METHOD FOR CARRYING OUT A POLYMERASE CHAIN REACTION AND DEVICE FOR CARRYING OUT THE METHOD

Abstract

A method for amplifying nucleic acids by a polymerase chain reaction in a reaction volume heated using electrical energy. In at least one of the passages of the amplification cycle of the polymerase chain reaction, the ratio of the electrical energy used in the denaturation step to heat the reaction volume to the size of the reaction volume is less than 20 Joule per milliliter. Further shown is a method of amplifying nucleic acids in a reaction volume by using a device that includes a reaction vessel and a heating means with at least one heating element in contact with the reaction volume where at least one heating element is conjugated to oligonucleotides. Also shown is a device for the amplification of nucleic acids in a reaction volume including a reaction vessel for receiving the reaction volume and a heating means consisting of at least one heating element contacting the reaction volume.

Claims

1. A method for amplifying nucleic acids by means of a polymerase chain reaction in a reaction volume, wherein the reaction volume is heated through the use of electrical energy, wherein the polymerase chain reaction comprises at least one amplification cycle and in at least one passage of the amplification cycle, the ratio of the electrical energy used in the denaturation step for heating the reaction volume to the size of the reaction volume is less than 20 Joule per millilitre.

2. A method for amplifying nucleic acids by means of a polymerase chain reaction in a reaction volume, wherein a heating means consisting of at least one electrically contacted heating element in contact with the reaction volume, heats the reaction volume, and wherein the polymerase chain reaction comprises at least one amplification cycle and in at least one passage of the amplification cycle, the heating means supplies to the reaction volume less heat generated in the denaturation step than C.sub.R*5 C., wherein C.sub.R is the heat capacity of the reaction volume during the heating by means of the heating means.

3. A method for amplifying nucleic acids by means of a polymerase chain reaction in a reaction volume, wherein a heating means consisting of at least one electrically contacted heating element in contact with the reaction volume, heats the reaction volume, and wherein the polymerase chain reaction comprises at least one amplification cycle and in at least one passage of the amplification cycle, the maximum increase of the average temperature of the reaction volume, taking place through the denaturation step, is less than 10 Celsius.

4. A method for amplifying nucleic acids by means of a polymerase chain reaction in a reaction volume, wherein a heating means consisting of at least one electrically contacted heating element in contact with the reaction volume, heats the reaction volume, and wherein the polymerase chain reaction comprises at least one amplification cycle and in at least one passage of the amplification cycle, the heating means supplies to the reaction volume less heat generated in the denaturation step than C.sub.R*5 C., wherein C.sub.R is the heat capacity of the reaction volume during the heating by the heating means and, while the heating means is heating the sample, a temporally stable temperature is not established on at least 10% of the contact area of the heating means with the reaction volume

5. The method of claim 2, wherein at least one of the heating elements is a heating resistor.

6. The method of claim 2, wherein the ratio between the surface of the heating element(s) in contact with the reaction volume, and the reaction volume is greater than 0.1 per metre.

7. The method of claim 2, wherein the heat supply through the heating means varies during the polymerase chain reaction.

8. The method of claim 1, wherein in at least one of the passages of the amplification cycle of the polymerase chain reaction, the cycle duration t.sub.c is shorter than 60 seconds.

9. The method of claim 1, wherein the duration of the polymerase chain reaction tpcR is shorter than 45 minutes.

10. A method for amplifying nucleic acids in a reaction volume, comprising: using a device comprising a reaction vessel for receiving the reaction volume; and heating the reaction volume with a heating means, which consists of at least one heating element in contact with the reaction volume, wherein at least one heating element is conjugated to oligonucleotides.

11. A device for amplifying nucleic acids in a reaction volume comprising a heating means, which consists of at least one heating element in contact with the reaction volume in order to heat it.

12. The device of claim 11, wherein at least one of the heating elements is conjugated to oligonucleotides.

13. The device of claim 11, wherein the device further comprises a light source and a light sensor.

14. A device for amplifying nucleic acids in a reaction volume comprising: a heating means, which consists of at least one heating element for heating the reaction volume using electrical energy, and a means for transferring the electrical energy into the device, wherein the device is configured so that its electrical power consumption during a polymerase chain reaction does not exceed 50 Watt at any point in time.

15. The device of claim 14, wherein the device further comprises an electricity storage that keeps available electrical energy greater than 0.1 J/mL.

16. A device for amplifying nucleic acids in a reaction volume comprising: a reaction vessel for receiving the reaction volume, a heating means consisting of at least one heating element for heating the reaction volume using electrical energy, and a means for transferring the electrical energy into the device, wherein the device is configured so that the ratio between the electrical power consumption of the device during a polymerase chain reaction and the capacity of the reaction vessel does not exceed 1 Watt per millilitre at any point in time.

17. A device for amplifying nucleic acids in a reaction volume by means of a polymerase chain reaction comprising: a reaction vessel for receiving the reaction volume, a heating means consisting of at least one heating element, and a control means, which applies electrical current to the heating means in order to heat the reaction volume, wherein the polymerase chain reaction comprises at least one amplification cycle and the control device is configured so that, in at least one passage of the amplification cycle, the ratio between the electrical energy applied by the control means to the heating element in the denaturation step, and the capacity of the reaction vessel is less than 40 Joule per millilitre.

18. A device for amplifying nucleic acids in a reaction volume by means of a polymerase chain reaction comprising: a reaction vessel for the reaction volume, a heating means consisting of at least one heating element that heats the reaction volume, and a control means that controls the heat emission of the heating means to the reaction volume, wherein the polymerase chain reaction comprises at least one amplification cycle and the control means is configured so that, in at least one passage of the amplification cycle, the ratio between the amount of heat emitted by the heating means in the denaturation step to the reaction volume, and the capacity of the reaction vessel for receiving the reaction volume, is less than 20 Joule per millilitre, and at least one heating element of the heating means has an expansion of more than 1.5 micrometres in at least one direction.

19. The device of claim 18, further comprising an electricity storage, and wherein the device is designed so that the electrical energy kept available in the electricity storage is less than 100 Joule per millilitre.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0099] FIG. 1A shows schematically a heating element functionalised to primers, through which there is no current flow, as the switch is open. There is a free DNA sequence in the vicinity of the heating element'.

[0100] FIG. 1B shows schematically a DNA-functionalised heating element, through which there is no current flow, as the switch is open. Duplexes of primers and target nucleic acid have formed on the heating element and have already partially been elongated.

[0101] FIG. 1C shows schematically a DNA-functionalised heating element, through which there is a current flow, as the switch is closed. A denaturation of the double strands has taken place on the heating element, with the result that only the elongated primers remain.

[0102] FIG. 2A shows the spatial and time-based progression of the temperature in the cooling phase of a previously heated cylindrical heating element.

[0103] FIG. 2B shows the time-based progression of the surface temperature of cylindrical heating elements of different diameters during heating.

[0104] FIG. 2C shows the time-based progression of the surface temperature of cylindrical heating elements of different diameters during cooling.

[0105] FIG. 2D shows the spatial and time-based progression of the temperature in the cooling phase of a previously heated gold film on an unheated cylindrical carrier.

[0106] FIG. 3A shows an electronic circuit for generating current pulses.

[0107] FIGS. 3B, 3C, 3D show possible embodiments of reaction vessels that contain heating elements.

[0108] FIG. 3E shows the global temperature increase in aqueous solution with different current flows, i.e. heating of the heating elements.

[0109] FIG. 4 shows a real-time PCR with local heating with genomic template.

[0110] FIGS. 5A, 5B, 5C show a real-time PCR with local heating with genomic template with the use of different voltages for operation of the heating means.

[0111] FIGS. 5D, 5E, 5F, 5G show a real-time PCR with local heating with genomic template with the use of many different heating elements.

[0112] FIG. 6A shows a honeycomb structure that can be electrically heated.

[0113] FIG. 6B shows details of the honeycomb structure that can be electrically heated.

[0114] FIG. 6C shows a real-time PCR with local heating with genomic template with the use of the honeycomb structure for local heating.

[0115] FIG. 7 shows a real-time PCR with local heating and heating element, on which both forward and also reverse primers are functionalised.

