WO2011117414A2

WO2011117414A2 - Device for carrying out pcr, method for producing a device for carrying out pcr and pcr method

Info

Publication number: WO2011117414A2
Application number: PCT/EP2011/054672
Authority: WO
Inventors: Joachim Nurnus; Axel Schubert; Fritz Volkert; Daniel Hoeferlin
Original assignee: Micropelt Gmbh
Priority date: 2010-03-26
Filing date: 2011-03-28
Publication date: 2011-09-29
Also published as: DE102010003365A1; WO2011117414A3

Abstract

The invention relates to a device for carrying out PCR, comprising a sample holder (2) for holding sample vessels (3), wherein the sample holder (2) has a plurality of holder openings (21), in each of which a sample vessel or an insert (3) for holding a sample vessel can be arranged; a plurality of Peltier elements (5), a plurality of temperature sensors (4) for determining the temperature of a sample present in a sample vessel (3), wherein each of the holder openings (21) is assigned at least one of the temperature sensors (4); and a plurality of restoring force generating means, wherein each of the holder openings (21) is assigned at least one restoring force generating means which interacts with the temperature sensor (4) assigned to the holder opening (21) in such a manner that said means holds the temperature sensor (4) in thermal contact with a sample vessel arranged in the holder opening (21) or with an insert for holding a sample vessel, wherein at least one of the restoring force generating means has an elastic element (6) on which the temperature sensor (4) is arranged, wherein the elastic element (6) projects beyond an edge of the holder opening (21) or into the holder opening (21), so it is bent down and the temperature sensor (4) is in this manner prestressed against the sample vessel or the insert (3) after a sample vessel or an insert (3) is arranged in the associated holder opening (21). The invention further relates to a method for producing a device for carrying out PCR and to a method for carrying out PCR.

Description

Devices for carrying out the PCR, methods for producing a device for carrying out the PCR and PCR methods

The invention relates to devices for carrying out the PCR according to claims 1 to 3, a method for producing a device for carrying out the PCR according to claim 1 1 and PCR method according to claims 16 and 20.

Devices and methods for carrying out the PCR, ie the polymerase chain reaction for amplifying DNA material (polymerase chain reaction), are known from the prior art. Known PCR devices have in particular a heating unit (thermocycler) for the cyclic temperature control of the DNA material to be amplified. The problem underlying the invention is to provide a device which is as simple to produce as possible and a method by which multiple DNA samples can be duplicated and / or analyzed simultaneously and individually in the most efficient manner possible can be. Furthermore, the simplest possible method for producing a device for carrying out the PCR should be specified.

These problems are solved by the devices having the features according to claims 1 to 3, by the method having the features according to claim 11 and by the methods having the features according to claims 16 and 20, respectively. Further developments of the invention are specified in the dependent claims.

Thereafter, a device for performing the PCR is provided with

a sample receptacle for receiving sample containers, the sample receptacle having a plurality of receiving openings, in each of which a sample container or an insert for receiving a sample container can be arranged;

a plurality of Peltier elements, each receiving opening being associated with at least one of the Peltier elements, so that the sample vessels or inserts (that is to say the DNA samples arranged in the sample vessels) arranged in the receiving openings can be tempered individually;

a plurality of temperature sensors for determining the temperature of a sample placed in the sample vessel, each of the receiving openings being associated with at least one of the temperature sensors; and

- A plurality of Rückstellkrafterzeugungsmitteln, each of the receiving openings is associated with at least one Rückstellkrafterzeugungsmittel which cooperates with the receiving opening associated temperature sensor (in particular biased) that it the temperature sensor in thermal contact with a arranged in the receiving opening sample vessel or with an insert for Holding a sample vessel, wherein

at least one of the restoring force generating means has an elastic element on which the temperature sensor is arranged, wherein the elastic element projects beyond an edge of the receiving opening or protrudes into the receiving opening, so that it is bent after arranging a sample vessel or an insert in the associated receiving opening and the temperature sensor is biased in this manner against the sample vessel or the insert.

According to one embodiment of the invention, the elastic element is arranged with a first portion on the sample holder and protrudes with a second portion, where one of the temperature sensors, so into the receiving opening, that the second portion during insertion of a sample vessel or an insert for Recording a sample container bent into the receiving opening and against the Sample vessel or the insert is biased so that the temperature sensor comes to a side wall of the sample vessel or the insert to the plant. For example, the first portion of the elastic element is fixed to a side wall of a receiving opening, while the second portion projects projecting from the side wall into the receiving opening.

According to another variant, the elastic element with the first section is arranged (in particular fixed) on an upper side of the sample receptacle and protrudes before arranging a sample vessel or an insert for receiving a sample vessel in the receiving opening with the second section on which the Temperature sensor is arranged, over an edge of the receiving opening, which extends in the top of the sample holder, addition, wherein the second portion when inserting a sample vessel (or the insert) bent into the receiving opening and biased against the sample vessel (or against the insert) is so that the temperature sensor on a side wall of the sample vessel (or the insert) comes to rest.

In particular, the temperature sensor is arranged on one side of the elastic element, which points away from the receiving opening before insertion of the sample vessel (or the insert). The "top side" of the sample holder is one side of the (eg plate-like) sample holder, via which the sample container (or the insert) is introduced into the receiving opening, and which faces away from the Peltier element associated with the receiving opening, as already explained above ,

In addition, the Peltier element can also be pressed via the restoring force generating means against a side wall of a sample vessel inserted in the receiving opening or an insert for receiving a sample vessel. In particular, the Peltier element is fixed together with the temperature sensor to the restoring force generating means. For example, the restoring force generating means are each in the form of an elastic element, wherein both a temperature sensor and a Peltier element can be connected to the elastic element. Of course, separate elastic elements for the temperature sensor and the Peltier element can be used.

