WO2005094981A1

WO2005094981A1 - Cyclic pcr system

Info

Publication number: WO2005094981A1
Application number: PCT/EP2004/050392
Authority: WO
Inventors: Eric Gottwald; Tobias Preckel
Original assignee: Agilent Technologies, Inc.
Priority date: 2004-03-29
Filing date: 2004-03-29
Publication date: 2005-10-13

Abstract

A cyclic reactor system for amplifying nucleic acids using the polymerase chain reaction (PCR) process is provided, wherein the reaction chamber (l) is partitioned in reaction chamber portions (2a,2b,2c). The entity of all reactor chamber portions (2a,2b,2c) is arranged in a cyclic manner thereby providing a circular fluid flow path (12), which permits a continuous and circulating processing of the amplification. This reactor system is operatively connected to an analysis device (15) and allows nearly real-time analysis without interrupting the synthesis process. Furthermore, an embodiment of the cyclic reactor system is chip sized and therefore can be integrated with a plurality of cyclic reactor systems on a PCR reactor chip, which provides additionally an analysis device (15) in an analysis level (117), and fluid support sources in a support level (19). A method for amplifying DNA using the polymerase chain reaction (PCR) is conducted by the use in this reactor system.

Description

DESCRIPTION CYCLIC PCR SYSTEM

[0001 ] The present invention relates generally to the field of devices for conducting biochemical and chemical processes, more particularly it relates to a novel reactor design - respectively reactor system - for conducting nucleic acid amplification using the polymerase chain reaction, or "PCR" BACKGROUND ART

[0002] Small dimensions of chemical and biochemical apparatus are still objects of development engineers in the analytical instruments fabricating industry Miniaturized sizes provide systems requiring a small volume of the agents, respectively samples and solvents, which is an important advantage when the sample material is rare and/or expensive Science is still aiming at apparatus and instruments helping to improve the performance of synthesis and analysis with respect of an efficient time/money to product ratio Thus the reduction of the number of experiments basing on a custom tailored analysis, again based on support with sampling matenal that frequently has to be synthesized before, is still leading to the development of promising approaches An example of a mmiatuπzed analysis system can be found in US 5,571 ,410

[0003] The invention disclosed herein generally refers to the field of bioanalysis, respectively biosynthesis, more precisely to the field of genomics One of the most important techniques in this field is the polymerase chain reaction (PCR) As a result of this powerful tool, it is possible to start with otherwise undetectable amounts of DNA and create ample amounts of the material for subsequent analysis (The technique is descπbed in U S Pat No 4,683,195 to Mulhs et al and related U S Pat Nos 4,683,202, 4,800,159, and 4,965,188 to Mul s et al ) Automated systems for performing PCR are known, as descπbed, for example, in U S Pat Nos 5,333,675 and 5,656,493 to Mul s et al PCR uses a repetitive series of steps to create copies of polynucleotide sequences located between two initiating sequences, the primers One starts with a template, two primers of about 15-30 nucleotides in length, PCR buffer, free deoxynucleoside tπ-phosphates (dNTP's), and thermostable DNA polymerase, then the components are mixed and heated in order to separate the double stranded DNA or to perform "denaturatioπ" During the subsequent cooling step, the pπmers are annealing to complementary sequences on single stranded DNA molecules containing the sequence for amplification Replication of the target sequence is then accomplished by the DNA polymerase, which produces a DNA strand that is complementary to the template

[0004] Now, the cycle of replicating the sequence of interest is completed To obtain a noticeable amount of the desired product, this process has to be performed repeatedly, each time doubling the number of the available double stranded DNA's and therefore leading to an exponential multiplication of the target sequence

[0005] In the prior art the process needs to be conducted in a device providing at least three temperatures, so called ther o cyclers (See US 2003/0169799 A1) The first step of denaturing is conducted at about 94 °C, while the second step, the "annealing", is performed at a temperature in the range of 40-65 °C and the third step of synthesis requires a temperature of approximately 72 °C Thus, repeating the process in order to obtain the desired amount of product means performing repeated cycling between lower and higher temperatures PCR devices withstanding the steady changes of temperature while additionally difficult conditions, as harsh reagents, extremes of pH or the like, are influencing its efficiency, are described for example in US 6,613,560 B1 to Tso et al