DETAILED DESCRIPTION OF THE INVENTION BY REFERENCE TO A PLURALITY OF EMBODIMENTS

Course of the Method According to the Invention

[0116] By way of example, the first passage of the amplification cycle of the PCR can be carried out as follows in a method according to the invention: After the addition of nucleic acid molecules (hereinafter described as target nucleic acids) to the reaction volume (and possibly the denaturation thereof by global heating) and the hybridisation thereof to forward primers bonded gebunden To one or more heating element, a polymerase elongates the forward primers and thereby produces complementary strands for the target nucleic acid. The denaturation, i.e. the separation of the molecules of the target nucleic acid from the elongated forward primers, is not realised by global heating of the whole reaction volume, but instead through a heat pulse, which is brought about by a current pulse through the heating element(s) of the heating means.

[0117] The subsequent second passage of the amplification cycle of the PCR can be realised in a similar way. The molecules of the original target nucleic acid hybridise again to forward primers bonded to one or more heating element(s) and the polymerase elongates the forward primers and hereby produces complementary strands for the target nucleic acid (or at least for a proportion of the target nucleic acid), In parallel, reverse primers (which are either freely suspended or also heating element-bonded) can bind to the elongated parts of the elongated heating element-bonded forward primers produced in the first passage of the amplification cycle of the PCR (the forward primers now constitute complementary strands to at least a proportion of the target nucleic acid) and the reverse primers are subsequently correspondingly elongated by the polymerase. In this way, for the first time genuine copies of at least a part of the original target nucleic acid are produced. The denaturation, i.e. the separation of the double strands produced through elongation by the polymerase (the double strands are in any case again bonded to the heating means) is realised once again through a heat pulse caused by the current pulse through the heating means. With effect from the third passage of the amplification cycle of the PCR, both the original target nucleic acid and also the nucleic acid strands produced by elongation of the primer sequences through the polymerase (depending on the embodiment: freely suspended in the reaction volume or heating element-bonded) as a template for the further amplification. They are amplified by hybridisation to corresponding primers (according to the embodiment: freely in solution of bonded to a heating element), there is subsequent elongation by through the polymerase and then denaturation by means of a local heat pulse, which is brought about by a current impulse through the heating means. The last described passage of the amplification cycle of the PCR is repeated several times in order to produce further copies at least of parts of the target nucleic acid in each further passage of the cycle. The passages are repeated until as often as necessary until a sufficiently high number of copies at least of parts of the target nucleic acid are present in order to be able to carry out a detection of the amplification carried out or the original presence of the target nucleic acid in the sample. Using one of the methods described above, for example fluorescence method, the thus generated amplicons can be detected.

[0118] In a further exemplary embodiment of the invention a plurality of different target nucleic acids are amplified in parallel (also described as multiplex-PCR). For this, a plurality of primer pairs that are different from each other (in each case: forward and reverse primers) are necessary for each amplicon (wherein a primer can also serve as a primer for two amplicons, for example of different lengths, therefore being part of two primer pairs). A heating element can carry a plurality of primer pairs or in each case (at least) one primer made up of a plurality of primer pairs. However, a plurality of primers or primer pairs can also be distributed in such a way that different sub-portions of the heating element each carry only one primer pair or each carry only one partner of a primer pair. In an exemplary embodiment one (possible only one per primer pair or even each) primer sort or primer sequence can be present in both a heating element-bonded form and also freely suspended form in the reaction volume.

[0119] A detection can be realised for example by using different dyes in such a way that different colour signals are produced (with different wavelengths) can be assigned to the formation of different amplicons. Alternatively, however, different amplicons can also produce the same colour signals, which cannot be differentiated. Different amplicons can also be differentiated, for example using gel electrophoresis or other methods.

Setting, or Establishing, a Global Reaction Temperature

[0120] The (global) elongation and hybridisation temperature is kept constant in a preferred exemplary embodiment during the whole course of the PCR, for example by means of a conventional external heater, for example a temperature-regulating block, or through a constant (or regulated) excitation of the heating element by means of a constant (or regulated) offset current through the heating element.

[0121] The regulation of the (global) elongation and hybridisation temperature or the heating temperature can be realised on a temperature sensor in the reaction volume, in one of a plurality of reaction volumes, individually for each individual reaction volume through a respective sensor in the respective reaction volume, by a sensor outside of the reaction volumes or through a sensor in the heater or a recording device for the reaction volume. In one embodiment the (global) elongation and hybridisation temperature for all reaction volumes is the same, in a further embodiment the (global) elongation and hybridisation temperature can differ for the different reaction volumes.

[0122] In a further embodiment the (global) elongation and hybridisation temperature can be varied or changed during the duration of the PCR or before or after the PCR. In one embodiment the heater can also consist of a plurality of parts, for example from a bottom part and a top part, wherein the top part has a somewhat higher temperature than the bottom part in order to avoid condensation on the walls of the reaction volumes. The temperature difference between the top and bottom part of the heater is preferably between 1 C. and 30 C., particularly preferably between 2 C. and 20 C. and most particularly preferably between 3 C. and 15 C.

[0123] In one embodiment the global heating of the reaction liquid in the reaction vessel through electrical heating of the heating means can account for a part of the heat input or all of the heat that is required to reach the desired elongation and hybridisation temperature. This can for example be achieved by the duty cycle and/or the continuous electrical current (or voltage at) the heating element(s) of the heating means being selected so that the global heating of the reaction liquid in the reaction vessel in thermal equilibrium or at the end of the PCR or at the start of the PCR or during a large part of the duration of the PCR leads to the desired elongation and hybridisation temperature in the reaction liquid. The external heating (i.e. the element for the heat input that does not come through the heating element) can thereby have smaller dimensions or be completely omitted.

[0124] Preferred temperatures according to the invention for the combined hybridisation and elongation temperature are preferably between 30 C. and 85 C., particularly preferably between 40 C. and 80 C., particularly preferably between 50 C. and 75 C. and most particularly preferably between 55 C. and 72 C.

[0125] In one embodiment, a global heating step (with a global temperature greater than the later hybridisation and elongation temperature) can take place before the first actual passage of the PCR cycle, wherein said global heating step can serve for initial denaturation of the double-stranded present target nucleic acid (DNA or RNA or other nucleic acid) and/or for thermal activation of other reaction partners of the PCR such as for example (hot-start) polymerases and/or for deactivation of constituent parts of the reaction volume, which are to be active before the PCR but not during the PCR (such as for example the enzyme Uracil-DNA-glycosylase).

[0126] In one embodiment, a further global heating step with a lower global temperature than in the abovementioned global heating step can take place before this global heating step, wherein the further global heating step can be utilised for example, an enzyme being given off, a reaction taking place before the PCR (such as for example the overwriting of RNA into DNA by a transcriptase enzyme).

Spatial Heat Spreading During and after the Denaturation Step

[0127] As soon as the current flow through the heating element in the denaturation step has begun, the heating element begins to heat up. As most current-conducting materials (in particular metals) also convey heat very well, the heating element heats approximately homogeneously over the duration of the heat pulse. At the surface of the heating element, which is surrounded in a preferred exemplary embodiment by the aqueous reaction volume, the heat is transferred to the reaction volume, where it spreads. The spreading of a thermal field is realised in the reaction volume through heat diffusion, for which a root-form rule applies.

d{square root over (D.Math.t)}Equation 1

wherein d describes the path distance covered by a heat front after a time t along a spatial direction in a reaction volume with temperature conductivity D and is to be referred to below as heat diffusion range. This means, that for a heating duration of for example 100 s, the heat generated in the heating element can diffuse far into the reaction volume with a typical temperature diffusivity (also known as temperature conductivity) of D1.6.Math.10.sup.7 m.sup.2/s in terms of value

[00001]$d\sqrt{\frac{1.6.Math.{10}^{-7}.Math.m^2}{s}}.Math.{10}^{-4}.Math.s4.Math..Math.\mathrm{.Math.m}$

[0128] In other words, the heat generated in the heating element for example by ohmic losses has spread after 100 s into the reaction volume surrounding the heating element, namely with a magnitude in the range of 4 m.