It should be noted that the formulation according to which the first section of the elastic element is arranged "at the top of the sample holder" does not necessarily mean that the first section is in direct contact with the top of the sample holder It must also be located between the top of the sample holder and the first portion of the elastic element one or more layers of material or material webs (eg, a conductor track).

For example, the elastic element in the form of an elastic sheet-like element is formed (for example made of a metal or a plastic), wherein a first portion of the sheet-like element is fixed to the top of the sample holder and another portion of the sheet-like element on which the temperature sensor is arranged , overlaps with the receiving opening before arranging a sample vessel. In particular, the planar element is in the form of a strip (eg a metal strip), one end of which is fixed to the top of the sample holder and the other end, on which the temperature sensor is arranged, before arranging a sample vessel (or a Insert for receiving a sample vessel during assembly of the device) in the receiving opening with the receiving opening overlaps, d. h., The end with the temperature sensor is located before the insertion of the sample vessel (or the insert) (eg centrally) above the receiving opening and is during insertion of the sample vessel (or the insert) through the sample vessel (or through the insert) in particular pushed by its bottom to the side so that it rests te with a side on a side wall of the receiving opening and the temperature sensor on an outer side of the sample vessel (or the insert).

It should be noted that a "strip" refers in particular to a shape which has a substantially flat extension, although a certain extent (thickness) perpendicular to the surface may also be present, which, however, is significantly smaller than the surface extent In addition, a strip extends in particular along a main extension direction in which it has its largest dimension (length), wherein the length is for example greater its dimension (width) in a direction perpendicular to the longitudinal direction (eg 3 to 10 times greater). For example, a strip has a substantially rectangular shape, although the elastic flat element may also have a different geometry, for example oval.

The elastic element may also be a component of the temperature sensor; For example, a connecting line (or several connecting lines), via which the temperature sensor is electrically contacted, realizes the elastic element. Furthermore, it is conceivable that the temperature sensor on a printed circuit board (eg in the form of a flexible Possibilities of using a printed circuit board for contacting the temperature sensor are described below (or the plurality of temperature sensors) is contacted via a rigid circuit board, the circuit board, however, has a flexible region on which the temperature sensor is arranged.

It is also conceivable for a receiving opening to be associated with a plurality of temperature sensors, which are e.g. on different elastic elements, i. at different positions with respect to the receiving opening (and thus at different positions with respect to a arranged in the receiving opening sample vessel) are arranged. Thus, the temperature can be determined at different positions of the sample vessel, e.g. can be used to control the temperature control. For example, a plurality of spatially distributed Peltier elements, which are controlled in dependence on the temperatures measured by the plurality of temperature sensors, e.g. to produce the most homogeneous temperature distribution or any other predeterminable spatial temperature profile.

In a second aspect, the invention relates to a device for carrying out the PCR, with

- A plurality of Peltier elements, wherein each receiving opening is associated with at least one of the Peltier elements, so that arranged in the receiving openings sample vessels or inserts can be tempered individually;

a plurality of temperature sensors for determining the temperature of a sample placed in a sample vessel, wherein each of the receiving openings is associated with at least one of the temperature sensors; and

- A plurality of Rückstellkrafterzeugungsmitteln, each of the receiving openings is assigned at least one Rückstellkrafterzeugungsmittel, which cooperates with the receiving opening associated temperature sensor so that it holds the temperature sensor in thermal contact with a arranged in the receiving opening sample container or with an insert for receiving a sample vessel , in which - At least one of the restoring force generating means in the form of an elastically shaped portion of a side wall of the receiving openings is formed, which presses the temperature sensor against a arranged in the receiving opening sample vessel or against an arranged in the receiving opening insert for receiving a sample vessel.

Thus, there is no separate restoring force generating means, but a portion of the specimen receptacle in a region adjacent to the receiving opening itself has elastic properties to press the temperature sensor against the sample vessel (or insert). In particular, the temperature sensor is fixed to the elastic portion, e.g. taped.

For example, the elastic portion of the side wall (i.e., in particular, a wall which is substantially parallel to the longitudinal axis of the receiving opening, i.e., the direction of insertion of the sample vessel) is formed in the form of a projection which projects from the side wall. It is also possible that the elastic portion is formed as a survey, which rotates the receiving opening, so that the receiving opening in the region of the circumferential elevation has a smaller diameter than outside the survey. For example, the diameter in the region of the circumferential elevation is somewhat smaller than the maximum outside diameter of the sample vessel (or of the insert).

As elastic material for the elastic portion of the side wall, for example, a plastic, a rubber or a rubber-like material comes into question. In particular, it is possible that the material of the elastic wall portion is different from the remaining material of the sample holder. For example, a rubber material or an elastic plastic may be inserted in a recess of the sample receptacle (e.g., otherwise integrally formed) to form the elastic wall portion. In particular, it is also conceivable that such a sample receptacle is produced by means of a two-component injection molding process. It is of course also possible that the elastic portion is formed integrally with the rest of the sample recording.

For example, the receiving opening may also taper towards a bottom area or to a lower edge of the receiving opening, wherein the elastic portion on which the temperature sensor is arranged, in the bottom portion or in the region of the lower edge (from a lower side of the sample receiving is trained, the for example, facing the associated Peltier element) is provided. Thus, the lower, adjacent to the bottom portion and the lower edge portion of the receiving opening has a smaller diameter than an adjacent to the upper edge of the receiving opening area. Thus, a sample vessel (or an insert for receiving a sample vessel) through the larger upper diameter of the receiving opening can be easily inserted into the receiving opening, wherein the temperature sensor nevertheless (completely insert into the receiving opening) sample container (or insert) against the outside of Sample vessel (or the insert) is pressed.