[0006] All of the above-descπbed systems operate batch-wise This means, that the repetition of performing the cycle of replicating the target sequence is determined by the reactor system Using a meander shaped PCR chip comprising a certain number of meanders, as described in WO 99/41015 A1 , permits a certain number n of repetition cycles and therefore 2" replicates of the target sequence To obtain some more product another chip has to be fed In the experimental stadium, when the perfect parameter combination is not yet determined to perform an optimal product which meets all requirements for the subsequent use, one actually does not need 2" replicates for subsequent analysis to examine the quality of the product, but less Obviously, it is desirable to provide a device permitting to perform analysis of the product being in process, while the process itself has not to be stopped In case the result of the analysis is as desired, the process ought to be continued until the foreseen amount of product has been produced When the result is not as desired the process should be stopped, thus preventing a waste of time, money and matenal DISCLOSURE OF THE INVENTION

[0007] It is an object of the invention to provide an improved amplifying of nucleic acids using the polymerase chain reaction PCR This object is solved by the independent claims Preferred embodiments are shown by the dependent claims

[0008] Embodiments of the present invention address the aforementioned needs in the art and provides a PCR reactor system for the amplification of nucleic acids

[0009] In its simplest embodiment, the invention consists of a reactor system for amplifying nucleic acids using the polymerase chain reaction (PCR) process, wherein the central improvement in comparison to conventional systems is the design of the reaction chamber, being partitioned in reaction chamber portions The entity of all reactor chamber portions is arranged in a cyclic manner thereby providing a circular fluid flow path, the components of said reactor system thus forming a cyclic PCR reactor system This reactor system design permits on the one hand to perform a continuous amplification process, the amplification steps of denaturation, annealing and replication being carried out repeatedly, on the other hand the reactor system design, providing at least one inlet port and at least one outlet port communicating with the reaction chamber, allows withdrawing of fluid at any time in order to analyze it, respectively the product being contained

[00010] An additional embodiment of the invention is the cyclic PCR reactor system shortly sketched above, being operatively connected to at least one analysis device This permits conducting an analytical examination of the withdrawn fluid in nearly realtime conditions, thus offering the fastest possibility for obtaining results regarding the quality of the product and therefore regarding the parameters defining the performance of the experiment

[00011] The embodiments of the invention given above refer to cyclic PCR reactor systems of any size.

[00012] In a further embodiment the cyclic PCR reactor system is miniaturized to the size of a chip, thus permitting to make use of the advantages of the above embodiments, additionally enabling the user of the cyclic PCR reactor system to amplify extremely small quantities of DNA educt fluid.

[00013] An additional embodiment refers to the chip sized cyclic PCR reactor system being operatively connected to an analysis device being chip sized, too. This embodiment incorporates the advantages of the embodiments listed above.

[00014] In order to conduct more than one experiment at once, the invention introduced above is integrated in a PCR reactor chip, which usually comprises a certain number of cyclic reactor systems for amplifying nucleic acids using the polymerase chain reaction (PCR) process. A synthesis level, an analysis level and a support level are operatively attached one to the other, thus forming a sandwich structure. The system allows to conduct the amplification process continuously while product fluid can be withdrawn for analysis purposes without interrupting the process, at the same time the volume difference caused by the withdrawal can be compensated by introducing fluid (sample fluid) into the system, passing the inlet port. The analysis gives quick and reliable results while the process is still running. The user can decide at any time to stop the processing, to produce more product or to change parameters. The chip described in this embodiment operates as "lab on the chip" and therefore meets the needs in modern science.

