advantage of a very short processing time.

Description

The invention relates to methods for processing nucleic acids by means of a temperature control element and devices and devices for performing of these procedures.

In analytics, especially in medical diagnostics, more and more are beginning Establish methods based on detection and synthesis of nucleic acids. Nucleic acids are known as e.g. B. very specific detection means for organisms well suited for the diagnosis of diseases. During these verification procedures are various processing steps, such as denaturation, hybridization, syntheses and Immobilization of nucleic acids and their enzymatic treatment are common. A The problem with such methods has long been the small amount of nucleic acids in the samples.

A solution to this problem, used by a variety of analysts for evidence is made accessible, brought the amplification of nucleic acids. Such one The process is described in EP-A-0 200 362, namely the polymerase chain Reaction (PCR). This is done by multiple extension of primers in one Reaction solution made a variety of copies of the original nucleic acid. These can be achieved, for example, by hybridization with a labeled nucleic acid probe can be detected according to EP-A-0 201 184. The used here Reaction solutions are used to separate double strands and for the extension reaction heated to specific temperatures or cooled again at intervals. Because the volumes are relatively large, the times for the temperature regulation a relatively long time for the implementation of the whole Amplification process.

The so-called capillary PCR tries to remedy this. Is located the reaction mixture in glass capillaries with a small diameter. This can the time for performing an amplification sequence can be reduced to approximately 30 minutes. A problem with capillary PCR is the vulnerability of the glass to be heated the capillary and the difficult to handle sample application.

More and more amplification methods have also been described recently, at which the amplification reactions in a closed system, d. H. without feed of reagents during the cycles. So is in WO 92/07089 described a system in which the amplification mixture was used for as long as necessary a circulatory system by heating the reaction mixture again and again and is cooled. By choosing the diameter, the residence time of the mixture can be influenced in the individual zones of the system. EP-A-0511712 describes an amplification process described in which the amplification mixture cyclically on whole certain temperatures are brought to achieve relatively short cycle times. Sample handling is also difficult here.

The object of the present invention was, inter alia, the existing nucleic acid processing methods to improve and in particular to provide processes where the amplification can be completed in particular in time.

The invention therefore relates to a method for processing and in particular Worship of nucleic acids in a reaction mixture, characterized in that that the temperature of a surface adjacent to the reaction mixture and its immediate environment is regulated, however, the main space of the reaction mixture remains essentially isothermal.

The invention also relates to a device for processing and multiplication of nucleic acids with a temperature regulation element as well as a device, which contains this device.

For the purposes of the invention, nucleic acids are all types of modified nucleic acids or unmodified. For example, unmodified nucleic acids are natural occurring nucleic acids. Modified nucleic acids can be exchanged by Groups of natural nucleic acids are created by other chemical residues. Examples are nucleic acid phosphonates, or phosphothioates and on the sugar residues or the bases modified by chemical groups, which can also be detectable Nucleic acids.

The processing of nucleic acids according to the present invention preferably contains at least one at elevated temperature, but at least from the ambient temperature deviating temperature, ongoing reaction step. Such reaction steps are, for example, the thermal denaturation of partially or completely double-stranded nucleic acids. It is used to manufacture single strands or to Melt secondary structures, the nucleic acids to temperatures above the corresponding melting point heated. Another step in the processing of nucleic acids is the hybridization of mutually complementary nucleic acids in single-stranded Areas to a nucleic acid duplex (hybrid). This takes place at Temperatures below the melting point of the hybrid take place. Hybridization To achieve this, the reaction mixture must often be cooled will. Another one is used in particular in amplification or sequencing methods Processing step carried out, namely the extension of one to one so-called template nucleic acid (template) hybridized primers with the help of others Mono- or oligonucleotides. Here, the temperature is preferably set at the corresponding enzyme used has its optimum activity, or competitive reactions are reduced. This temperature can also be measured with the hybridization temperature be identical (2-stage PCR).

A special case of processing nucleic acids is the multiplication of nucleic acids. According to the present invention, practically all amplification methods, e.g. B. target sequence dependent amplifications (especially non-isothermal Amplifications such as the polymerase chain reaction, ligase chain reaction or similar) be performed. Also takes place during this propagation process at least one of the temperature-sensitive processing steps mentioned above take place.

A central feature of the present invention is the fact that the change in Temperature not in the entire reaction mixture, but only in a very small one Part of the reaction mixture takes place. This has the consequence that the heating or Cooling down of this relatively small area can happen very quickly and thus the Processing of the nucleic acids is greatly accelerated. To carry out the invention The process is therefore the reaction mixture with a temperature adjustable Brought into contact. By changing the temperature of the surface the immediate surroundings of this surface are heated, adjusted or cooled, depending on the processing step and temperature of the reaction mixture. One can distinguish several cases here.

In the event that the temperature of the reaction mixture is higher than the melting temperature of the nucleic acids to be processed, the surface can be cooled to to achieve hybridization of the nucleic acids in the adjacent environment.

In the event that the temperature of the reaction mixture is lower than the melting temperature and the temperature required for a primer to extend to the nucleic acid is necessary, the surface and thus the immediate surroundings can initially are heated to a temperature at which the extension of the primer takes place can. The temperature can then be raised above the melting point of the nucleic acids be, thereby denaturing and strand separation of the double-stranded Nucleic acids can take place. This can be a cooling down to a Connect the temperature at which the single strands can hybridize with new primers. This cycle can be run through several times.