[0129] Through the spatial spread of the heat corresponding to the above equation the amount of heat brought is distributes over an increasingly large volume so that, perpendicular to the surface of the heating element, which is hotter by a temperature T than the global average temperature, a temperature gradient of the value T/d results, which facilitates the heat transport. A more precise estimation of the spatial heat expansion during and after the heat pulse for the respective geometry of the heating element can be achieved by finite element methods such as, for example, with commercial solutions like for example Comsol, which facilitate a numerical solution of the heat diffusion equation. The reaction volume within a layer with the thickness of a heat diffusion range is preferably heated around the heating means by at least more than 10 C.

[0130] In a preferred embodiment of the invention the heat diffusion range in at least one, preferably in at least three, particularly preferably in at least 10, particularly preferably in at least 20 of the passages of the amplification cycle of the PCR, at the end of the denaturation step is preferably between 0.05 m and 200 m, particularly preferably between 0.2 m and 100 m, particularly preferably between 0.5 m and 50 m and most particularly preferably between 1 m and 25 m. It is an achievable advantage of this embodiment of the invention that on the one hand a sufficient spatial expansion of the heated area perpendicular to the surface of the heating element can be achieved and that the PCR amplicons formed on the heating element, which typically have a length of between 0.02 and 3 m (correspondingly roughly between 60 and 10000 base pairs), can be heated as homogeneously as possible and thus denatured, and that the heat diffusion range is not so large that the volume ratio of the heating-up zone to the unheated passive volume becomes too low.

[0131] In the sense of the present invention the heating-up zone is the part of the reaction volume, in which the heat can diffuse during the denaturation step. The expansion of the expansion of the heating-up zone perpendicular to the surface of the heating element can be estimated approximately through the heat diffusion range defined above. The volume of the reaction liquid that is not in the heating-up zone is referred to as unheated passive volume. This means for example in the case of a cylindrical heating element (for example a heating wire) that the heating-up zone can be estimated as the volume located at the distance of a heat diffusion range d from the cylinder surface (i.e. the cylinder shell with thickness d). If the heating element is for example an elongate cylinder with radius r and length l (for example a wire), the volume of the heating-up zone can be roughly estimated as

V.sub.AHZ.Math.I.Math.((r+d).sup.2r.sup.2).Equation 2

[0132] In a preferred embodiment of the invention the volume ratio of the heating-up zone to the unheated passive volume in at least one, preferably in at least three, particularly preferably in at least 10, particularly preferably in at least 20 of the passages of the amplification cycle, at the end of the denaturation step is less than 10%, preferably less than 5%, particularly preferably less than 2%, particularly preferably less than 1%, particularly preferably than 0.5%, particularly preferably less than 0.25% and most particularly preferably less than 0.1%. With this embodiment of the invention a high localisation of the heat can be achieved, which means that the amount of heat brought in the denaturation step can spread after the denaturation step to the unheated passive volume. As the unheated passive volume is many times greater than the heating-up zone, the distribution of the amount of heat over the whole reaction volume (=heating-up zone+unheated passive volume) can lead to a preferably negligible global temperature increase of the whole reaction volume, so that a very rapid cooling of the heating-up zone is possible and in addition the cooling process is (extensively) independent from a discharge of the heat to outside of the sample

Estimation of the Local Temperature Increase in the Denaturation Step

[0133] For typical denaturation temperatures of the double-stranded nucleic acid of between 85 C. and 98 C., in one embodiment a local temperature lift with respect to the combined hybridisation and elongation temperature of roughly between 20 C. and 40 C. must be reached on the surface of the heating elements and over the length of the double-stranded nucleic acid to be denatured.

[0134] In a preferred embodiment of the invention the temperature of the area of the heating means that is in contact with the reaction volume is, in at least one, preferably in at least three, particularly preferably in at least 10, particularly preferably in at least 20 of the passages of the amplification cycle of the PCR, during the denaturation step, is between 70 C. and 250 C., particularly preferably between 75 C. and 150 C., particularly preferably between 80 C. and 120 C., most particularly preferably between 80 C. and 100 C.

[0135] In a preferred embodiment of the invention the average temperature of the area of the heating means that is in contact with the reaction volume is over 100 C. With this embodiment of the invention, a particularly rapid separation of the double strand is advantageously possible. This embodiment of the invention utilises the fact that a short-term overheating of the reaction volume is also possible on the surface of the heating means without vapour bubbles forming (inter alia, on account of the high Laplace pressure due to the curvature of the surface of the heating elementsee specialist literature for Young-Laplace equation.

[0136] In the case of complex geometries of the heating means and/or in order to ensure a high precision, the use of finite element methods (for example Comsol) is advisable, in order to determine the temperature of the heating element as a function of the electrical operating parameters. In such simulations, in the simplest case a constant volumetric heating density can be assumed or a current flow through the heating means can be simulated. In many cases, however, the temperature increase brought about by the heating means can be estimated by a simple calculation, which is set out by way of example below.

[0137] Firstly, the amount of heat that is released in a conductor with a current flowing through it is determined. The electrical power P, which is available during the electrical heat pulse for heating the heating element, is calculated from the resistance of the heating element R and the voltage U supplied at the heating element as P=U.sup.2/R. The amount of heat Q released in the heating element is then the electrical power P times the heating duration t.sub.heat,

Q=U.sup.2.Math.t.sub.heat/REquation 3

wherein, here, a temporally constant voltage and constant resistance were assumed over the time duration of the heat pulse. If this is not the case, then the following applies:

Q=.sub.0.sup.t.sup.heatU(t).sup.2/R(t).Math.dt

[0138] If the heating means is made up in one embodiment (in portions) of homogeneous conductors with constant cross-section area, then the resistance of a homogeneous conductor R can be calculated from its cross-section area A and its length l as well as the specific conductivity G of this conductor element as

[00002]$\begin{array}{cc}R=\frac{1}{A.Math.}& \mathrm{Equation}.Math..Math.4\end{array}$

[0139] Typical values for the specific conductivity are shown in tables in the specialist literature and are as follows for typical materials such as gold:

[00003]${}_{\mathrm{Au}}=4.6.Math.{10}^7.Math.\frac{A}{V.Math.m},$

for tungsten:

[00004]${}_W=1.9.Math.{10}^7.Math.\frac{A}{V.Math.m}$

and for V2A (stainless steel):

[00005]${}_{V.Math..Math.2.Math..Math.A}=1.4.Math.{10}^6.Math.\frac{A}{V.Math.m}$

[0140] If it is assumed that the duration of the heat pulse is so short that the energy in initially only heats the heating element(s) and the heating-up zone, the local temperature increase of the heating means, which is indeed to be heated to the denaturation temperature, can be estimated as follows:

[00006]$\begin{array}{cc}.Math..Math.T_{\mathrm{Local}}\frac{Q}{c_{\mathrm{MH}}.Math.m_{\mathrm{MH}}+c_{\mathrm{AHZ}}.Math.m_{\mathrm{AHZ}}}& \mathrm{Equation}.Math..Math.5\end{array}$

[0141] Here, C.sub.MH describes the specific heat capacity (per unit of mass xx) of the heating element and m.sub.MH describes the mass thereof and also c.sub.AHZ the (mass-related) specific heat capacity of the heating-up zone (which is c.sub.AHZ=4.2 J/( C..Math.g) for the aqueous PCR solution) and m.sub.AZ is the mass of the heating-up zone. The above approximation is all the more precise, the smaller the heat capacity of the heating-up zone in comparison with the heat capacity of the heating element. This is due to the fact that the above equation does not take into consideration that the temperature rapidly falls in the heating-up zone (i.e. a solid temperature gradient forms around the heating element).

[0142] If the heating duration is selected to be so short that the size of the heating-up zone remains very small (significantly smaller than the heating element itself) and therefore its heat capacity is negligible with respect to the heat capacity of the heating element, the above equation can be simplified to:

[00007]$\begin{array}{cc}.Math..Math.T_{\mathrm{Local}}\frac{Q}{c_{\mathrm{MH}}.Math.m_{\mathrm{MH}}}& \mathrm{Equation}.Math..Math.6\end{array}$

[0143] While Equation 6 can only be applied in special cases and for very short heating durations, Equation 5 can be used as an approximation for determining the local temperature on the surface of the heating element(s), wherein it must be checked in each case from the geometry and the actual current flow how, above all, the mass of the heating-up zone can be calculated. In many cases, the mass of the heating-up zone can be estimated from its volume by taking into consideration the geometry of the heating element and also the heat diffusion range (see above).