In a third aspect, the invention relates to a device for carrying out the PCR, with

a plurality of temperature sensors for determining the temperature of a sample placed in a sample vessel, wherein each of the receiving openings is associated with at least one of the temperature sensors;

- A plurality of Rückstellkrafterzeugungsmitteln, each of the receiving openings is assigned at least one Rückstellkrafterzeugungsmittel, which cooperates with the receiving opening associated temperature sensor so that it holds the temperature sensor in thermal contact with a arranged in the receiving opening sample container or with an insert for receiving a sample vessel ; and

- A separate to the sample holder support on which the Peltier elements are arranged.

The carrier is in particular cohesively and / or positively connected via fastening means with the sample receptacle.

In addition, the carrier may be formed as a heat sink and define a "cold side" of the Peltier elements .In particular, the carrier is made of a good heat-conducting material, such as a metal (in particular aluminum or copper) its side facing away from the Peltier elements) have structures for increasing the heat dissipation, eg ribs or other structures that increase the surface of the carrier. It is also possible for the carrier (in particular via its underside facing away from the Peltier elements) to form a heat pipe (heat removal tube) or to be connected to a heat pipe, in particular in order to produce a more homogeneous heat distribution in the underside.

According to another embodiment of the invention (according to the first, second and / or third aspect), the sample holder is integrally formed, wherein the receiving openings are arranged in particular grid-shaped. The receiving openings (at least some of the receiving openings) are formed approximately in the form of a through hole or in the form of a blind hole which extends from one side of the sample holder into the material of the sample holder, but has a depth which is less than the thickness of the sample holder.

The dimensions of the receiving openings are placed in particular on the male sample vessels, wherein the receiving openings in particular have an at least approximately circular cross-section. Of course, at least some of the receiving openings may also be designed differently, e.g. with a rectangular or other cross-section. The sample vessels to be inserted into the receiving openings, in which the PCR starting materials are filled, are e.g. to plastic vessels, in particular to so-called "Eppendorf Tubes".

For example, several interconnected sample vessels may also be used, e.g. in the form of commercially available microtiter plates. However, it is also conceivable that other types of sample vessels are used, for example, sample vessels, which are substantially hollow cylindrical and / or (eg in one piece) made of a good heat-conducting material (in particular a metal, such as aluminum), in order to obtain the best possible Thermal contact of the present in the sample vessel sample with the respective temperature sensor and the respective Pel- animal produce element.

According to a variant of the invention, in each case inserts (in particular permanently) are arranged in the receiving openings, into each of which a sample vessel can be inserted. The sample vessels (reaction vessels) are thus not inserted directly into the receiving openings of the sample holder, but into the inserts which are located in the receiving openings. In particular, the inserts to be arranged in the receiving openings are made of a thermally highly conductive material (in particular a metal such as aluminum) and designed so that they distribute heat generated by the Peltier elements, in each case a uniform temperature as possible in a sample vessel, the in the insert is arranged to allow sample submitted.

The respective restoring force generating means serve in accordance with this variant of the invention primarily to enable the simplest possible mounting of the inserts in the receiving openings and nevertheless to realize the best possible thermal contact between the temperature sensors and inserts.

In addition, the sample holder can be made of a thermally highly insulating material (in particular a plastic), whereby a "crosstalk" when tempering adjacent samples is avoided as possible, so that the tempering of the samples (which are each presented in a sample vessel, which in a By means of the Peltier elements and the determination of the current temperature of the samples by means of the temperature sensors, the temperature of the individual samples can be determined individually and very accurately, independently of the temperature of the other, in particular adjacent, samples The different (arranged at the common sample receiving) samples therefore different PCR temperature profiles (temperature cycles) can be specified, each of the samples individually associated with the Peltier elements and the feedback on the ebenfall s the individual samples individually assigned temperature sensors can be accurately generated.

Thus even with the use of sample vessels in the form of a conventional microtiter plate, different sample vessels (ie different "wells") of the microtiter plates with different heating rates and temperature profiles can also be processed for complex analyzes In particular, many different samples (eg using a microtiter plate in standard format It should be noted that the sample holder does not necessarily have to be formed in one piece from a thermally insulating material. would in principle be conceivable for any constructions which have a plurality of receiving openings, wherein the areas of the receiving openings are in particular thermally insulated from one another. For example, could be used as a sample receiving a structure of a wire having the receiving openings and which is at least partially provided with a thermally insulating material (eg, the wire surrounds).

In particular, resistance elements, thermocouples and / or semiconductor sensors are suitable as temperature sensors, although of course the invention is not defined for a particular type of temperature sensor.

The Peltier elements provided for temperature control (ie in particular for heating but possibly also for cooling) in the sample vessels are designed in particular in the form of microtechnologically produced Peltier elements, ie in the form of Peltier elements, each having dimensions (in particular thickness) in the millimeter. or micrometer range. Such a Peltier element is z. B. in the German patent application DE 198 45 104.0, to which reference is expressly made. The Peltier elements are in particular arranged such that they each come into mechanical and thus thermal contact with an outer side of a side wall or a bottom of a sample vessel inserted into a receiving opening. For example, the Peltier elements (ie at least some of the Peltier elements) each project into a lower region of one of the receiving openings. The "lower area" of a receiving opening opens into a lower side of the sample receptacle, which faces away from an upper side of the receiving opening, wherein an upper area of the receiving opening opens into the "upper side" (substrate surface), via which the sample vessel into the receiving opening is introduced. It is also possible if the receiving opening is designed in the form of a blind hole, that the associated Peltier element is arranged on an inner side of the bottom of the receiving opening, so that the bottom of the sample vessel rests on the Peltier element when the sample vessel is fully inserted into the receiving opening. However, it is also conceivable that the Peltier element is not arranged in a bottom region of the sample vessel, but in such a way that it comes into contact with a side wall of the sample vessel. An arrangement of the Peltier element laterally to the sample In particular, the carrying out of an optical measurement (in particular the transmitted-light PCR) of the sample located in the sample vessel through the bottom of the sample vessel is made possible by the use (ie in particular between a side wall of the sample holder and an outside of the sample container or of the insert) ,