In the event that the temperature of the reaction mixture between that for the extension optimal temperature and the melting temperature, the temperature at the Surface initially raised to separate double strands. Then the Temperature lowered to a temperature at which the single strands hybridize with primers. The optimum temperature for the extension is then set, if desired the cycle repeats.

The desired temperature can be maintained for a set period of time be, e.g. B. until the desired reactions on the surface expired are. For this purpose, the temperature of the surface can also be controlled by known means and connected control measures (reheating, cooling) kept constant will. The periods depend, as is customary in known methods, on the length of the to be processed nucleic acids and their homology as well as the special hybridization conditions from. However, the person skilled in the art can determine the optimum by simple experiments Determine time periods. Depending on the processing step, the periods are between Fractions of a second and a few seconds.

In the cases described above, the main space of the reaction mixture remains in the essential isothermal, while that in the immediate vicinity of the surface Adapts reaction mixture to the temperatures set on the surface.

To regulate the temperature and to set specific temperatures on the It is recommended to use the surface and its immediate surroundings as the surface Surface to choose a device, consisting of a heating element and a cooling element consists. The heating element preferably has a relatively large surface area in comparison low heat capacity. Metal foils, for example, have proven suitable proven that can be heated in a suitable manner, e.g. B. by electrical Electricity. Materials for the metal foil are good heat-conducting materials, e.g. B. Gold, preferred.

The cooling element should have a comparatively high heat capacity. As a cooling medium solid, liquid or gaseous substances are possible. Liquid ones are preferred Cooling media, e.g. B. water. Good thermal conductivity is an advantage.

The arrangement of the heating element and the cooling element can correspond to the temperature of the reaction medium are changed, depending on whether the device is more used to heat or cool the surface and its immediate surroundings shall be. In general, however, the heating element is in close proximity be localized on the surface. The surface of the heating element can also directly on the Adjacent reaction mixture. However, it is also possible to use a heating element separate the thin, thermally conductive layer from the reaction mixture.

The cooling element can in principle be positioned at any point with which cooling the surface and the immediate vicinity is possible. It has been preferred however, the cooling element was found to be on the side of the reaction mixture remote from the reaction mixture Position heating element. In the event that the heating element is low Has heat capacity is the fact that the heating element can also be cooled must not be a decisive disadvantage.

In the method according to the invention, the heating element is used for heating until the surface and its immediate surroundings are heated to the desired temperature. A strong temperature gradient will form near the surface, while the rest of the reaction mixture remains isothermal. This applies in particular if no remarkable liquid exchange is realized during the heating process, but also if a liquid exchange takes place. The heating process can be completed within fractions of a second, in favorable cases even milliseconds. The same applies to cooling. The reaction space, which is heated to the desired temperature, can be very small, preferably it is less than 0.2 mm, particularly preferably less than 0.5 µm deep. The area of the heating element directed onto the reaction mixture influences the depth of the temperature gradient and its rate of build-up. A preferred size of the surface can result from the desired applications of the method. As a rule, the surface area is <2 cm ² , particularly preferably between 0.2 cm ² and 0.2 mm ² . The surface can be smooth or rough. In the event that the surface is simultaneously used as a carrier for agents for processing the nucleic acids, it is advantageous to enlarge this surface by means of surface structures.

The volume of the reaction mixture is practically not great for the invention Meaning. It can be a drop with a volume of 20 µl, however can also be an arbitrarily large volume. It is an advantage of the invention Process that the device containing the heating and cooling element, for example can also be introduced into a large vessel with reaction mixture, whereby the essential reactions only in a very small border area of the surface take place, while the remaining reaction space practically no influence on the reaction exercises. On the other hand, the samples and reagent quantities available are practical Limit the size of the reaction mixture. The reaction volumes will increase therefore usually in a range between 1 ml and 30 ul, preferably between 100 ul and 50 µl, move, but can be done, for example, by applying the reaction mixture work on the temperature regulating surface even with much smaller volumes.

To process the nucleic acids, they are attached to the temperature-adjustable surface or their immediate surroundings. This can be done in several ways happen, for example mechanically or by diffusion. The are preferred Nucleic acids bound to the surface. This bond can be different Be kind. A chemical bond is preferred, either via adsorption or biospecific interactions. In the case of binding by adsorption, the Surface with a nucleic acid binding reagent. Biospecific Interactions can interact between nucleic acids and those to be processed Be nucleic acids (hybridization, complementary part of these nucleic acids), however, interactions between antigens or haptens with it directed antibodies and receptor-ligand interactions. A preferred species the nucleic acids to be processed are bound to the surface Oligonucleotides that are covalently bound to the surface and at least one Part of the nucleic acid to be processed are complementary. These oligonucleotides can, however, also via biospecific interactions, e.g. B. biotin streptavidin the surface be bound.

The binding of the nucleic acids to be processed is in accordance with the present invention preferably reversible. The nucleic acids can be carried out according to one of the methods Process dependent time by heating the surface and its immediate surroundings to be released again. Between binding and releasing the nucleic acid any steps can be taken, e.g. B. carrying out chemical reactions, however, a separation of the bound nucleic acids from the original one Reaction mixture and transfer to a new reaction mixture.