[0144] The case that is particularly relevant according to the invention will be considered below, wherein the heating means is formed at least in portions (approximately) cylindrically and at least in portions is homogeneous and has a constant cross-section. The following calculation is to be regarded as an example for a cylindrical geometry of the heating means and can very easily also be transferred to other geometries. The mass of the heating element in Equation 5 can then be calculated from its volume and density:

m.sub.MH=p.sub.MH.Math.A.Math.l,

wherein A is the cross-sectional surface area and l is the length of the heating element (or the portion thereof considered). The amount of heat Q can then be calculated with Equation 3 and Equation 4.

[0145] With Equation 2, which describes approximately the volume of the heating-up zone, which surrounds an (approximately) cylindrical conductor portion with radius r and length l, the mass of the heating-up zone can be estimated in Equation 5 as:

m.sub.AZ=V.sub.AHZ.Math.p.sub.AHZ=.Math.l.Math.((r+d).sup.2r.sup.2).Math.p.sub.AHZ,

wherein the density of the heating-up zone in the reaction volume is equal to the density of water (p.sub.AHZP.sub.H.sub.2.sub.O1 g.Math.cm.sup.3). Therefore, for a heating element that is (approximately) cylindrically formed and at least in portions is homogeneous and has a constant cross-section, the following estimation can be given using Equation 2, Equation 3, Equation 4, Equation 5:

[00008]$.Math..Math.T_{\mathrm{Local}}\frac{\frac{\frac{U^2.Math.t_{\mathrm{heat}}}{l}}{A.Math.}}{c_{\mathrm{MH}}.Math.p_{\mathrm{MH}}.Math.A.Math.l+c_{\mathrm{AHZ}}.Math..Math.l.Math.\left({\left(r+d\right)}^2-r^2\right).Math.p_{\mathrm{AHZ}}}$

[0146] For a cylindrical conductor portion, the cross-sectional area can be calculated form the radius A=r.sup.2, so that, together with Equation 1, the following simplification is given:

[00009]$\begin{array}{cc}.Math..Math.T_{\mathrm{Local}}\frac{U^2.Math.}{l^2}.Math.t_{\mathrm{heat}}.Math.\frac{l}{c_{\mathrm{MH}}.Math.p_{\mathrm{MH}}.Math.c_{\mathrm{AHZ}}.Math.p_{\mathrm{AHZ}}.Math.\left({\left(r+\sqrt{D.Math.t_{\mathrm{heat}}}\right)}^2-r^2\right)/r^2}& \mathrm{Equation}.Math..Math.7\end{array}$

[0147] With the above equation, the temperature to which an (approximately) cylindrical conductor is heated during the heating duration t.sub.heat can thus be approximately estimated, in order to preferably be able to determine the parameters for achieving the denaturation temperature. If only a portion of the heating element is taken into consideration (for example because the heating element consists of a complex geometrical series arrangement of conductors), it is obvious that, for the voltage, only the voltage that drops over the respectively considered conductor is relevant.

[0148] The first factor

[00010]$\frac{U^2.Math.}{l^2}$

in the above equation is thereby the volumetric heating density, which is described as q below, i.e.

[00011]$q=\frac{U^2.Math.}{l^2}.$

This in turn means that the temperature increase in Equation 8 is proportional to the volumetric heating density.

[0149] If the heating element(s) is/are for example designed as wires from gold (Au) with a length of the wire l=0.1 m and also a radius of the wire of r=12.5 m and the material parameters

[00012]$p_{\mathrm{MH}}=p_{\mathrm{Au}}=19200.Math.\frac{\mathrm{kg}}{m^3},.Math.c_{\mathrm{MH}}=c_{\mathrm{Au}}=125.Math.\frac{J}{\mathrm{kg}.Math..Math..Math.C.}.Math..Math.\mathrm{and}$$={}_{\mathrm{Au}}=4.6.Math.{10}^7.Math.\frac{A}{\mathrm{Vm}},$

based on a voltage of U=10.5V spacing before V and a heating duration of t.sub.heat=200 s according to Equation 7 this results in a local temperature increase on the heating element of T.sub.local14.4 C. (when using

[00013]$p_{\mathrm{AHZ}}=p_{H_2.Math.O}=1000.Math.\frac{\mathrm{kg}}{m^3},.Math.c_{\mathrm{AHZ}}=c_{H_2.Math.O}=4200.Math.\frac{J}{\mathrm{kg}.Math..Math..Math.C.}.,$

which are typical values for the reaction volume contained in the heating-up zone). From the operating parameters used, the volumetric power and heating density q can be calculated with

[00014]$q=\frac{U^2.Math.}{l^2}5.Math.{10}^{11}.Math..Math.W/m^3,$

so that a comparison with the results of the finite element simulations in FIG. 2B is possible. This shows, after 200 s heating durations for a cylindrical heating wire at the said heating density of 5.Math.10.sup.11 W/m.sup.3, a heating of just about 17 C.

Provision of the Electrical Power and Energy Density

[0150] In a preferred embodiment of the invention, in at least one, preferably in at least three, particularly preferably in at least 10, particularly preferably in at least 20 of the passages of the amplification cycle of the PCR, the average volumetric power density of the heating means is greater than 10.sup.9 W/m.sup.3, preferably greater than 10.sup.10 W/m.sup.3, particularly preferably greater than 10.sup.11 W/m.sup.3 It is an achievable advantage of this embodiment of the invention that the heating element is heated sufficiently quickly even with short duration of the heat pulse.

[0151] In a preferred embodiment of the invention, in at least one, preferably in at least three, particularly preferably in at least 10, particularly preferably in at least 20 of the passages of the amplification cycle of the PCR, the average specific power density of the heating means is preferably less than 10.sup.16 W/m.sup.3, particularly preferably less than 10.sup.15 W/m.sup.3 and particularly preferably less than 10.sup.14 W/m.sup.3. With this embodiment of the invention, damage to the heating elements can advantageously be avoided.

[0152] In the case of a heating resistor being used as a heating element, the specific power density q, which is generated in the heating element, is given by

[00015]$q=\frac{U^2.Math.}{l^2}$

i.e. a voltage U that is as high as possible, a conductivity that is as high as possible and a short length l, over which the voltage drops in the heating element lead to a high specific power density. The entire electrical power requirement resulting for the provision of the heat pulses is thus calculated from the required volumetric power density and the combined volume of all heating elements of the heating means, wherein it depends, inter alia, upon the reaction volume that is to be processed.

Global Heating of the Sample Through the Local Heating Step for the Denaturation

[0153] In a preferred embodiment of the invention, current pulses through the heating means are selected so that only the heating means and the reaction volume in the direct vicinity of the surface of the heating means are significantly heated, thus a merely local heating takes place. The amount of heat Q brought in the course of the whole denaturation step is produced locally in the heating means and is distributed initially over the heating means itself and the direct vicinity thereof, as the discharge of the heat through diffusion takes place only gradually, as explained below. This means that the amount of heat Q brought is an amount of energy that is initially distributed over a very small volume and, in time, spreads (flows) into the surrounding reaction volume. Provided that the amount of heat (often also only described just as the heat) is still spatially concentrated in the heating element and its direct vicinity, it brings about a substantial temperature increase there. As soon as this amount of heat is distributed over an increasingly large volume, however, it also brings about a temperature increase there, but which is correspondingly smaller, as the originally brought amount of heat naturally remains constant (if only the liquid volume of the reaction volume is considered, the temperature decreases inversely proportionally to the volume, over which the amount of heat is distributed).

[0154] It is only after a certain time, hereinafter referred to as the sample thermalisation time and defined in the following paragraph, that the amount of heat is distributed to the whole reaction volume and causes a global temperature increase there. Depending on how well insulated the reaction volume is, or how well it is coupled to an external thermic tank, the brought amount of heat can remain in the reaction volume (with very good insulation, which then leads to a gradual, slight increase in the global temperature of the reaction volume with each passage of the amplification cycle of the PCR) or, in the case of good thermic thermal contacting of the reaction volume, the heat flows away, so that the reaction volume goes back to the original temperature (before the heating step). In practice, it is mostly the case that a part of the brought amount of heat flows away in the time between two denaturation steps so that the global temperature of the reaction volume increases slightly (typically less than 3 C.) over the first passages of the amplification cycle of the PCR until an equilibrium state has formed and, for each cycle, the same amount of heat is brought in as the amount of heat that flows away.