[00015] In a still further embodiment of the invention, a method is presented for amplifying the quantity of a DNA molecule of interest introducing into a reactor system sample fluid and the reagents required to carry out an amplification process under application of the polymerase chain reaction. The method can be performed in any cyclic PCR reactor systems according to one of the above embodiments, it's particularly advantageous to use the method with a PCR reactor chip, including a plurality of cyclic PCR chips in order to conduct a series of experiments at once. BRIEF DESCRIPTION OF DRAWINGS

[00016] Other objects and many of the attendant advantages of embodiments of the present invention will be readily appreciated and become better understood by reference to the following more detailed description of preferred embodiments in connection with the accompanied drawings Features that are substantially or functionally equal or similar will be referred to with the same reference signs

[00017] Fig 1 shows a plan view of a PCR reactor system,

[00018] Fig 2 shows a schematic view of a PCR reactor system, illustrating the fluid flow, and the temperature zones, [00019] Fig 3 shows a plan view of a miniaturized PCR reactor system,

[00020] Fig 4 shows a cross-sectional side view of the PCR reactor system of fig 1 ,

[00021] Fig 5 shows a view of a miniaturized PCR chip, consisting of nine reactor systems, two gel support wells and one well containing a standard sample

[00022] Fig 6 shows a perspective view miniaturized PCR chip, pointing out the "feedline" between a reactor system, gel support well and a well containing a standard sample DETAILED DESRIPTION OF THE INVENTION

[00023] Before the invention is described in detail, it is to be understood that this invention is not limited to the particular component parts of the devices described or to process steps of the methods described as such devices and methods may vary It is also to be understood, that the terminology used herein is for purposes describing particular embodiments only and it is not intended to be limiting It must be noted that, as used in the specification and the appended claims, the singular forms of "a", "an", and "the" include plural referents until the context clearly dictates otherwise Thus, for example, the reference to "an isolating element" includes two or more such isolating elements [00024] In this specification and in the claims which follow, reference will be made to the following terms which shall be defined to have the herewith explained meanings

[00025] The term "cyclic" as a part of the expression "cyclic PCR reactor system" is used herein to indicate that said reactor is provided to perform a chemical reaction, which has to undergo a set of steps, or a cycle, repeatedly to obtain the desired amount of product

[00026] A "circular" fluid flow path is a flow path forming a closed system The fluid does not necessarily have to flow along a circular shaped line, it may as well flow along a rectangular, an oval or an otherwise designed line, as long as it is circulating [00027] Referring now to FIG. 1 , the reactor system A is shown in a simple embodiment The reactor system A, which has a reaction chamber 1 being subdivided in chamber portions 2a, 2b, 2c, each having an inlet end 5 and an outlet end 6, is heated by the heater 10 An inlet port 8 is foreseen in reaction chamber 2a in order to provide a possibility for filling the reactor with fluid To introduce liquid, respectively PCR reaction components from an external source into the reaction chamber 2a via the inlet port 8, a loading means not illustrated in the drawings is used This loading means shall charge the reactor system A with liquid that is substantially free of bubbles To keep the fluid bubble free after being introduced in the reactor chamber system A, deaerating means can be integrated in the reaction chamber (Not shown in FIG 1 ) The simplest embodiment, which is not shown in the figures, might be designed with only two reactor chamber portions

[00028] An outlet port 9 is mounted at the chamber portion 2c to permit taking liquid from the reactor chamber 1

[00029] Referring to FIG 1 and FIG.2, which shows a schematic view of the reactor system A introduced in FIG 1 , it can be seen that a circular fluid flow path 12 is defined by the design of the reactor chamber 1 After being introduced into the chamber 1 via the inlet port 8, thus firstly flowing along the first part 8a of the fluid flow path, the fluid enters the first reactor chamber portion 2a, then leaves the reactor chamber portion 2a by passing the outlet end 6, and flows via a transition portion 11 into the next reactor chamber 2b by passing its inlet end 5. Now, the fluid leaves this portion by passage of outlet end 6, flows via another transition port 11 into reactor chamber portion 2c. Since there is an outlet port 9 mounted at reaction reactor chamber portion 2c, samples of the fluid can be branched off by directing a certain volume of the fluid along the last part 9a of the fluid flow path 12. The fluid remaining within the reactor chamber continues flowing circularly and thus follows the fluid flow path 12, which is defined by the arrangement of the reaction chamber portions 2a to 2c.