In the event of a multiplication reaction of the nucleic acids, they can surface bound oligonucleotides as primers for elongation or extension under Use of the nucleic acid to be processed can be used as a template. Thereby an extended primer forms which is bound to the nucleic acid to be processed. The Nucleic acid used as a template can be removed from the Extension product can be solved and in the next temperature cycle for one new, not yet extended immobilized primers act as a template. On this way it is possible to have a large amount of surface immobilized To extend primers and thus copies of parts of the nucleic acid to be processed to manufacture. The extended (extended) surface primers in turn serve by means of complementary primers for an increase in those serving as a template Molecules. The reagents required for the reactions to be carried out are in keep the entire reaction mixture in stock or add it as needed. For a propagation reaction based on the principle of the polymerase reaction (EP-B-0 201 184) are therefore the deoxyribonucleotides, a DNA polymerase and a to keep additional primers and suitable buffer reagents in the reaction mixture. To display other nucleic acid types (e.g. RNA), other reagents, e.g. B. Ribonucleotides or RNA-dependent polymerases are provided. This step of the process can take special consideration of the working temperature of the processing enzyme to take.

However, the nucleic acids to be processed can also be done using physical methods be bound to the surface, e.g. B. by means of a magnet. To do this, the However, nucleic acids are bound to a magnetizable particle. The connection magnetic particles loaded with nucleic acid can thereby be made reversible that there is a magnet behind the surface or that it is induced. By Applying an alternating field can cause the magnetic parts to surface bound or removed from it.

Special embodiments, some of which have advantageous effects, are also conceivable. So the efficiency can be increased by the diffusion of the nucleic acids is enhanced in the reaction mixture by convection. It can also be beneficial be higher than the reaction mixtures usually used Use concentrations of the reactants. In addition, the one to be edited Nucleic acid at the start of the reaction by pre-hybridization on the surface be concentrated.

In the case of amplification or multiplication of the nucleic acids, it may be that the Number of cycles to go through, e.g. B. in a PCR, must be increased so enough amplification product is generated. Because of the very short cycle time However, this is not a disadvantage of the method according to the invention.

In the event that the reaction mixture is kept at a relatively low temperature , this means that less demands are made on the heat stability of the reagents than if the entire reaction mixture is heated several times. A Therefore, thermostable polymerase is not essential for a multiplication reaction required and yet new enzyme does not have to be added to every amplification cycle Pipette reaction mixture.

Any further processing of the nucleic acids according to the invention can be used Connect steps. The nucleic acids can either be bound to the surface Condition or in the released state examined or with others Reagents are processed.

With the help of the described propagation method according to the invention can thus a method for the detection of nucleic acids in a sample in a simple manner For this purpose, the surface to which the oligonucleotides functioning as primers are formed are brought into contact with the sample liquid. After that the Brought the temperature of the surface and its immediate surroundings to a temperature which is above the melting point of the double-stranded contained in the sample Nucleic acid. After cooling the surface and the immediate area the nucleic acid to be detected will hybridize with the oligonucleotide. By As described above, the cycle is carried out with the aid of the nucleic acid to be detected made a variety of copies that were sent to the end of the propagation reaction Surface can remain bound. By hybridization with labeled nucleic acid probes and their detection with the help of the label can reduce the amount of nucleic acids be determined on the surface. For procedures that provide direct evidence of nucleic acid hybrids without labeling or of extended single strands Allow (primers) (e.g. according to the principle of surface plasmon resonance) direct evidence is also possible. Used for the propagation reaction labeled primers or mononucleotides, the hybridization with a detectable omitted labeled nucleic acid probe. An approach is also conceivable in which an approach higher temperature is maintained and the surface necessary for further steps assumes lower temperatures.

Since the inventive use of the method for the amplification of the nucleic acids practically on a surface, we carry out this propagation reaction the name 2D amplification.

The invention also relates to a device which can be used for use in the above-mentioned machining method. In particular, the subject of the invention is a device for processing nucleic acids by means of a temperature regulating element, this element being suitable for regulating the temperature of the surface and the immediately adjacent surroundings, and wherein means for processing the nucleic acids can be bound to this. These agents can be oligonucleotides and serve, for example, as primers. This device preferably contains a cooling element and a heating element, the arrangement preferably being as in the processing method described above. This device is preferably very small. For example, it can have a thickness of <5 mm and an area of <10 cm ² . The elements therein, such as the cooling element or the heating element, are simple components. Therefore, this device is excellently suitable for single use (disposable), which can reduce the inherent contamination risk for reusable. If the device is to be suitable for multiple use, it is also possible to separate the heating element from the space containing the reaction mixture by means of a very thin component and to make this component separable from the device. A new component can then be used for a further processing stage.

The invention also relates to a device for processing nucleic acids, which is a control element for a time-dependent temperature regulation and an inventive one Includes device. The control element can be a control element so-called thermal cyclers according to EP-A-0 236 069 are used, is preferred however, control based on the principle of inkjet printers, since it has a higher Show control speed. The control must be able to Heat the heating element at predetermined intervals until the surroundings of the Surface has a sufficient temperature. Likewise, it must be able to the cooling element at predetermined intervals for cooling on the surface For this purpose, for example, a coolant can be passed through the device Provided that the heat capacity of the heating element is low, but the heat output is relatively high, continuous cooling is also possible. As a surface you can use different shapes in an ohmic temperature version of this device, which heats depending on the voltage and the duration of the current flow will. The surface to be tempered is in a different version on a preferably black base, preferably a plastic film, the from the surface to be tempered distal side by a laser beam, preferably an infrared laser. Both the surface to be tempered, as well as the heat absorbing and heat conductive material a support surface, preferably infrared-transmissive glass.

The device according to the invention can have a wide variety of embodiments. For example it can be designed for immersion in a vessel. However, it can also be designed so that the liquid containing the nucleic acid to be processed on the Dripped surface or poured into a vessel formed by the surface This reaction space can also be closed after the liquid has been poured in so that contamination problems can be reduced.