Of course, it is also conceivable that in each case one receiving opening a plurality of Peltier elements are assigned, of which, for example, a first on the bottom portion and a second on a side wall of a sample container used in the receiving opening is present. When using inserts for holding sample vessels, the Peltier elements are arranged in particular so that they each rest with their warm side on the insert. For example, the inserts are each cup-shaped, wherein the Peltier elements are in particular arranged so that they each abut on an Au ßenseite a bottom portion and / or laterally on a side wall of the insert.

It is also conceivable that between the inserts and the Peltier elements in each case a compensating material, such as a TIM (Thermal Interface Material), is present, e.g. Graphite, indium and / or silicone. It is additionally or alternatively also possible that the Peltier elements are each connected to the inserts via an adhesive.

In addition, the inserts can also be latched via a latching mechanism in the receiving openings of the sample holder. For example, the inserts for this purpose have latching structures (for example hooks or grooves), which in each case engage in corresponding latching structures of the receiving openings, which are e.g. are formed on side walls of the receiving openings, engage.

According to another development of this variant of the invention (ie the use of "inserts"), means are also provided which press the inserts against the Peltier elements, for example, these means have an attachment (eg in the form of a plate) which is placed on the sample holder and For example, the attachment presses in each case against an edge (eg in the form of a projection) of a cup-shaped insert such that a bottom region of the insert is pressed against the Peltier element between the insert (in particular its bottom area) and the Peltier element be provided an elastic material (eg in the form of an elastic TIM).

According to, in particular, the first and second aspects of the invention, at least some of the temperature sensors are respectively positioned between a side wall of the receiving opening and a sample vessel (or an insert for receiving a sample vessel) arranged in the receiving opening, so that in each case, in particular, a temperature-sensitive area of the temperature sensors the outer wall of the sample vessel (or the insert) is applied. For example, the temperature sensor extends at least approximately parallel to a side wall (which extends at an angle, in particular perpendicular, to the substrate surface) of the receiving opening and / or the outer wall of the sample vessel (or insert) inserted into the receiving opening. It is, of course, also conceivable for the temperature sensor to run between a bottom region of the receiving opening and a bottom of the sample vessel (or the insert) or for a plurality of temperature sensors, of which there are e.g. one on one side wall and another on a bottom of the sample vessel (or insert).

The temperature sensor is biased against the sample vessel (or insert) by the restoring force generating means so that the temperature sensitive area of the temperature sensor is in direct mechanical contact with the outside of the sample vessel (or insert) to maintain the temperature of the temperature in the Sample vessel material located to determine as accurately as possible. In particular, the temperature sensor is fixed to the elastic member (e.g., materially bonded). It is also conceivable that an additional temperature sensor is applied to a bottom of the sample vessel (or the insert).

When using inserts for holding sample containers, it is also possible that the temperature sensors are each connected to an outside of the inserts by means of an adhesive (in particular highly heat-conductive) in order to achieve a particularly stable, permanent connection between the temperature sensors and the (in particular metal) inserts and, for example, to compensate for any unevenness of the insert. For example, the elastic element in the form of a spring (such as a coil spring) is formed, which in particular with one end to a portion of the sample would, which is adjacent to the receiving opening, abuts (eg there is fixed) and the other end is coupled to the temperature sensor.

According to another embodiment of the invention, the temperature sensors are provided with a conductor track arranged on an upper side of the sample holder, e.g. in the form of a printed circuit board (PCB), are electrically connected. The guide plate could also serve to push the inserts for receiving a sample vessel against the respective Peltier elements at the same time. The Peltier elements are, for example, also electrically connected to a conductor track, wherein the conductor track is arranged in particular on a side of the carrier facing the sample receiving means, in particular the conductor track is designed for contacting the Peltier element in the form of a printed circuit board. The invention also includes a method for producing a device for carrying out the PCR, comprising the steps:

- Providing a sample receptacle having a plurality of receiving openings;

- Providing at least one temperature sensor and at least one restoring force generating means in the form of an elastic element on which the temperature sensor is arranged;

Arranging the elastic element on the sample receptacle such that it projects beyond an edge of one of the receiving openings or projects into one of the receiving openings;

- Arranging an insert for receiving sample containers in the receiving opening, so that the elastic element is bent and the temperature sensor is biased against the insert.

According to a variant of this method, the elastic element and / or the temperature sensor is connected to the insert in a material-locking manner. For example, the elastic element, the temperature sensor and / or the insert are provided with an adhesive prior to arranging the insert in the receiving opening in order to ensure a permanently good thermal contact after installation. Accordingly, the invention also relates to a device which was initially designed like the above-described device according to the first aspect of the invention and in which at least one insert is then inserted according to the above-mentioned method was, so that the elastic element with a portion bent between a side wall of the receiving opening and a side wall of the insert extends, for example, the temperature sensor can be materially connected to the side wall of the insert.