[00030] Of course, this embodiment may be varied in different ways: One might, for example, wish to conduct the chemical reaction in a reactor consisting of more than three reaction chamber portions, what can be realized easily. Furthermore, it's possible to mount outlet ports on several parts of the reactor chamber, only to name two more embodiments.

[00031] Since the amplification of DNA is the central process to be performed with the reactor system A described in the present invention, temperature zones are required within the reactor system in order to carry out the necessary steps of denaturation of the DNA double strand, followed by annealing and replication. For very fast reactions a number of two temperature zones might be sufficient, or future research results might teach better to conduct the reaction by the use of more than three temperature zones, but generally three temperature zones are foreseen in devices for DNA amplification.

[00032] Referring still to the above figures, heater 10 are indicated in order to provide temperature zones 14a, 14b, 14c which are controlled autonomously by temperature controllers. Heating can be done by any means suitable for this application. Thermoelements manufactured from an isolating material could be used for example. To keep the temperature once achieved constant, isolating elements 13a can be placed vertically in between two temperature zones. An isolating element 13a, 13b (see fig. 4) may surround a part or an entire reactor chamber portion or it may be placed horizontally above or below the level, which is formed by the arrangement of the reaction chamber portions. [00033] In order to cause the fluid to flow along the fluid flow path 12, a motive force has to be applied Therefore, a conventional pumping means can be used as well as a fluid pump basing on piezo technology or a fluid moving system based on electro- kinetic movement For the application of technologies like the piezo technology an elastic matenal is required or the reactor chamber has to be at least partly elastic in order to allow compression of the fluid containing body, respectively the reaction chamber 1 To prevent back stream effects as well as mixing effects, which may occur as a result of the pumping, particularly in those sections of the reaction chamber where the fluid is carried from one to another reaction step as in the transition portions 11 , fluid flow controllers can be provided A valve, for example, can serve as such a controller

[00034] FIG. 3 refers to another embodiment of the reactor system A In a miniaturized version the reactor system B has a size of about 2-3 mm, which is the appropriate size to integrate the system on a chip In the embodiments of FIG 3 three temperature zones are indicated The isolating elements 13a are placed vertically between the reactor chamber portions 2a, 2b and 2c The reaction chamber 18 is provided by a capillary that extends from the inlet port 8 to the outlet port 9 and thus forms as well the reaction chamber portions 2a, 2b and 2c as the transition portions The capillary portions, which are reaction chamber portions generally, have a larger diameter than those capillary portions, which are transition portions [00035] Referring to FIG.4, the system of any size can be operatively attached to an analysis device 15 This can be achieved by connecting an analysis means such as an electrophoresis capillary directly to the outlet port 9 An indirect connection of the analysis device 15 with the reaction chamber 1 is achieved when a feedlme 23 (see FIG 6) is placed between the outlet port 9 and the analysis means, which is, for example, an electrophoresis capillary The direct connection as well as the indirect connection with the analysis device 15 allows withdrawing product of the reaction chamber at any time and without wasting time The taken fluid is analyzed immediately and thus the combination of synthesis device a nd analysis device 15, particularly when both are chip sized and therefore the ways inherent the system are very short, thus allowing to conduct experiments nearly within real time conditions [00036] Referring to FIG. 5, an embodiment of a PCR reactor chip C is shown Of course, the PCR reactor chip can be designed as a micro fluidic chip, which comprises then at least one inlet permitting the introduction of fluid and at least one micro fluidic channel, which stands in fluid communication with the reaction chambers [00037] The embodiment of the chip shown in FIG. 5 comprises a number of nine cyclic PCR reactor systems B for amplifying DNA Herein the entity of the substantially horizontally arranged components of said reactor systems compose a synthesis level 16 which is operatively attached to an analysis level 17 being composed of substantially horizontally arranged components of an analysis device 15 The synthesis level 16 is operatively attached to a support level 19 The support level 19 compπses support sources such as wells 20 containing DNA sample fluid, gel or other chemical reagents containing wells 21 or DNA standard solution containing wells 22 Normally, at least two different solution containing wells will be provided In order to perform the reaction and to keep it running, each reactor system B is charged with fluid deriving from at least one of the wells 20, 21 or 22