[0155] The sample thermalisation time is the time until the brought amount of heat has spread from the heating means to the whole reaction volume. The sample thermalisation time can be estimated by initially determining the point(s) at the greatest distance d.sub.max from the nearest heating element (typically, in many cases, these points lie on the surfaces that delimit the reaction volume). The sample thermalisation time is then the time taken until the heat diffusion range is equal to d.sub.max, i.e. in terms of image, until the heat that is generated in the heating elements has diffused into the last corner of the reaction volume. If, for example, the reaction volume is cylindrical with a radius of 1.01 mm and the heating element consists of a single cylindrical wire with a radius of 0.01 mm, which runs concentrically through the axis of the cylinder, the maximum distance that a point in the reaction volume can be from the nearest (in this case the only) heating element is d.sub.max=1 mm. With Equation 1, a heat diffusion range of 1 mm is produced after 6.3 s, so that in this special case the sample thermalisation time is approximately 6.3 s.

[0156] The MGTE, which is the maximum increase in the average temperature taking place through the denaturation step, can be estimated as follows from amount of heat Q and the heat capacitance that is brought through the heating step into reaction volume, with

[00016]$\mathrm{MGTE}\frac{Q}{C}.$

With the density p of the reaction volume, its volume V, its specific heat capacity c and with the aid of the correlation C=c.Math.p.Math.V it can be estimated that

[00017]$\mathrm{MGTE}\frac{Q}{c.Math.p.Math.V}.$

The less-than sign is substantiated in that the heat capacity of the heating means and the reaction container is not taken into consideration here. The density and heat capacity of the reaction volume is generally substantially that of water, i.e. p=1 g.Math.cm.sup.3 and c=4.2 J.Math. C..sup.1.Math.g.sup.1. The amount of heat Q can be determined from the electrical operating parameters: If the heat pulse t.sub.Heat continues and if the voltage U and the current I during the heat pulse are constant, then Q=U.Math.I.Math.t.sub.heat. (Insofar as current and voltage are changeable over time, the following applies:

Q=.sub.0.sup.t.sup.heatU(t).Math.I(t).Math.dt).

[0157] This means in the first case that the upper limit for the MGTE can be determined by the equation

[00018]$\begin{array}{cc}\mathrm{MGTE}\frac{U.Math.I.Math.t_{\mathrm{heat}}}{4.2.Math..Math.J.Math.{\mathrm{cm}}^{-3}.Math.V}& \mathrm{Equation}.Math..Math.8\end{array}$

(the volume V is then to be indicated in millilitres). Here, of course, only the voltage U that drops via the heating means in the reaction volume V is to be considered, and also the current I that actually flows through the heating means in the reaction volume V. (This means: the voltage drop in inlet lines would for example not have to be considered.)

[0158] The MGTE can be experimentally determined by the temperature of the reaction volume being taken before and after a single denaturation step, wherein in the latter case it is only after the sample thermalisation time that the physical measurement of the temperature in the reaction volume is carried out. The difference between the two measured temperatures is then equal to the MGTE. This procedure is advantageous according to the invention, as a complete thermalisation of the reaction volume, i.e. an even distribution of the heat in the reaction volume is ensured and the temperature sensor does not detect the temperature of the heating-up zone, for example randomly). This measurement of the MGTE can for example in practice be detected with a temperature sensor, preferably with a particularly small heat capacity of its own, which is brought into the reaction volumes.

Example of a Heating Means

[0159] FIG. 1A shows in a schematic illustration a heating element forming a heating means, which can be connected to a voltage source 2, wherein a device for generating electrical pulses 3 changes the temporal current pattern 4. Here, the device for generating electrical pulses 3 is a switch, which is open in the state shown, whereby in the temporal/time-based-related current pattern 4, no pulse can be seen. The heating element 1 is functionalised with primers 5, onto which free target nucleic acids 6 can hybridise. On the right-hand side of the drawing, the spatial progression of the temperature distribution in and around the heating element is shown. At the point in time of the open switch the heating element is not heated with respect to the surrounding liquid and the temperature profile is therefore planar.

[0160] FIG. 1B shows once again in a schematic illustration the heating element 1. After hybridisation of the primer 5 with the target 6, this primed target nucleic acid 7 can be elongated by the polymerase to form a nucleic acid double strand 8.

[0161] FIG. 1 C shows how, after the elongation of the nucleic acid, the device for generating electrical pulses 3 becomes active (the switch shown here is now the closed state), whereby in the temporal current pattern 4 an electrical pulse can be seen. This leads to a heating of the heating element 1 and the local vicinity thereof (shown in the spatial progression of the temperature distribution in and around the heating element on the right hand side of the drawing), whereby, in the case of sufficient local heating, the nucleic acid double strands are denatured and, once again, free targets and amplicons 6 are formed and primers 9 elongated on the heating element remain. Both free targets and amplicons and also the elongated primers can serve in the subsequent amplification cycles as a template for further amplification.

Simulated Temperate Patterns

[0162] FIG. 2A shows a finite element simulation for the spatial temperature pattern in a heating wire (which stands for example for a heating element) and the vicinity thereof at different point in times, namely 50, 100, 200, 400, 800 and 1600 s after the start of a heat pulse. On the vertical axis, the temperature increase is recorded in relation to the value prior to the start of the heat pulse. On the horizontal axis, the distance from the cylinder axis of the wire is recorded. It is assumed here that it is a wire made of gold with a diameter of 25 m, which is heated for 50 s with a volumetric power density of 2.Math.10.sup.12 W/m.sup.3 (corresponds according to the equation

[00019]$q=\frac{U^2.Math.}{l^2}$

to a voltage of 210 V per metre of heating wire length) in an aqueous vicinity.

[0163] It can be recognised that after 50 s a temperature increase of approximately 25.5 C. is reached on the surface of the wire, but the temperature increase already falls already after a few micrometres from the surface. After 1600 s (i.e. 1550 s after the end of the heat pulse) on the other hand the heat has already diffused around 10 m away from the surface of the heating wire and thus fills out a far larger volume. This leads to the temperature on the surface of the wire being only approximately 3 C. warmer than prior to the heat pulse.

[0164] It should be pointed out that the curve progression is scaled proportionally to the volumetric power density. This means for example that, for a 4 power density (corresponds to double the voltage with constant wire length) the temperature lift of the whole spatial temperature pattern multiplies by four.

[0165] FIG. 2 B shows a finite elements simulation of the time-based temperature pattern on different heating wires (which stand for example for a heating element) since the start of the heat pulse, i.e. the heating-up behaviour of the heating wires is shown. On the vertical axis the temperature increase is recorded with respect to the value before the start of a heat pulse. On the horizontal axis the time since the start of the heat pulse is recorded. It is assumed here that there were full wires made of gold with diameters of 10, 25, 50 and 100 m, which are heated with a volumetric power density of 5.Math.10.sup.11 W/m.sup.3 (corresponds to a voltage of 104 V per metre of heating wire length). It is to be noted here that the constant volumetric power density leads to the wires with larger diameter being heated with a higher power (as they have a larger volume), which is ultimately due to its lower electrical resistance and thus the higher current flow with constant voltage.

[0166] It can be seen that initially (in the first microseconds) the temperature increase on the wire surface increases approximately linearly with the heating duration and then above all, with small wire diameterflattens out and increases less than linearly. This effect is due to the fact that, with small diameters of the heating element, the heat capacity of the heating-up zone plays a greater role, or, in other words, that the transport carrying away removal of the heat through diffusion due to the higher volume/surface ratio for small wire diameters is of consequence at an earlier point in time. It should be pointed out that the curve pattern is scaled proportionally to the volumetric power density. This means, for example that, for a 4 fourfold power density (corresponds to double the voltage with constant wire length) the temperature reached on the surface of the wire also increases fourfold.