One embodiment of the invention is a method for multiplying nucleic acids. As in example 3, e.g. B. a as a polymerase chain reaction designated amplification reaction with subsequent detection.

a) eine genaue Auswertung der tatsächlichen Hybridisationstemperatur vorzunehmen,

b) eine genaue Konzentration der zu analysierenden Nukleinsäuren im Reaktionsgemisch vorzunehmen,

c) auf das Vorhändeseein kreuzhybridisierender Sequenzen zu testen.

Another embodiment of the method according to the invention is a method for enriching nucleic acids e.g. B. by hybridization. In the case of primers adsorbed covalently to the surface with sufficient specificity, a hybridization temperature can be specified by preselecting the hybridization temperature, with a known composition (ionic composition, concentration of reagents) of the reaction solution to be analyzed, such that a specific binding of the nucleic acid to be analyzed to that for hybridization used primer covalently bound to the surface takes place. This method can be used for this

a) to carry out an exact evaluation of the actual hybridization temperature,

b) to carry out an exact concentration of the nucleic acids to be analyzed in the reaction mixture,

c) test for the presence of a cross-hybridizing sequence.

This short list is not exhaustive.

This method can also be used to isolate nucleic acids from a solution to convince another. Then a hybridization takes place in the first vessel (lower Temperature) and a denaturation (higher temperature) takes place in the second vessel.

Another embodiment of the present invention is a sequencing method of nucleic acids. One way of applying the invention in context with sequence analysis is the so-called mini-sequence analysis. It can in each case the exact sequence determination of the nucleic acid nucleotide adjoining the primer be carried out by providing for sequencing in the solution only dideoxynucleotides and no deoxynucleotides for sequencing be added. The four possible dideoxynucleotides ddATP, ddCTP, ddGTP and ddTTP are labeled with different fluorescent markers. In each In this case, the incorporation of the next nucleotide leads to the termination of the sequence reaction and after purification of the extended primer located on the surface can analyze the sequence of the on the primer by analyzing the specific fluorescence Nucleotides can be determined.

A special case of the possibilities of using the present invention is a Procedure for sequencing nucleic acids previously in a PCR reaction have been amplified. After amplification, the increased product lies above the Primer covalently bound in double-stranded form on the reactive surface. By briefly going through an altitude temperature phase, this is denatured Single-stranded amplificate can be obtained by transferring it to a washing solution clean and is then again by transferring this time to a sequencing reaction can be used for subsequent sequencing. This sequencing is particularly successful efficient, since there is now only single-stranded matrix material to which one Bind sequencing pair particularly efficiently. Due to the sequencing reaction then again a double-stranded molecule whose labeled (sequenced) Half is now available for analysis by gel chromatography. When using different labeled dideoxynucleotides (labeled with different fluorescent markers) all four reactions necessary for a sequence analysis can be Perform once, with the conventional method, this analysis must be done four times be repeated.

Figur 1 zeigt das Aufbauschema einer erfindungsgemäßen Vorrichtung (1), welche in ein mit Reaktionsgemisch (2) gefülltes Reaktionsgefäß (3) eintaucht. Die Vorrichtung enthält ein Kühlelement (4) und ein mittels Strom heizbares Heizelement (5). An die Oberfläche des Heizelement sind Oligonukleotide (6) kovalent gebunden. Sowohl das Kühlwie das Heizelement sind über Anschlüsse (7; 8) an Steuereinheiten regulierbar.

In Figur 2 ist ein beispielhafter Temperaturverlauf für eine Vermehrungsreaktion nach der Art der PCR gezeigt. In einer ersten Phase (A) werden die Nukleinsäuren bei einer Temperatur von etwas unterhalb 100 °C denaturiert. In einer Phase (B) findet bei etwa 50 °C die Hybridisierung der zu vermehrenden Nukleinsäuren mit den immobilisierten Oligonukleotiden an der Festphase statt. In einer dritten Phase (C) findet bei ca. 70 °C die Extension der Primer unter Verwendung der Nukleinsäure als Templat statt.

In Figur 3 sind die unmittelbarer Nähe der Oberfläche ablaufenden Reaktionen (Phasen A bis C) gezeigt. Für Phase (A) sind drei Isothermen in ihrem schematischen Verlauf, für Phase (B) und (C) jeweils nur eine Isotherme angezeigt. In Phase (A) werden Nukleinsäuren denaturiert und diffundieren über eine gewisse Zeit einzelsträngig, in Phase (B) hybridssieren Vorlagen und Primer und in Phase (C) werden Primer entlang der Matrizen extendiert.

Figur 4 zeigt einen möglichen Prototypen einer für die erfindungsgemäße Anwendung geeigneten Oberflächentemperierung. Die Heizeinheit, die die auswechselbaren beheizten Goldoberflächen sowie die physischen Voraussetzungen zur Kühlung bzw. Erwärmung (Kühlmittelanschluß, Stromanschluß) enthält, ist über Steckverbindungen (7; 8) an die eigentlichen Meßeinheiten (1) mit den ebenfalls daran angebrachten Halterungen für Reaktionsgefäße (11) angebracht, in denen die für die Durchführung der Reaktion notwendigen Matrizenmoleküle, Pufferbestandteile und Primer in Lösung sind sowie die Polymerase vorliegen.Die Meßfühler (9), hier in Form einesTeststreifens, enthalten in FIG 4 beispielhaft Goldoberflächen (5), die nicht mit denselben Primern (6) beschichtet sein müssen.