The invention further relates to a PCR method, in particular using a device as described above, the method comprising the steps:

Arranging at least a first and a second DNA sample in a common sample receptacle;

- Cyclic tempering of the first DNA sample according to a first temperature profile and cyclic tempering of the second DNA sample according to a second temperature profile, wherein

- the first temperature profile according to a first number of cycles and the second temperature profile according to a second number of cycles, which is different from the first number of cycles, repeatedly traversed.

In particular, the sample holder for carrying out the method, such. described above, Peltier elements and temperature sensors, which are each assigned to a receiving opening, in each of which a sample vessel with the DNA sample, the sample holder. For example, a device as described above is used to carry out the method. Thus, it is possible for the first and second DNA samples to be individually, i. especially according to different temperature profiles to temper. Of course, the method can be carried out with more than two DNA samples, with the samples e.g. differ in the type of DNA material and / or the amount of DNA material.

Moreover, it is possible to temper the first and second DNA samples with different temperature profiles so that a PCR with different samples, e.g. different primer DNA-material combinations can be carried out simultaneously, although the samples are arranged in the same sample holder.

In particular, the invention allows the number of temperature cycles to be performed depending on the amount of DNA material so that, for example, not the sample with the least amount of DNA material determines the total number of cycles for all samples but the number of cycles individually for each sample can be chosen. Here- By the duration of the PCR can be reduced in particular for samples that have a larger amount of DNA material. In particular, different PCR-based diagnostic tests (eg HIV, HCV, etc.), which require different numbers of cycles and temperature profiles, can be carried out jointly, ie in one and the same sample intake.

In a further development of the method according to the invention, the first and / or the second DNA sample are labeled with a dye and while performing the PCR in each case determines the time behavior of the fluorescence of the dye as a measure of the amount of material generated by the PCR. In particular, the first and / or the second number of cycles is selected as a function of whether a predefinable limit value of the fluorescence intensity has been reached. Thus, based on the fluorescence signal, the PCR z. B. in only one of the two samples are terminated when it appears from the fluorescence signal that a sufficient amount of duplicated DNA material is present.

According to a further aspect, the invention also relates to a PCR method with the steps:

Arranging at least a first and a second DNA sample in a common sample receptacle, wherein each DNA sample is assigned a temperature sensor; - cyclic tempering of the first DNA sample according to a first temperature profile and cyclic tempering of the second DNA sample according to a second temperature profile;

after the cyclic tempering, increasing the temperature of the first DNA sample according to a first temperature ramp and simultaneously increasing the temperature of the second DNA sample according to a second temperature ramp (which may be different from the first temperature ramp) to the melting point of the first and second temperature ramps to determine the second DNA sample.

In particular, the first and the second DNA sample can be labeled with different dyes (eg "SYBR green" or ethidium bromide), wherein in each case the change of the fluorescence intensity of the corresponding dye with the temperature as a function of the melting point of the respective DNA sample Temperature is determined, ie, the derivative d (RFU) / dT of a normalized fluorescence signal RFU is determined according to the temperature T. Characteristic of reaching the melting point is that the change of the fluorescence signal with the temperature, ie the derivative of the fluorescence signal according to the temperature In this respect, different melting curves can be analyzed in parallel. In particular, with melting point determination, it is possible to identify single nucleotide polymorphisms (SNP) of the amplified DNA material or perform genotyping experiments, and the determination of the melting point also enables the detection of "Mispriming" or " For example, primer-dimer artefact due to unwanted amplification of primer compounds results in a lower melting point compared to the melting point of the DNA sequence to be amplified.

With the method according to the invention, which is carried out in particular using a device as described above, it is e.g. possible to apply to the samples different temperature ramps, e.g. differ with regard to the start and / or the end temperature and / or the time course. For example, the temperature ramps are selected with respect to different alleles of a DNA sequence having different melting temperatures.

For example, the following β-globin alleles have different melting points T, to which the respective applicable temperature ramp could be adapted:

Locus Allele Pairing T _m (° C)

Hb C / Hb S Wild type T-Mismatch 55.6

Hb C G-T & T T mismatch 50.1

Hb S T-A matsch 62,7

The invention is explained in more detail below with reference to embodiments with reference to the figures. FIG. 1 A shows a device for carrying out the PCR according to one exemplary embodiment of the invention; FIG.

FIG. 1B shows a device for carrying out the PCR according to an embodiment of the invention; FIG.

FIGS. 2A-2C show examples for operating a device according to the invention;

Fig. 3 is an example of a fluorescence signal of one labeled with a dye

DNA sample as a function of the number of cycles; and 4 shows the change of a fluorescence signal of a dye-labeled DNA sample as a function of the temperature of the DNA sample. FIG. 1 A shows a device 1 according to the invention for carrying out the PCR during its assembly. The device 1 has a sample receptacle 2 for receiving sample containers (not shown). The sample vessels to be arranged in the sample receptacle 2 are in particular plastic reaction vessels in which, in particular, at least one DNA sample is arranged, which is to be duplicated by means of the PCR.

In the sample holder 2, which consists for example of a one-piece piece of material made of a thermally insulating material, a plurality of receiving openings 21 is introduced in the form of through holes. The receiving openings 21 are arranged, for example, in a grid (for example, as shown in Figures 2A to 2C) and each have a circular cross-section. In the receiving openings cup-like inserts 3 are inserted, each serving to receive a PCR sample vessel. In particular, the inner diameter of the inserts 3 is adapted to the outer diameter of the PCR sample vessels. A temperature sensor 4, which serves to determine the temperature of the sample present in the sample vessel used in the respective insert 3, is assigned to each of the receiving openings 21.