[00038] Referring to FIG.6, which shows the functioning of the PCR reactor chip C in closer detail, the fluid is carried via feedline 24 from a supporting well into a PCR reactor system B

[00039] Still referring to FIGS 5 and 6, the analysis 17, synthesis 16 and support levels 19 are located horizontally one upon another, thus forming a sandwich structure The sandwich structure is a perfect design to place isolating layers which are consisting of isolating elements 13b (figure 4) between two of the chip constituting levels and therefore this design guarantees an optimal temperature control Furthermore the small dimensions of each of the reactor systems B and the small size of the entire PCR reactor chip C provides a temperature sensitive system which allows to adjust the temperature setting of the temperature zones 14a, 14b, 14c in steps of at least 0 1 °C

[00040] The method of embodiments according to the present invention for amplifying the quantity of a DNA molecule of interest, contained in a volume of sample fluid using the polymerase chain reaction process, can be performed in any reactor system according to the claims appended and according to the accompanying figures The sample fluid which contains the DNA molecule of interest in double stranded form, a first and a second primer molecule complementary to opposing strands of the DNA molecule, thermostable DNA polymerase, deoxynucleoside tnphosphate and PCR buffer, is introduced via an inlet port into the reactor system which comprises a reaction chamber consisting of at least two reaction chamber portions having an inlet and an outlet end, each of them constituting a temperature zone The fluid that is introduced in the reactor system can derive from an external source, or from a support well as shown in FIG 5 [00041] The introduction into the system has to be performed carefully in order to avoid bubbles to enter the system A loading means helps to carry out the charging of the reactor In case bubble building has not been avoided completely, the fluid may be deaerated by the use of deaeratmg means 25 which are foreseen in the device Once introduced, the fluid circulates continuously through the reaction chamber portions and the transition portions provided adjacent to the reactor chamber portions According to the conditions existing in each reaction chamber portion, the amplification process proceeds

[00042] After once having passed each temperature zone, one amplification cycle is completed If it is desired to control the quality of the product at this early stage or at any later stadium, parts of fluid volume can be branched of the circulating substance Depending on the status of the expeπment one may as well withdraw the entire volume by permitting it to exit the synthesis level and to enter the analysis device, which is part of the analysis level in the embodiment of a PCR reactor chip The fluid can be carried via a feedline from the outlet port to the electrophoresis capillary and to an analysis device This arrangement is useful when several expeπments are earned out at the same time and have to be detected respectively analyzed by one and the same analysis device, as it's the case on a PCR reactor chip The fluid can be analyzed by use of an analyzing method, which may be based on electrophoresis, mass spectrometry or else During the process, the fluid can be moved by a pump, for example basing on piezo technology, or it is moved by a fluid moving system based on electro-kinetic technology To avoid turbulences in the reaction chamber portions, which can occur when a pumping method is applied, the fluid flow can be controlled within a transition portion by installing valves or the like in these sections. In order to provide the appropriate temperatures for denaturation, annealing and replication of the DNA strand, the temperatures of the temperature zones are adjusted in steps of at least 0.1 °C.

Claims

CLAIMS A reactor system for amplifying nucleic acids using a polymerase chain reaction - PCR - process, comprising at least one reaction chamber (1 ) comprised of at least one first and at least one second reaction chamber portions (2a, 2b), each reaction chamber portion