Figur 5 zeigt Vergrößerungen der Meßeinheiten mit auswechselbaren Teststreifen aus FIG 4, wobei die Meßeinheit (1, bestehend aus 9 und 10) aus Elektronikanschluß und Kühlaggregat (oberer, grauer Teil der Abbildung) und dem Teststreifen (= Meßhülle) besteht. Ein Querschnitt durch diese Meßeinheit ist im unteren Teil der Abbildung dargestellt, auf dem man den zur Kühlung verwendeten Kühlkreislauf (4) mit Rührmechanismus (12) und Kühlflüssigkeit (13) sowie die Reaktionsoberflächen (5, 6) mit Elektronikanschluß erkennen kann. Im rechten Teil der unteren Abbildung ist eine weitere Vergrößerung des Kathodenanschlusses der Goldmembran (5) mit Unterstützungs- und Trennfilter (14) sowie Haftschicht für die Oligonukleotide (6) erkennbar.

Figur 6 besteht aus einer Schemazeichnung des sich in unmittelbarer Umgebung der Haftschicht mit Oligonukleotiden ausbildenden Temperagradienten und dem zu erwartenden Temperaturprofil, hier am Beispiel einer 3stufigen PCR, bei der die Temperaturen 96 ° für 0,1 Sekunden, 54 °für 0,5 Sekunden und 72 ° für ebenfalls 0,5 Sekunden dargestellt sind.

The present invention is explained in more detail with reference to the accompanying figures:

FIG. 1 shows the construction diagram of a device (1) according to the invention, which is immersed in a reaction vessel (3) filled with reaction mixture (2). The device contains a cooling element (4) and a heating element (5) which can be heated by means of electricity. Oligonucleotides (6) are covalently bound to the surface of the heating element. Both the cooling and the heating element can be regulated via connections (7; 8) on control units.

FIG. 2 shows an exemplary temperature profile for an amplification reaction according to the type of PCR. In a first phase (A) the nucleic acids are denatured at a temperature slightly below 100 ° C. In a phase (B) the hybridization of the nucleic acids to be amplified with the immobilized oligonucleotides takes place on the solid phase at about 50 ° C. In a third phase (C) the primers are extended at about 70 ° C using the nucleic acid as a template.

The immediate vicinity of the surface reactions (phases A to C) are shown in FIG. For phase (A) three isotherms are shown in their schematic course, for phase (B) and (C) only one isotherm each. In phase (A) nucleic acids are denatured and diffuse single-stranded over a certain period of time, in phase (B) templates and primers hybridize and in phase (C) primers are extended along the matrices.

FIG. 4 shows a possible prototype of a surface temperature control suitable for the application according to the invention. The heating unit, which contains the exchangeable heated gold surfaces and the physical requirements for cooling or heating (coolant connection, power connection), can be connected to the actual measuring units (1) by means of plug connections (7; 8) with the holders for reaction vessels (11) also attached to them. attached, in which the matrix molecules, buffer constituents and primers necessary for the implementation of the reaction are in solution and the polymerase is present. The sensors (9), here in the form of a test strip, contain, in FIG (6) must be coated.

FIG. 5 shows enlargements of the measuring units with exchangeable test strips from FIG. 4, the measuring unit (1, consisting of 9 and 10) consisting of an electronic connection and cooling unit (upper, gray part of the illustration) and the test strip (= measuring sleeve). A cross section through this measuring unit is shown in the lower part of the figure, on which you can see the cooling circuit (4) used for cooling with stirring mechanism (12) and cooling liquid (13) as well as the reaction surfaces (5, 6) with electronic connection. In the right part of the figure below, a further enlargement of the cathode connection of the gold membrane (5) with support and separation filter (14) and adhesive layer for the oligonucleotides (6) can be seen.

FIG. 6 consists of a schematic drawing of the temperature gradient which forms in the immediate vicinity of the adhesive layer with oligonucleotides and the temperature profile to be expected, here using the example of a 3-stage PCR in which the temperatures are 96 ° for 0.1 seconds, 54 ° for 0.5 seconds and 72 ° are also shown for 0.5 seconds.

The following examples are intended to illustrate the invention:

Example 1: Production of a cyclically temperable surface (variant a).

An exemplary cyclic gold surface can be obtained in that in a thin circuit board, two approx. 1 mm wide and 3 mm long slots at a distance of 3 mm parallel to each other. On the one hand, that of the solution facing and is therefore called the proximal side, these elongated holes are through Evaporating with gold conductively connected. One becomes on the distal side this elongated holes over the PCB tracks with the anode, the other also over the printed circuit board paths are connected to the cathode.