Furthermore, the device has a plurality of Peltier elements 5, wherein in each case one of the Peltier elements 5 is associated with one of the receiving openings 21. The Peltier elements serve to temper the sample vessels, i. the sample arranged in the sample vessels. In particular, the device 1 also comprises a controller, via which the Peltier elements are controlled in dependence on a signal of the associated temperature sensor.

Furthermore, the device 1 comprises a plurality of restoring force generating means, each in the form of an elastic strip of material 6 (which is formed for example of a metal) are designed. The material strip 6 is fixed with a first end 61 on an upper side of the sample holder 2, wherein the "upper side" is formed by a side of the sample holder 2, which faces away from the Peltier elements 5. The other end 62 of the material strip 6, however, projects beyond an upper edge 21 1 of the receiving opening 21, which is formed in the upper side 22 of the sample holder 2, so that the end 62 and the temperature sensor 4 arranged at this end 62 with the receiving opening 21 overlap, ie the temperature sensor 4 is aligned with the receiving opening 21st In particular, the end 62 is above the receiving opening 21 that an insert 3, which is inserted during assembly of the device according to the invention in one of the receiving opening 21, presses against the end 62 so that it is bent from the remaining strip of material 6 and at completely inserted insert 3 is parallel to a side wall 212 of the receiving opening 21, as shown in Figure 1 for the right of the two inserts 3 shown. The inserted into the left of the two receiving openings 21 insert is not yet mounted so that the left of the two receiving openings associated material strip 6 protrudes just beyond the edge of the left receiving openings 21. The thus bent material strip 6 thus biases the temperature sensor 4 against the insert 3, so that a temperature-sensitive region of the temperature sensor 4 on an outer side of a side wall 32 of the insert 3, which in this case extends parallel to the side wall 212 of the receiving opening 21, rests. The prestressing of the temperature sensor 4 via the bendable material strip 6 produces a lasting good thermal contact between the temperature sensor 4 and the sample vessel 3 and thus to the sample vessel arranged in the insert 3. In addition, an adhesive may be used to permanently connect the temperature sensors to the inserts 3. It should be noted that the invention also covers a device which has no inserts 3, but the PCR sample vessels can be inserted directly into the receiving openings 21. According to this variant of the invention, the material strips 6 are each bent when inserting the sample vessels into the receiving openings, so that the temperature sensors are each pressed against an outer wall of the sample vessel.

The Peltier elements 5 are arranged on a side of the sample holder 2 facing a heat sink 7 formed as a carrier, wherein in each case a Peltier element 5 is associated with one of the receiving openings 21. The sample receptacle 2 and the heat sink 7 are separately formed parts (as shown in FIG. 1), which however are connected to one another, for example adhesively bonded or non-positively or positively (for example, by way of example). screwed). After connecting the sample holder 2 with the heat sink 7, the Peltier elements 5 protrude from below, ie, through a region of the receiving opening 21 which is delimited by a lower edge 213 formed in a lower side 214 of the sample holder 2, at least in sections into the receiving opening 21, so that a completely inserted into a receiving opening 21 insert 3 is in thermal contact with the Peltier element 5 via its bottom portion 31, that is, in particular with the bottom portion 31 on the hot side of the Peltier element 5 abuts. In addition, the inserts 3 can also latching elements (not shown), over which they lock with the receiving openings 21 so that they are held in contact with the Peltier elements. Furthermore, a TIM and / or an adhesive may be arranged between the inserts 3 and the Peltier elements 5 in order to produce the most stable possible thermal coupling of the inserts 3 with the respective Peltier elements 5.

In particular, a compensation material in the form of a TIM is arranged prior to arranging the inserts 3 on the heat sink 7 and / or on the respective Peltier element, which serves as a filler to compensate differences in height between the particular Peltierelemen- and the top of the heat sink 7. As TIM comes here in particular an elastic material in question; e.g. a silicone-based material that has a comparatively high elasticity with acceptable thermal conductivity. After insertion of the inserts 3 into the receiving openings 21, the (in particular kneading) TIM is deformed and adheres to a bottom region of the inserts on the one hand and to the heat sink 7 and / or the Peltier elements on the other hand.

The Peltier elements 5 are electrically contacted via a printed circuit board 8, which is arranged on an upper side 71, the heat sink 7. Similarly, a printed circuit board 80 is provided on the upper side 22 of the sample holder 2, via which the temperature sensors 4 are electrically contacted.

It should be noted that the arrangement of the Peltier elements in a lower region of the receiving openings is not mandatory, but at least some of the Peltier element can also be arranged on a side wall of the receiving openings, so that they each have a side wall and not a bottom of the sample vessels in thermal contact with the sample stand. This allows, as mentioned above, For example, a transmitted-light PCR and in particular the measurement of the fluorescence of a marker dye through the bottom of the sample vessel through.

According to the exemplary embodiment of FIG. 1B (which shows the device according to the invention in the fully assembled state), the Peltier elements 5 are not arranged on the heat sink 7 in such a way that they each come into contact with a bottom region of the inserts 3, but with the outside of a side wall 32 of the inserts 3.

The sample holder 2, which is in particular formed integrally from a thermally insulating material, has an at least approximately L-shaped cross section in the region of its receiving openings 21, so that they each have a bottom section 21 1 1 and a side wall 21 12 extending perpendicularly to the bottom section 21 1 1. On the side walls 21 12 each a Rückstellkrafterzeugungsmittel is arranged in the form of an elastic material strip 6, wherein the material strip 6 is fixed in each case with one end to the side wall 21 12 of the sample holder and the other ("free") end of the strip of material 6 before placing the inserts 3 protrudes from the side wall and protrudes into the receiving opening 21. Analogously to FIG. 1A, the material strip 6 is bent each time the inserts 3 are inserted into the receiving openings 21 in the direction of the side wall 21 12, so that the end with the temperature sensor opens the spring properties of the material strip 6 presses against the side wall 32 of the inserts 3.