[0167] FIG. 2C shows a finite element simulation of a standardised time-based temperature pattern on different heating wires (which stand for example for a heating element) after the end of a heat pulse with a duration of 200 s, i.e. the cooling behaviour of the heating wires is shown. On the vertical axis, the still present standardised temperature increase is recorded with respect to the value before the start of the heat pulse. On the horizontal axis, the time since the end of the heat pulse is recorded. It was assumed here that there were full wires made of gold with diameters of 10, 25, 50 and 100 m, which had been heated for 200 s. It can be seen that, for all wire diameters, already after 10 ms the original temperature increase (at the end of the heat pulse) has fallen to a fraction. For wire diameters of 10 and 25 m, the remaining temperature increase has even decreased to less than 5% of its initial value, which shows the potential of the invention, namely in that in the case of a wire of 10 m diameter, the next heat pulse (i.e. denaturation step or PCR cycle) is possible already after 10 ms. The smaller the wire diameter is, the higher is the surface/volume ratio and the quicker the cooling takes place after the heat pulse.

[0168] FIG. 2D shows a finite elements simulation of the temporal temperature pattern of a heating element since the start of the heat pulse, i.e. its heating-up behaviour is shown. The heating element consists of a 200 nm (nanometre) thick gold film, which has been applied to a non-conductive PMMA cylinder with a diameter of 200 m. On the vertical axis, the temperature increase is recorded with respect to the value before the start of the heat pulse. The values on the horizontal axis indicate, with their amount, the distance from the cylinder axis. It was assumed here that there was a 200 nm thick cylinder sheath made of gold, which is heated with a volumetric power density at the level of 2.2.Math.10.sup.13 W/m.sup.3 (corresponds to a voltage of 692 V per metre of cylinder sheath length) for the duration of 200 s, whereas, inside the cylinder (which is made of plastic) in the same way as in the surrounding aqueous reaction volume, no heat is generated (i.e. the volumetric power density there is 0 W/m.sup.3. After 200 s of heating duration the temperature distribution (in the radial direction) is still strongly localised around the gold film and the heat cannot spread in the shortness of time in the aqueous outer region or in the middle of the cylinder. After the end of the pulse (t>200 s) the gold film cools and the heat spreads into the aqueous outer region and the inside of the cylinder. After approximately 6.4 ms it can be seen that the thermal fields generated in the cylinder sheath run together in the middle of the cylinder so that, here, the temperature initially increases in the further progression. In the outer region on the other hand it can be seen that, already after 25 ms, the temperature increase is only less than 2 C. Two advantages of the use according to the invention of thin metallic films as a heating element can be seen here, namely that they can be applied on a large surface (here a cylinder with a diameter of 200 m) and at the same time have a very small volume (and thus thermic mass), so that they cool out again almost completely in very short times (1 s).

Experimental Conversion

[0169] FIG. 3A shows the example of an electrical circuit 10 as a control device for generating electrical pulses in order to apply electrical current to the heating means.

[0170] The circuit is constructed so that between earth (GND) and U+, a voltage (in this document always indicated as U) is supplied (for example between 30 and 100 V), with which the heating means is to be heated. At the point R3 Load, the heating means is arranged, so that R3 is the resistor of the heating means. The power MOSFET Q (IRFP4468, International Rectifier), used by way of example, produces a low ohmic connection between the contact T2 and contact T3, in the connected-through, in such a way that a current flows through the heating means R3. Between earth and the gate (contact T1) of the MOSFET, a control voltage, which is provided for example by a pulse or frequency generator or a digital-analog converter, is supplied via the control terminal FET GND rt/ge. Particularly suited are pulses with a level of 5 V and a duration of for example between 10 and 1000 s, which allow a clean connection of the MOSFET. At the point C1, a capacitor with sufficient capacitance, example 4 mF, and lowest possible ESR value is provided, which allows, even with low ohmic heating meansresistance of all heating elements together typically less than 1 (Ohm)the supplied voltage to be maintained for the duration of the heat pulse. The resistors R1, R2, R7 and R9, for example, have resistance values of 1, 100, 24 and 24 k(kiloohm).

[0171] FIG. 3B shows schematically and in a simplified manner the cross-section of an embodiment of the invention, wherein the heating means is formed by portions of a continuous wire 12, which is connected to a voltage source 11. To simplify the illustration, the device for generating electrical pulses has been omitted. In addition and the drawing is not true to scale. The wire runs through a plurality of reaction vessels, separate from each other, in the form of sample liquid chambers (also known as wells) in a sample plate 13, which is located between a two-part temperature-regulating block 14. The temperature-regulating block 14 has the function of bringing the reaction volume in the sample liquid chambers to hybridisation/elongation temperature and holding it there. In the embodiment shown here, in the lower part of the temperature-regulating block 14 below each sample liquid chamber, there is an excitation light source (in this case in the form of a light emitting diode 15 with an optical low pass filter) for exciting a dye in the respective reaction volume, and, in the upper part of the temperature-regulating block 14 over/above each sample liquid chamber, there is a photodiode 16 as a light sensor for detecting the fluorescence of the excited dye in the respective reaction volume (with an optical high pass filter, which allows fluorescent light to pass through).

[0172] FIG. 3C shows schematically and in a simplified manner the cross-section of a further embodiment of the invention, which differs from the exemplary embodiment of FIG. 3B in that the heating elements are designed as coils composed of a wire 12 connected to a voltage source 11. To simplify the illustration, the device for generating pulses has also been omitted here. The heating elements in the form of wire wound up into coils are in contact with the reaction volume in the respective reaction vessel. Contrary to how they are shown in the figure, they are preferably completely surrounded by the reaction volume. The reaction vessels in this exemplary embodiment are a plurality of sample liquid chambers, separated from each other, in the form of reaction tubes, which are located in a temperature-regulating block 14 in order to bring the reaction volumes to hybridisation/elongation temperature and to keep them there. In the embodiment shown here, in the lower part of the temperature-regulating block 14 below each sample liquid chamber, there is a light emitting diode 15 as an excitation light source for exciting a dye in the reaction volume, and, above each sample liquid chamber, there is a photodiode 16 as a light sensor for detecting the fluorescence of the excited dye in the reaction volume.

Production of a Sample Plate with Wire Heating Elements

[0173] FIG. 3D shows schematically components, from which a sample plate of a device according to the invention can be created with wire heating elements. The heating elements used here are portions of a gold-plated sheathed wire 12 of 25 m in diameter (23 m tungsten core with approximately 1 m gold sheath, LUMA METALL AB, Kalmar, Sweden). This is wound around an acrylic glass plate 17 having a thickness of 0.5 mm (middle, lighter plate). In the plate there are seven openings (6 mm6 mm), through which the sample liquid chambers (wells) are defined. Through the winding, there are two parallel layers in each sample liquid chamber, each layer having fifteen parallel heating elements; the two layers of the heating means are at a distance of 0.5 mm due to the plate, the heating elements within a layer have a distance of approximately 0.4 mm. From both sides, by means of doubled-sided adhesive foils 18 (shown darkened, 100-250 m thick VHB adhesive tape of 3M) with corresponding receptacles for the sample liquid chambers, a further acrylic glass plate 19 (thickness of the lower plate 0.5 mm and thickness of the upper plate 3 mm) with equal openings is stuck to the plate 17, and pressed according to the manufacturer's indications of the adhesive tape 18. From below, the wells are each closed with a thin foil 20 (lighter in the illustration, adhesive PCR foil seal, 4titute), which are stuck to the bottom acrylic glass plate. In this way a sample plate with seven wells is formed, through which parallel wires 12 can be pulled. The wires 12 are connected to each other at the two outer-lying ends of the plate (i.e. all wires/heating element are connected in parallel) and in electrical contact. It is made possible in this way for current pulses to be sent in series through all the wells. The openings of the sample plate (at the top here) can then be closed with a thin foil 21 (shown in a light colour). The sample plates have a width of 20 mm and a length of 90 mm (so that the voltage of the heat pulses drops essentially over a length of approximately 96 mm if the 3 mm overhang of the wires at the ends are considered, which are required for contacting purposes).

Measuring the Global Temperature Increase

[0174] FIG. 3E shows the result of a measurement of the global temperature increase in a reaction volume of an exemplary embodiment of the invention according to FIG. 3D.

[0175] The wells of a sample plate produced as described in FIG. 3D are each filled with 100 l of water. A PT100 sensor as a temperature sensor is additionally inserted into one of the middle wells. If a constant current of 3 A is sent through the wires, a temperature increase of approximately 47 C. is observed over a time of 200 s in the water sample (curve with hashes). If on the other hand, as provided according to the invention, current pulses of 80 A are sent with 70 s duration through the wires every 3 s (voltage 32 V supplied, load resistance the wires, 0.4), merely a global temperature increase of the water sample of less than 1 C. can be measured with this measurement method (curve with circles).