The gold layer on the proximal side is applied by a mask in the desired size, in this case 3 x 3 mm flush, over the two longitudinal surfaces is applied. The inside of the longitudinal surface is galvanically flush to the surface coated with copper. In the following vapor deposition process, which like industry-standard, the two electrically conductive elongated holes are now coated with a gold layer a few µm thick. The coating process is described below:

The gold surface was produced by vapor deposition on "Lexan" polycarbonate foils (manufacturer: General Electric, thickness 0.75 mm) with the dimensions 8x8 cm over a metal mask (d = 0.5 mm, aluminum) in a Leybold high vacuum coating system (Univex 450). The surfaces lie at regular intervals, which allow the steamed plate to be broken down into several units. The layer thickness of the money is 300 nm. In a further step, the multi-gold sports plate was coated with "diluted" biotinthiol binding layers to form a hydrophobic SAM layer. The biotin thiol compound (HS-C12-DADOO-biotin; N, N '- (12-mercaptododecylyl-biotinylyl) -2,2'-diaminooethyl-glycol diether; 2.94 mg; 5x1 ^0-5 m) and the diluent (12-mercaptoundecanol, 9.2 mg, 4.5x10 ^-4 m) were dissolved in 100 ml of ethanol pa The freshly coated polycarbonate films were immersed in this solution. After 4 hours the plates were removed, washed twice with ethanol pa and immediately coated with streptavidin. For this purpose, the gold spots and the SAM-coated foils were immersed in a streptavidin solution for one hour (concentration streptavidin: 0.5 mg / ml in 0.05 M potassium phosphate buffer pH 7.2). The surfaces treated in this way were then treated with a washing solution (50 mM potassium phosphate buffer pH 7.2, 2% sucrose, 0.9% NaCl, 0.3% bovine serum albumin fraction II) and then dried for 20 hours (25 ° C. and 40% atmospheric humidity).

The very thin thickness of the gold layer results in a very small conductive gold cross section (3 mm x 3 µm over a distance of 3 mm). This gold layer therefore represents a relatively high resistance compared to the conductor tracks.

The conductor tracks are now connected to a power supplier that is computer-based allows a control of the ohmic induced temperature of the surface of the oil.

Production of a cyclically temperable surface (variant b)

In a variant for producing a surface that can be cyclically heated, the covering layer, which isolates the measuring meander from platinum, one generally available on the market Platinum temperature sensor (PT 100) coated with a gold layer. One closes this measuring sensor to a regulator-controlled voltage source, this can be Also use the sensor as a thermal source for the evaporated gold layer. It is for that necessary, current and voltage at the platinum sensor via an analog divider to a controller forward. The controlled variable of the controller is the resistance of the heated platinum element, which allows precise temperature control when normalizing the gold surface temperature with the temperature of the platinum meander.

Example 2: An exemplary device for measuring the surface temperature of the gold foil (Option A).

A gold foil according to Example 1, variant a, is covered with a mask that Evaporation of the gold foil with an approx. 1 mm wide gold thread that runs over this gold surface protrudes and can be connected to a measuring point. Another Brand, the steaming with a second thread, e.g. B. made of nickel, bismuth or another alloy suitable for temperature measurement is also provided. The metal thread lies on the same place on the gold surface as the gold thread at (200 nm thick), but separates in its course when leaving the tempering gold surface and leads as a separate metal thread (300 nm thick) a second measuring point. This arrangement of the measuring probes made of gold and another, metal or alloy suitable for temperature measurement allows the measurement of Thermal voltage (2.2 mV / 100 ° Kalvin) in the area of the gold foil where the two Probe threads overlap. The temperature at the cold junction is measured with a thermal precision PT 1000 element (accuracy> 1%) and standardized to 10 V (0 V corresponds to 0 °, 10 V corresponds to 100 °), only amplified and also standardized to 10 V. The sum of both is in a sum amplifier Temperauren formed and for electronic control of the respective surface temperature used.

Example 2b An exemplary device for measuring the surface temperature of the gold surface from example 1, variant b.

For the exact determination of the surface temperature of the gold layer, there is the possibility the surfaces of two gold-vaporized as described in Example 1, variant b To press PT 100 measuring sensors together in such a way that each half of the gold-coated Surfaces of the heating sensor and the measuring sensor cover each other and themselves the remaining area is in the surrounding medium. Now only one Sensor for measurement, the other uses for heating, so that can be exactly Calculate the temperature of the gold surfaces. The temperature on the surface of the sensors corresponds exactly to the mean of the temperature of the heated and the non-heated Sensors, both of which can be precisely defined using resistance measurements. To this A whole can be done by means of a temperature sensor used for the measurement Range of temperable surfaces calibrated and for carrying out PCR reactions to get prepared. The temperature gradient of two surfaces arranged in this way between the heated and unheated surface is approx. 3 ° C.

Example 3: Carrying out a two-stage PCR with a surface-fixed primer.

und ein Gegenprimer in der Konzentration von zwischen 0,5 µM bis 2 µM. Die Reaktion findet in handelsüblichem PCR-Puffer (Boehringer Mannheim, Beipackzettel zum Enzym 8. Ansgabe, Identnummer 1146165) bei 1,5 mM Mg²⁺ statt. Als Polymerase wurde eine händelsübliche Taq-DNA-Polymerase (Boehringer Mannheim, Identnummer 1146165) in der Konzentration von 20 nM eingesetzt. Der in Lösung vorhandene Gegenprimer (5'-GGGTGGGGTGGTTGGGTGGTGGTG-3') war an seinem 5'-Ende Digoxigenen-markiert (Patent EP 0324474).An exemplary implementation of a polymerase chain reaction uses a surface-fixed, biotin-labeled primer (5'-GAAGGGAGGAAGGAGGGAGCGGAC-3 '). This primer is coupled to a surface streptavidin or gold streptavidin surface via its biotin group located at the 5 'end. The molar amount of streptavidin or biotin / square millimeter is approximately 0.2 pmol / mm ² . The solution contains nanogram quantities or less of the following double or single-stranded template molecule

and a counter primer in the concentration of between 0.5 µM to 2 µM. The reaction takes place in commercially available PCR buffer (Boehringer Mannheim, package insert for enzyme 8th entry, ID number 1146165) at 1.5 mM Mg ²⁺ . A commercially available Taq DNA polymerase (Boehringer Mannheim, ID number 1146165) was used as the polymerase in a concentration of 20 nM. The counterprimer present in solution (5'-GGGTGGGGTGGTTGGGTGGTGGTG-3 ') was digoxigen-labeled at its 5' end (patent EP 0324474).