At the same time, due to their spring action, the material strips 6 can press the inserts 3 against the Peltier elements 5 arranged on an opposite side of the inserts 3, so that both the temperature sensors 4 and the Peltier elements 5 are kept in thermal contact with the side wall 32 of the inserts 3 ,

The Peltier elements 5 are arranged with their cold side on partitions 72, which are in particular integrally connected to the heat sink 7, wherein the partitions 72 extend perpendicular with respect to the inserts 3 facing top 71 of the heat sink 7. The sample receptacle 2 has recesses 25 through which partitions 72 extend.

The lateral arrangement of the Peltier element 5 makes it possible, in particular, to carry out the transmitted-light PCR, in which case in particular recesses in the bottom region 31 of FIG Inserts 3, in the bottom region 21 1 1 of the sample holder 2 and corresponding recesses in the heat sink 7 are formed.

FIGS. 2A-2C show a device according to the invention (for example according to FIG. 1A or 1B) from above, wherein a plurality of receiving openings 21 are arranged in a grid. The temperature sensors (not shown) associated with each receiving opening 21 and Peltier elements (not shown) allow, in particular, the carrying out of an individual PCR for each of the samples arranged in the receiving openings 21. For example, as shown in FIG. 2A, it is possible for different samples to go through a different number of temperature cycles.

Furthermore, it is conceivable that different samples are tempered differently, i. each undergo different temperature profiles (Figure 2B). Of course, it is also possible that some of the sample images (shown bright in Figure 2C) are inactive, i. the associated temperature sensors and Peltier elements are turned off.

For example, it is also possible to monitor the amount of each generated in the individual sample vessels DNA material during PCR by individual real-time PCR. For this purpose, the starting samples are labeled with a dye and the fluorescence signal of each sample is detected individually over time. In particular, the respective fluorescence intensity is proportional to the amount of DNA material present in each case. FIG. 3 shows an example of such fluorescence curves for different DNA samples as a function of the number of cycles performed in each case. It will be appreciated that as the number of cycles, i. As the amount of DNA material increases, the fluorescence intensity increases.

With the device according to the invention, it is in particular possible, for example after performing a PCR, to simultaneously determine the melting point of different samples. In this case, for each sample which is marked with a dye marker, the change in the fluorescence intensity with the temperature as a function of the temperature is determined individually, ie each DNA sample is subjected to an individual temperature ramp and the respective derivative -d (RFU) / dT of a normalized fluorescence signal RFU determined. The result of such a measurement is shown for various DNA samples in FIG. The derivative -d (RFU) / dT of the fluorescence signal points at the respective Melting temperature to a maximum, so that by determining the respective maximum, the respective melting temperature can be determined. On the basis of the exact melting temperature, as explained above, in particular impurities or artifacts of the amplified DNA material can be determined.

LIST OF REFERENCE NUMBERS

1 PCR device

2 sample intake

3 use

4 temperature sensor

5 Peltier element

6 elastic strips of material

7 heat sink

8, 80 printed circuit board

21 receiving opening

22 top of sample holder

31 ground use

32 sidewall insert

61 first end

62 second end

71 Upper side heat sink

72 partition wall

21 1 upper edge

212 side wall

213 lower edge

214 Bottom sampling

21 1 1 Bottom area Sampling

21 12 Sidewall Sample holder

Claims

claims

1 . Apparatus for carrying out the PCR, with

- A sample receptacle (2) for receiving sample containers, wherein the sample receiving means (2) has a plurality of receiving openings (21), in each of which a sample vessel or an insert (3) for receiving a sample vessel can be arranged;

- A plurality of Peltier elements (5), wherein each receiving opening (21) is associated with at least one of the Peltier elements (5), so that in the Aufnahmeöff- openings (21) arranged sample containers or inserts (3) can be tempered individually;

- a plurality of temperature sensors (4) for determining the temperature of a sample placed in a sample vessel, each of the receiving openings (21) being associated with at least one of the temperature sensors (4); and

- A plurality of Rückstellkrafterzeugungsmitteln, each of the receiving openings (21) each having at least one restoring force generating means associated with the receiving opening (21) associated with the temperature sensor (4) so that it the temperature sensor (4) in thermal contact with a in the Holding receptacle (21) arranged sample vessel or with an insert (3) for receiving a sample vessel, wherein

- At least one of the restoring force generating means comprises an elastic member (6) on which the temperature sensor (4) is arranged, wherein the elastic element (6) projects beyond an edge of the receiving opening (21) or projects into the receiving opening (21), so that after a sample vessel or insert (3) has been arranged in the associated receiving opening (21), it is bent and the temperature sensor (4) is thus prestressed against the sample vessel or the insert (3).

2. Apparatus for carrying out the PCR, in particular according to claim 1, with

- A sample receptacle (2) for receiving sample containers, wherein the sample receptacle (2) has a plurality of receiving openings (21), in each of which a sample vessel or an insert (3) for receiving a sample vessel can be arranged;

- a plurality of Peltier elements (5), wherein each receiving opening (21) is associated with at least one of the Peltier elements (5), so that in the receiving openings (21) arranged sample containers or inserts (3) can be individually tempered; - a plurality of temperature sensors (4) for determining the temperature of a sample placed in a sample vessel, each of the receiving openings (21) being associated with at least one of the temperature sensors (4); and

- At least one of the restoring force generating means in the form of an elastically shaped portion of a side wall of the receiving openings (21) is formed, the temperature sensor (4) against a arranged in the receiving opening sample vessel or against an arranged in the receiving opening insert (3) for receiving a sample vessel suppressed.