PCR with Genomic Nucleic Acid and Background

[0176] FIG. 4 shows the results of polymerase chain reactions with genomic nucleic acid and control measurements, respectively in a reaction volume of an exemplary embodiment of the invention according to FIG. 3D. The heating elements in the wells of a sample plate produced as described in FIG. 3D are functionalised with forward primers. For the functionalisation, the forward primer ID1 is used. Thiol serves for bonding to the gold surface, the first five thymine bases serve as a spacer sequence in order to obtain more space, or distance, between an actual primer sequence and gold surface and thus to prevent for example possible steric obstacles hindrances xx. Prior to functionalisation, the protecting group of the thiol modification of the thus formed oligonucleotide is de-protected by the oligonucleotide being incubated in a concentration of 0.5 m in PBS buffer (5 mM phosphate buffer, 10 mM NaCl, 0.01% Tween20, 1 mM EDTA, pH 7.5) for 15 min. with 1 mM tris-(2-carboxyethyl)phosphine is incubated. Subsequently NaCl (5M) is to be added in order to reach a final concentration of NaCl of 500 mM for the functionalisation. After 3 hours of incubation of the wires with a suspension of the de-protected oligonucleotides, 2 washing steps with PBS buffer are carried out, in order to remove excess non-bonded oligonucleotides. The plates prepared in this way are now available for the amplification reaction.

[0177] The amplification reaction was carried out in 90 l total volume per well. The reaction mix consists of 36 L, H.sub.2O; 9 L MgCl.sub.2 120 mM; 18 l 5 Aptataq genotyping master (Roche); 9 l reverse primer ID2 5 m; 9 L TaqMan probe, oligo ID3 2 m. Added to this, is 9 l sample, which, depending on the well, contains either boiled genomic nucleic acid or only water. Forward and reverse primers and also the TaqMan probe were selected so that the resistance gene MecA is amplified and detected, whereby this arises for example in the genome of the methicillin resistant Staphylococcus aureus (MRSA).

[0178] The plate is placed between two temperature-regulating blocks made of aluminium, the temperature of the lower block is 65 C. and the temperature of the upper block is 70 C. The temperature difference serves for avoiding condensate formation on the upper covering foil. The heating means comprising the heating elements is arranged corresponding to FIG. 3A at the position R3 of the circuit, which is operated here with a voltage of U+=32 V. The wires form a load resistance of 0.4, through which, during the following PCR, electrical pulses with the length of 70 s are sent every 3 s.

[0179] For the real-time detection in TaqMan format, there are excitation light emitting diodes in the lower temperature-regulating block, with a corresponding bandpass filter with 478 nm central wavelength and FWHM of 29 nm (ET480/30, Chroma Inc, USA) and, also in the upper temperature-regulating block, photodiodes and optical filters with a passage area of 515-700 nm (ET510lp, Chroma Inc. USA).

[0180] The result of the above-described PCR can be seen in FIG. 4, where the percentage change of the fluorescence signal I during the PCR process (with respect to the fluorescence base signal I.sub.0 at the start of the PCR) is shown. In the first well, 10.sup.6 copies of genomic nucleic acid isolated from MRSA were used as a sample. As this genomic nucleic acid contains the MecA gene and thus the target sequence of the PCR, the corresponding fluorescence curve (solid curve) increases after approximately 190 s PCR duration, as at this time a sufficiently large amount of amplicon has formed (and thus, through exonuclease activity of the polymerase, a sufficiently large amount of FAM dye has been released from the TaqMan probe), in order to generate a signal that can be differentiated from the base fluorescence. In the second well, 100 less target nucleic acid from the same organism was used, the corresponding fluorescence curve (roughly broken curve) increases later here, from 220 s PCR duration onwards. The method thus allows (as is usual in the qPCR) a correlation between target concentrations used and the time of the increase of the fluorescence curve. In the third well, in addition to the reaction mix, only water was added as a negative control (finely broken curve), in the fourth well 10.sup.6 copies of genomic nucleic acid isolated from Escherichia coli (E. coli, of which the genome does not contain the MecA gene) was used as a sample in order to demonstrate the specificity of the PCR (dotted line). Neither of the fluorescence curves (water: finely broken line, E. coli: dotted line) shows a significant increase. In the fifth well, finally, both 10.sup.4 copies of genomic nucleic acid isolated from MRSA and also 10.sup.4 copies of genomic nucleic acid isolated from E. coli were used as samples. The corresponding fluorescence curve (dashed-dotted curve) behaves like the fluorescence curve of the sample from the second well, which contains only 10.sup.4 copies of genomic nucleic acid isolated from MRSA. This shows that the presence of E. coli nucleic acid does not supress the amplification of the MRSA nucleic acid. In addition, in a comparable amplification experiment, an external quantification of the amplicon amount produced was carried out with the aid of a conventional thermocycler (Roche Lightcycler 1.5). For this, the above-described PCR process was interrupted shortly after recognisable increase in the fluorescence curve, the sample was removed, 1:100 diluted with water and 1 l of this diluted sample was inserted into a qPCR, which uses the same primer sequences (forward primer here but without thiol and 5T spacer sequence), as in the case of the PCR described above. The comparison with a dilution series sequence of known target concentrations led to the result that at the point in time of the clear signal increase, there was an amplicon concentration of approximately 3 nM (nanomolar, or nanomol per litre) in the reaction. This is also a typical area for the signal that can be differentiated, fluorescence base all in the case of other thermocyclers, and this additionally clearly shows that during the PCR carried out, the desired amplicon was produced in very large amount. The comparison with FIG. 3E shows that, under the given conditions (supplied voltage 32 V, load resistance of wires 0.4), only a global temperature increase of the reaction volume of <1 C. can be detected within 200 s can be detected, and, therefore, reaching the denaturation temperature (approximately 95 C.) of the reaction volume can be ruled out. Through the functionalisation with thiol-modified forward primer, however, the amplicon is still bonded to a heating element. Obviously, the temperature there locally during the heating steps and during the electrical pulses is sufficiently high in order to achieve a denaturation of the amplicon. (Not shown here: If free unmodified forward primers with sequence ID4, which are added to the reaction mix but do not bond to the wires, are used instead of the thiol-modified forward primers, but under otherwise identical conditions, no amplification can be observed. The amplification reaction is thus, in the case of local heating, preferably localised on a locally heating element, whereby the forward primer is immobilised on the heating element).

Measurements in the Case of Different Volumetric Power Densities

[0181] FIGS. 5A to 5C show, with the aid of exemplary embodiments of the invention, the dependence of the PCR performance upon the supplied voltage, which changes the volumetric heating density according to the equation

[00020]$q=\frac{U^2.Math.}{l^2}.$

Here, a measurement series with different voltages supplied to the heating means was carried out. The parameters and reaction mix compositions are identical to those in the exemplary embodiment of FIG. 4, whereby merely by way of sample in a well, water was used as a negative control (broken fluorescence curve) and also, in two wells, a synthetic target nucleic acid with the sequence ID5 was used, so that the final concentration at the start of the reaction was respectively 100 fM (Femtomolar or Femtomol per litre) of the target nucleic acid (positive controls with solid fluorescence curves). The target nucleic acid is the cut-out from the MecA gene, which can be amplified by the selected primer pair. In the first measurement (fluorescence curves in FIG. 5A) the supplied voltage is 26 V and hardly any signal lift can be seen. In the second measurement (fluorescence curves in FIG. 5B) the supplied voltage is 28 V, the positive controls here show a weak, flatly increasing, late arising signal. In the third measurement (fluorescence curves in FIG. 5C) the voltage is 36 V, the two positive controls here increase clearly and sharply steeply with effect from approximately 180 s PCR duration, the negative control does not thereby show a signal. This measurement series shows that the heat brought by the heating means into the reaction volume must be sufficiently great in order to reach the required denaturation temperature of the amplicon locally on the heating elements.