The PCR reaction took place at a constant solution temperature of 68 ° and one between 96 and 68 ° cyclically varying primer-coated surface instead. The Cyclical duration of the higher temperature varied between in our example 0.1 seconds, 10 seconds and 1 minute that the lower temperature between 0.5 seconds, 20 seconds and 1 minute.

After the cyclic temperature control had ended, the solution was cooled to room temperature, only the complementary elongated biotin and elongated digoxigenin-labeled primers hybridizing with one another and forming double-stranded DNA fragments. After washing with PCR buffer solution, the surfaces were reacted with anti-Dig-POD, incubated for 30 minutes, washed again and treated with ABTS (staining protocol according to package insert item 3 for the Boehringer Mannheim reverse transcriptase assay, non-radioactive, ID number 1468120). The resulting colored product was quantified in an ELISA photometer at a wavelength of 405/490 nanometers. The extinction values obtained demonstrate an amplification of a region of the template, measured as an extended counterprimer, which hybridizes to the primer coupled to the solid phase. In typical experiments, we obtained extinction values around 0.4 when measured in an elisa reader and after correction for the digoxigenin-free blank. In control experiments without a template, the values were 0.04 after correction of the blank value, and in controls without polymerase or without increasing the temperature, the values were 0.07 in the complete reaction mixture. Even heating the surface for several minutes (5 minutes) did not raise the ambient temperature in the constant temperature solution by more than 7 C, even if a significantly (100 times) larger surface (3 cm ² ) was heated.

Example 4: Production of a device according to the invention

1. die in das flüssige Reaktionsmillieu eindringende heizbare und temperaturregulierbare Oberfläche,

2. um die zur Heizung und Kühlung notwendigen Bauteile, die an

3. die elektronische Steuerung angeschlossen sind.

An exemplary device according to this invention consists of three structural subunits. These three structural units are:

1. the heatable and temperature-adjustable surface penetrating into the liquid reaction environment,

2. order the components necessary for heating and cooling

3. the electronic controls are connected.

The element surface described in this example consists of a thin gold foil with a thickness of less than half a millimeter, that with a streptavidin layer is firmly connected. Affinity coupling allows this dextran layer again the biotin marked necessary for the further processing steps Attach oligonucleotides. These oligonucleotides are then above their 5'-phosphate end covalently coupled to the surface and thus have a reactive, accessible for enzymes 3 'end.

This gold foil, which is structurally stabilized by a supporting plastic net can, and which has a reactive surface of about 5 x 5 mm, also serves as Heating element, since it is connected to an electrical power source, when the current flows leads to a spontaneous heating of this gold foil. Distal to the reaction approach, behind the gold foil, there is the cooling element. In this case, the cooling element consists of a plastic channel with a 7 mm wide border on the gold foil Surface. The channel is 2 mm deep and extends the entire length of the here Sensor designated part of the device, in this case the heating and cooling elements as well as the reactive surface. This channel extends from one end of the Probe to the other end of the probe on which the reactive surface is attached, and back by including a web separating the two channels. In this component, which is referred to as a cooling loop, is a suitable cooling liquid, e.g. B. water circulated. The entire unit is cast in hard plastic and recyclable, d. H. both the gold foil and the plastic are recyclable.

The control of both the heating of the reactive surface and the circulation speed and pre-cooling temperature of the coolant is controlled by an electronic third part, which is attached outside the sensor, made. This third The unit also includes the storage container and the connection connections for the coolant. In this third unit there is also a micropump that is used for the Circulation of the coolant is responsible. By flanging the coolant hoses to the measuring unit and by connecting the power supply to the reactive one The surface of the sensor is ready for use. Via an input unit on the Surface of the control unit is attached, which has a digital display both the individual temperature ranges and the duration of their influence on them Selectable surface.

Example 5: Carrying out a propagation reaction using the device according to the invention.

Acting here as an example of an application of the device according to the invention it is an amplification reaction of polymerase chain reaction a predetermined nucleic acid sequence. There are two for this nucleic acid sequence Primers with a length of 16 bases each, spaced 4 nucleotides apart encode. The sequence of one primer is identical, that of others complementary to the analyzing sequence. The one identified here as identical The primer is reactive via the biotin label present at the 5 'end Surface coupled. The other, complementary primer and all other reagents, that are usually used in a so-called PCR reaction, are added to the reaction mixture. By entering the appropriate reaction temperatures which should prevail in the immediate vicinity of the reactive surface, and by immersing the reactive surface or the sensor (see example 4) in the Reaction approach, the reaction is started. The temperature changes in the reactive surface are programmed to carry out 50 heating and Cooling cycles can be carried out in about 1 minute. By subsequent Immersing the sensor in a solution with distilled water and briefly heating, the surface is cleaned of all non-covalently bound reaction partners. The surface cleaned in this way, which now only extended and primer molecules Contains primer molecules for analysis, together with the probe, in one device introduced that according to the principle of plasmon resonance, a direct determination of the Quantity of the extended product allows.

Reference list

1.

device according to the invention, measuring unit

2nd

Reaction mixture

3rd

Reaction vessel

4th

Cooling element

5.

Heating element / surface

6.