Apparatus for carrying out the PCR, in particular according to claim 1 or 2, with

- A plurality of Peltier elements (5), wherein each receiving opening (21) is associated with at least one of the Peltier elements (5), so that in the receiving openings (21) arranged sample containers or inserts (3) can be individually tempered;

- a plurality of temperature sensors (4) for determining the temperature of a sample placed in a sample vessel, each of the receiving openings (21) being associated with at least one of the temperature sensors (4);

- A plurality of Rückstellkrafterzeugungsmitteln, each of the receiving openings (21) each having at least one restoring force generating means associated with the receiving opening (21) associated with the temperature sensor (4) so that it the temperature sensor (4) in thermal contact with a in the Holding receiving opening (21) arranged sample vessel or with an insert (3) for receiving a sample vessel; and

- A to the sample holder (2) separate carrier (7) on which the Peltier elements (5) are arranged.

Device according to one of the preceding claims, characterized in that the sample holder (2) is formed from a thermally insulating material.

5. Device according to one of the preceding claims as far as dependent on claim 2, characterized in that the receiving opening (21) in the insertion, along which a sample container or during assembly of the device an insert (3) for receiving a sample vessel in the receiving opening ( 21) is inserted, tapered and the elastic portion adjacent to a lower edge (213) of the receiving opening (21) or a bottom portion of the receiving opening.

6. Device according to one of the preceding claims as far as dependent on claim 1, characterized in that the elastic element (6) with a first portion (61) on the sample holder (21) is arranged and with a second portion (62), where the temperature sensor (4) is located projects beyond the edge of the receiving opening (21) or protrudes into the receiving opening (21) so that the temperature sensor (4) after arranging the sample vessel or the insert (3) in the associated receiving opening (21) on a side wall (32) of the sample vessel or the insert (3) comes to rest.

7. The device according to claim 6, characterized in that the elastic element (6) with the first portion (61) on an upper side (22) of the sample holder (21) is arranged.

8. The device according to claim 7, characterized in that the elastic element (6) is designed in the form of a sheet-like element, wherein the first portion of the elastic element (6) is formed by a first portion of the sheet-like element which at the top ( 22) of the sample receptacle (2) is arranged, and the second portion of the elastic element (6) through a second portion of the sheet-like element on which the temperature sensor (4) is arranged, wherein the second portion of the sheet-like element before arranging the sample vessel or the insert (3) with the receiving opening (21) overlaps.

9. Device according to one of the preceding claims, characterized in that the temperature sensors (4) with a on an upper side (22) of the sample holder (2) arranged conductor track (80) are electrically contacted. 10. Apparatus according to claim 9 as far as dependent on claim 3, characterized in that the Peltier elements (5) with a on one of the sample holder (2) facing side (71) of the carrier (7) arranged conductor track (8) are electrically contacted.

1 1. Method for producing a device for carrying out the PCR, comprising the steps:

- Providing a sample receptacle (2) having a plurality of Aufnahmeöffnun- gene (21);

- Providing at least one temperature sensor (4) and at least one restoring force generating means in the form of an elastic element (6) on which the temperature sensor is arranged;

- Arranging the elastic element (6) on the sample holder (2) such that it protrudes beyond an edge of one of the receiving openings (21) or projects into one of the receiving openings (21);

- Arranging an insert (3) for receiving sample containers in the receiving opening (21), so that the elastic element (6) is bent and the temperature sensor (4) is biased against the insert (3).

12. The method according to claim 1 1, characterized in that the elastic element (6) and / or the temperature sensor (4) to the insert (3) are materially connected. 13. The method according to claim 12, characterized in that the elastic element (6), the temperature sensor (4) and / or the insert (3) before placing the insert (3) in the receiving opening (21) are provided with an adhesive ,

14. The method according to any one of claims 1 1 to 13, characterized by

- Providing a support (7) on which a plurality of Peltier elements (5) is arranged, and

- Connecting the sample holder (2) with the carrier (7), so that each of the receiving openings (21) is associated with at least one Peltier element. 15. The method according to claim 14, characterized in that prior to arranging the insert (3) in the receiving opening (21) a compensating material on the carrier (7) and / or the receiving opening associated Peltier element (5) is applied.

PCR method, in particular using a device according to the preceding claims, comprising the steps: Arranging at least a first and a second DNA sample in a common sample receptacle (2), wherein each DNA sample is assigned a temperature sensor (4);

17. The method according to claim 16, characterized in that the first and / or the second DNA sample labeled with a dye and during the implementation of the PCR, respectively, the fluorescence intensity of the dye as a measure of the amount of material generated by the PCR over time is determined.

18. The method according to claim 17, characterized in that the first and / or the second number of cycles are selected depending on whether a predefinable limit value of the fluorescence intensity is reached. 19. The method according to any one of claims 16 to 18, characterized in that the first temperature profile is different from the second temperature profile.

20. PCR method, in particular according to one of claims 16 to 19, with the steps:

Arranging at least a first and a second DNA sample in a common sample receptacle (2), wherein each DNA sample is assigned a temperature sensor (4);

cyclic tempering of the first DNA sample according to a first temperature profile and cyclic tempering of the second DNA sample according to a second temperature profile;

after the cyclic tempering, raising the temperature of the first DNA sample according to a first temperature ramp and simultaneously increasing the temperature of the second DNA sample according to a second temperature ramp to determine the melting point of the first and second DNA samples.

21. A method according to claim 21, characterized in that the first and the second DNA sample are labeled with a dye and for determining the Melting point in each case the change in the fluorescence intensity with the temperature of the dye as a function of the temperature is determined.