[0182] FIGS. 5D to 5G show the results of a series of measurements, in which the sample plates were produced precisely in the manner as set out above in the description of FIG. 3D. However, in each sample plate, a different number of wires was used. In FIG. 5D, 3 wires were used, in FIG. 5E, 5 wires were used, in FIG. 5F, 10 wires were used and, in FIG. 5G, 30 wires were used (connected in parallel over the length of the sample plate: resistances of 3700, 2100, 2210 and 400 m (milliohms). To carry out the PCR, corresponding chemicals and buffers were used as in the text section relating to FIG. 4. To generate the heat pulses, one of voltage U+=40 V, a pulse duration of 40 s and a pulse repetition rate (i.e. PCR cycle duration) of 3 s was used. The temperature of the lower temperature-regulating block was brought at to 63 C. and that of the upper block at 68 C. By way of target nucleic acid, synthetic DNA ID5 was used, which was present at the start of the reaction in a concentration of 1 fM (solid line). No target was used in the negative controls (broken line). It can be seen that, when using only three wires in the sample plate (first measurement, fluorescence curves in FIG. 5D), even in the positive control, no signal lift (i.e. no TaqMan signal, i.e. no amplification) can be seen. In the case of five wires in the sample plate (FIG. 5E), the positive control exhibits a weak signal. In the case of ten wires in the sample plate (FIG. 5F), the signal is significantly higher, but still significantly weaker than in the fourth measurement, where a usual number of wires (namely 30 is used (FIG. 5G).

Heating Means with Honeycomb Structure

[0183] FIGS. 6A to 6C show an embodiment of the heating means in honeycomb structure.

[0184] For the production thereof, a honeycomb structure is produced through photochemical fine etching methods from a stainless steel foil, and subsequently the honeycomb structure is coated with gold. In the exemplary embodiment, it is a hexagonal lattice, but naturally other lattices are also conceivable. The current flows through the structure along the length thereof xx, wherein, as shown in FIG. 6A, only in the area of the sample chambers, thus where the foil forms the heating elements, was a honeycomb structure etched. The length f of a heating element is for example 8.2 mm, the distance g between the heating elements is for example 3.8 mm. The sample chambers are preferably arranged centrally above the heating elements and preferably have smaller dimensions (for example 6 mm6 mm) in order to use only the area of the heating elements, which is tempered as evenly as possible. The whole length h of the foil is for example 100 mm, i.e. the electrical contacting takes place at the short sides, so that the voltage drops over a length of approximately 100 mm. The webs of the honeycomb structure heat up due to the current passing through them and can denature the double-stranded nucleic acid bonded to them.

[0185] FIG. 6B is an enlarged illustration of the honeycomb structure of a heating element of FIG. 6A with adjacent edge. In the example honeycomb structure, the web widths are configured so that overall in the honeycomb structure a current density that is as even as possible and thus volumetric heating density is achieved. In the exemplary embodiment this is achieved in that the width d of the longitudinal webs is precisely double the width b of the transverse webs. Examples for the dimensions are 0.87 mm for the honeycomb diameter a, 0.065 mm for the width b of the transverse webs, 0.5 mm for the web length c, 0.13 mm for the width of the longitudinal webs d and 0.57 mm for the width of a long edge 3. The long edge a serves, above all, for the mechanical stability and experiences a different current density than the honeycomb structure.

[0186] FIG. 6c shows, as in FIG. 4, the percentage change of the fluorescence signal I during the PCR process (with respect to the fluorescence base signal I.sub.0 at the start of the PCR), wherein now, in contrast with the example of FIG. 4, the heating element used is a stainless steel foil coated with approximately 0.5 m gold (foil thickness 20 m) was used with the structure shown in FIGS. 6A and 6B. This gold-plated lattice is stuck between two acrylic glass plates with seven well openings, as were also used for the exemplary embodiment in FIG. 3D (thickness of the lower plate 0.5 mm and thickness of the upper plate 3 mm). There is thus also here, as already in the exemplary embodiment in FIG. 3D, a sample plate, which is traversed by the heating means. The honeycomb structures are electrically contacted at the two outer-lying ends of the plate. It is thereby made possible for current pulses to be sent in series through all wells. The openings of the sample plate can subsequently be closed with a thin foil. The functionalisation of the honeycomb structures is realised similarly to that of the tungsten-gold sheathed wires of FIGS. 3D and 4. The reaction volume is now 60 l per well, the composition and concentrations of the reaction mix correspond to those of FIG. 4. The voltage supplied is 41 V with load resistance of 0.35. Here, electrical pulses with the duration of 150 s were sent through the heating elements during the PCR every 10 s. As stainless steel is a significantly poorer current conductor than tungsten, from which the core of the sheathed wire is made in the previous examples, when using heating elements of stainless steel in this example, both a higher voltage and also a longer heating duration are used. In FIG. 6C, the fluorescence curves from three wells can be seen. The negative control with water as a sample does not show a signal increase (dotted fluorescence curve), the sample with a high initial concentration (100 fM) of synthetic nucleic acid target with the sequence ID5 shows an increase of the fluorescence signal with effect from 400 s PCR duration (solid fluorescence curve) and the sample with a lower starting concentration (1 fM) of synthetic nucleic acid target with the sequence ID5 shows a later increase of the fluorescence signal (broken fluorescence curve).

Bridge PCR

[0187] In the exemplary embodiment of FIG. 7, both primers (forward primer and reverse primer) are on the surface of the heating means. The procedure for this exemplary embodiment corresponds essentially to that for the exemplary embodiment of FIG. 4.

[0188] For the functionalisation of the heating means, however, a mixture of one part of forward primer ID6 and three parts of reverse primer ID7 was used (total concentration of the two primers during the de-protection 0.5 m). Both primers carry a thiol modification, which serves for immobilisation on the surface of the heating means. The forward primer carries, in addition to a spacer sequence, also two abasic modifications spacer 9 between spacer sequence and primer sequence, which prevents the overwriting of the spacer sequence by the polymerase. As the reverse primer is already present on the heating element, it no longer needs to be present in the reaction volume; the correspondingly missing volume is replaced there by water. The fluorescence curves in FIG. 7 who the signal pattern progression for a negative sample with water (broken line) and a positive sample with a starting concentration of 100 fM of synthetic nucleic acid target with the sequence ID5 (solid line). Indeed the fluorescence signals here are significantly smaller than in the preceding exemplary embodiments, but with effect from 450 s PCR duration onwards, it can also be clearly seen here that the signal of the positive sample increases in comparison with the negative sample. This exemplary embodiment illustrates that a PCR process, of which the primers are both fixed on a surface (Bridge PCR, see Kawashima et al., WO 1998/044151 A1 and Adams et al., U.S. Pat. No. 5,641,658 A) functions in the method according to the invention.

[0189] The features disclosed in the above description, the claims and the drawings, can be of importance both individually and also in any desired combination for the realisation of the invention in its different embodiments.

LIST OF REFERENCE SYMBOLS

[0190] 1 Wire-form heating element of the heating means [0191] 2 Voltage source [0192] 3 Device for generating electrical pulses [0193] 4 Illustration of the time-based current progression [0194] 5 Primer [0195] 6 Free target nucleic acid [0196] 7 Target nucleic acid bonded to a primer [0197] 8 Nucleic acid double strand [0198] 9 Elongated primer [0199] 10 Electrical circuit [0200] GND Earth connection of the electrical circuit [0201] U+ Voltage supply connection of the electrical circuit [0202] Q1 MOSFET of the electrical circuit [0203] T1 Gate terminal of the MOSFET Q1 [0204] T2 Drain terminal of the MOSFET Q1 [0205] T3 Source terminal of the MOSFET Q1 [0206] C1 Capacitor of the electrical circuit [0207] FET GND Control terminal of the electrical circuit [0208] R1 Resistor of the electrical circuit [0209] R2 Resistor of the electrical circuit [0210] R3 Heating means [0211] R7 Resistor of the electrical circuit1 [0212] R9 Resistor of the electrical circuit [0213] 11 Voltage source [0214] 12 Wire [0215] 13 Sample plate [0216] 14 Temperature-regulating block [0217] 15 Light-emitting diode [0218] 16 Photodiode [0219] 17 Acrylic glass plate [0220] 18 Double-sided adhesive films [0221] 19 Acrylic glass plate [0222] 20 Thin film (bottom) [0223] 21 Thin film (top)