(2) having an inlet (5) and an outlet end (6) and constituting a temperature zone (14a, 14b, 14c), wherein at least one transition portion (11) is provided between adjacent reaction chamber portions (2a, 2b, 2c), and wherein the entity of all reactor chamber portions (2a,2b,2c) is arranged in a cyclic manner thereby providing a circular fluid flow path (12), the components of said reactor system thus forming a cyclic PCR reactor system The reactor system of claim 1 , wherein the reaction chamber (1 ) comprises at least one inlet port (8) and at least one outlet port (9) communicating with the reaction chamber (1 ), the inlet port (8) enabling the passage of a fluid into the first reaction chamber portion (2a) and the outlet port (9) permitting withdrawal of fluid from at least one of at least one second reactor chamber portion (2b), therefore defining a fluid flow path (12) from said first reaction chamber portion (2a) to said at least one second reaction chamber portion (2b) The reactor system of claim 1 or any of the above claims, compπsi ng a heater (10) adapted for heating the temperature zones (14a,14b,14c) The reactor system of the above claim, wherein a temperature controller controls the heater (10) The reactor system of claim 1 or any of the above claims, comprising a source for moving the fluid along the fluid flow path (12) The reactor system of claim 1 or any of the above claims, comprising at least one outlet for withdrawing fluid from the outlet port (9) The reactor system of claim 2 or any of the above claims, wherein the fluid derives from an external fluid support source The reactor system of claim 1 or any of the above claims, wherein the cyclic PCR reactor system is operatively connected to at least one analysis device (15) The reactor system of claim 1 or any of the above claims, wherein the cyclic PCR reactor system is chip sized The reactor system of claim 1 or any of the above claims, wherein the components of said reactor system are substantially horizontally arranged on a synthesis level (16) and wherein the components of the analysis device (15) are arranged horizontally on an analysis level (17) The reactor system of claim 8 or any of the above claims, wherein said outlet is adapted to be connected with the analysis device (15) in order to permit fluid to exit said synthesis level (16) for entering said analysis level (17) The reactor system of claim 8 or any of the above claims, wherein the analysis device (15) is a detection instrument based on electrophoresis The reactor system of claim 1 or any of the above claims, wherein the reaction chamber (1 ) comprises at least one deaerating means (25) to deaerate the fluid The reactor system of claim 1 or any of the above claims, wherein the reaction chamber (1) is comprised of a capillary extending from said inlet port

(8) through at least two temperature zones to said outlet port (9) The reactor system of claim 1 or any of the above claims, wherein said reaction chamber portions (2a,2b,2c) are first capillary portions and said transition portions (11 ) are second capillary portions wherein the first capillary portions have a larger diameter than the second capillary portions The reactor system of claim 4 or any of the above claims, wherein the temperature controller is adjusting the temperature setting in steps of at least 0 1 °c The reactor system of claim 1 or any of the above claims, wherein isolating elements (13a) for isolating the reaction chamber portions (2a,2b,2c) are provided The reactor system of claim 1 or any of the above claims, wherein at least one first isolating element (13a) is at least partly surrounding said reaction chamber portions (2a, 2b, 2c) included in a temperature zone (14a, 14b, 14c) The reactor system of claim 1 or any of the above claims, wherein at least one second isolating element (13b) is placed horizontally between the synthesis level (16) and the analysis level (17) The reactor system of claim 5 or any of the above claims, wherein the source for moving the fluid is a fluid pump basing on piezo technology The reactor system of claim 5 or any of the above claims, wherein the source for moving the fluid is a fluid moving system based on electro-kinetic movement The reactor system of claim 1 or any of the above claims, wherein at least one transition portion (11) comprises at least one fluid flow controller The reactor system of claim 22 or any of the above claims, wherein said fluid flow controller is a valve The reactor system of claim 8 or any of the above claims, wherein said analysis device (15) comprises an electrophoresis capillary The reactor system of claim 1 or any of the above claims, wherein said analysis device (15) comprises a feedline (23), carrying fluid from the outlet port (9) to the electrophoresis capillary A PCR reactor chip, comprising at least one reactor system of claim 1 or any of the above claims, wherein the PCR reactor chip is a micro fluidic chip, comprising at least one inlet port (8), at least one outlet port (9), at least one reaction chamber (1 ) and at least one micro fluidic channel, wherein the micro fluidic channel is in fluid communication with the reaction chamber (1) The PCR reactor chip of claim 26 or any of the above claims, wherein a detection area is provided The PCR reactor chip of claim 26 or any of the above claims, wherein