Oligonucleotides

7.

Connections for cooling element

8th.

Connections for heating element

9.

exchangeable sensor

10th

Electronics connection and cooling unit

11.

Bracket for reaction vessel

12th

Stirrer

13.

Coolant

14.

Support and separation filter (separation of the coolant)

Claims (41)

1. Method for processing nucleic acids in a reaction mixture, wherein the temperature of the surface adjoining the reaction mixture and its immediate vicinity is regulated but the main space of the reaction mixture remains essentially isothermal.
2. Method as claimed in claim 1, in which at least one processing step has a temperature that differs from the temperature of the reaction mixture.
3. Method as claimed in claim 2, wherein a temperature-changing step serves for thermal denaturation.
4. Method as claimed in claim 2, wherein a temperature-changing step serves to hybridize nucleic acids.
5. Method as claimed in claim 2, wherein a temperature-changing step serves to extend a template molecule.
6. Method as claimed in claim 2, wherein various processing steps serve to target sequence-dependently amplify a template molecule.
7. Method as claimed in claim 1, wherein reactants are present bound to a surface and thus are suitable for detection of the processing and amplification results.
8. Method as claimed in claim 1, wherein agents for processing nucleic acids are bound to the surface.
9. Method as claimed in one of the claims 1 and 8, wherein these agents are oligonucleotides.
10. Method as claimed in one of the claims 1, 7 and 8, wherein agents for processing nucleic acids are present in the reaction mixture.
11. Method as claimed in one of the claims 1 and 6, wherein specific sequences are detected after hybridization with labelled nucleic acids or with nucleic acids that can be labelled.
12. Method as claimed in claim 6, wherein a non temperature-resistant DNA polymerase is used for the amplification.
13. Method as claimed in claim 6, wherein a temperature-resistant DNA polymerase is used for the amplification.
14. Method as claimed in claim 6, wherein an RNA-dependent RNA polymerase is used for the amplification.
15. Method as claimed in claim 6, wherein an RNA-dependent DNA polymerase is used for the amplification.
16. Method as claimed in claim 6, wherein a DNA ligase is used for the amplification (processing).
17. Method as claimed in one or several of the claims 1, 6, 8, 9, 10, wherein agents for processing nucleic acids are applied to the surface.
18. Method as claimed in one or several of the claims 1, 8, 9, 10, 16, 17, wherein at least one of the reactions involved in the processing that proceeds at an increased temperature takes place on a surface.
19. Method as claimed in claim 1 and 6, wherein the measuring solution is largely independent of the volume i.e. this method for processing and amplifying nucleic acids can be carried out using very small as well as very large volumes.
20. Method as claimed in claim 1 wherein the diffusion of the nucleic acids in the reaction mixture is increased by convection or motion.
21. Device for processing nucleic acids with the aid of a temperature regulating element, therein the device contains a surface whose temperature can be regulated on which agents are bound for processing the nucleic acids.
22. Device as claimed in claim 21 for use in methods as claimed in one of the claims 1 to 20.
23. Device for processing nucleic acids as claimed in one of the claims 21 and 22, wherein the surface that protrudes into the reaction medium can be cooled and/or heated.
24. Device as claimed in one of the claims 21 and 22, wherein the temperature-regulating surface has been prepared for the uptake of reactants for processing nucleic acids by chemical or physical treatment.
25. Device as claimed in claim 21, wherein it additionally has a control unit to regulate the temperature of the surface.
26. Device as claimed in claims 21, 22 or 24, wherein a time-coordinated regulation of the temperature changes is carried out.
27. Device as claimed in one of the claims 21, 22 and 24, wherein it has a heating element with a low heat capacity and a cooling element with a high heat capacity.
28. Device as claimed in one of the claims 21 and 22, wherein the surface is to directly analyze the processing and amplification results.
29. Device as claimed in claim 24, wherein the temperature-regulated surface is composed of a foil made of heat-conducting material preferably a metal and particularly preferably gold.
30. Device as claimed in one of the claims 21, 22 and 24, wherein the temperature-regulated surface is cooled on the side distal to the reaction mixture.
31. Device as claimed in one of the claims 21, 22 and 24, wherein the heating element is identical to the reactive surface and this surface is specially treated.
32. Device as claimed in one of the claims 21, 22, 24, 30 and 31, wherein nucleic acids can be immobilized on the surface of the heating element.
33. Device as claimed in claim 32, wherein the immobilization on the heating element is achieved by means of mediating structures.
34. Device as claimed in claim 33, wherein this immobilization via mediating structures can be achieved by chemical as well as by biospecific binding.
35. Device as claimed in one of the claims 21 and 22, wherein nucleic acids only temporarily associate or remain in the immediate vicinity of the surface.
36. Device as claimed in claim 35, wherein this association of nucleic acids on the reactive surface is ensured by binding these nucleic acids to magnetic beads or adsorbing these nucleic acids onto magnetic beads.
37. Device as claimed in claim 36, wherein an inducible magnetic field serves to attract said magnetic beads.
38. Method as claimed in claim 21 and 22, wherein the device that contains the heating and cooling elements can only be used once in order to prevent contaminations.
39. Device as claimed in claim 21 or 22, wherein the entire device can be used several times and only the surface or its holder has to be replaced for individual processing steps.
40. Device as claimed in claim 24, wherein the temperature-regulated surface is heated ohmically.
41. Device as claimed in claim 24, wherein the temperature-regulated surface is heated by rays preferably infrared rays and in turn preferably by laser-generated infrared rays.

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