- the entity of the substantially horizontally arranged components of said reactor systems composes a synthesis level (16),

- the synthesis level (16) is operatively attached to an analysis level (17) being composed of substantially horizontally arranged components of at least one analysis device (15),

- said synthesis level (16) is operatively attached to a support level (19) comprising at least one fluid support source, and wherein said analysis (17), synthesis (19) and support levels(19) are located one upon another, said analysis level (17) preferably being the bottom level and the support level (19) preferably being the top level, with isolating elements (13a, b) placed in between two of said levels, the aforesaid arrangement resulting in a chip design The PCR reactor chip of claim 26 or any of the above claims wherein said outlet is adapted to be connected with the analysis device (15) in order to permit fluid to exit said synthesis level (16) for entering said analysis level (17) The PCR reactor chip of claim 26 or any of the above claims, wherein at least one first fluid support source is a well which contains samples (20), one second fluid support source is a well which contains chemical reagents (21 ) such as gel and one third fluid support source is a well which contains a DNA standard fluid (22) The PCR reactor chip of claim 26 or any of the above claims, wherein said analysis device (15) comprises a feedline (23), carrying fluid from the outlet port (9) to the electrophoresis capillary The PCR reactor chip of claim 26 or any of the above claims, wherein said support level (19) comprises at least one feedline (24), carrying fluid from the at least one fluid support source to the reactor system A method for amplifying the quantity of a DNA molecule of interest contained in a volume of sample fluid using the polymerase chain reaction process, comprising introducing into a reactor system, in particular a reactor system of claim 1 or anyone of the above claims sample fluid containing DNA molecule of interest in double stranded form, a first and a second primer molecule complementary to opposing strands of the DNA molecule, thermostable DNA polymerase, deoxynucleoside tπphosphate and PCR buffer, the reactor system comprising at least one reaction chamber (1) comprised of at least one first and at least one second reaction chamber portions (2a, 2b, 2c), each reaction chamber portion having an inlet (5) and an outlet end (6) and constituting a temperature zone (14a, 14b, 14c), wherein the fluid flows continuously and in a circular manner, thus circulates through the reaction chamber portions (2a,2b,2c) and transition portions (11 ) provided adjacent to the reactor chamber portions A method of claim 33 or anyone of the above claims, wherein said fluid is permitted to exit said synthesis level (16) to enter the analysis level (17) in order to be introduced into an analysis device (15) A method of claim 33 or anyone of the above claims, wherein the reaction chamber (1 ) is deaerated A method of claim 34 or anyone of the above claims, wherein the temperature of each temperature zone (14a, 14b, 14c) is autonomously controlled and the temperature is adjustable in steps of at least 0 1 °C A method of claim 34 or anyone of the above claims, wherein fluid is moved by a fluid pump basing on piezo technology

38. A method of claim 34 or anyone of the above claims, wherein fluid is moved by a fluid pumping system based on electro-kinetic technology.

39. A method of claim 34 or anyone of the above claims, wherein the fluid flow is controlled in at least one transition portion (11 ). 40. A method of claim 34 or anyone of the above claims, wherein the fluid is carried to said analysis device (15) via a feedline (23) from the outlet port (9) to the electrophoresis capillary.

41. A method of claim 34 or anyone of the above claims, wherein sample fluid is moved by a motive force along the fluid flow path(12) along through the reaction chamber portions, thus through the temperature zones (14a, 14b, 14c).

42. A method of claim 34 or anyone of the above claims, wherein temperatures of the temperature zones (14a, 14b, 14c) are adjusted in order to provide the appropriate temperatures for denaturation, annealing and replication of the DNA strand. 43. A method of claim 33 or anyone of the above claims, wherein the sample fluid is moved through the reaction chamber portions (2a, 2b, 2c) repeatedly, thus being moved cyclic through the reactor chamber (1 ).

44. A method of claim 34 or anyone of the above claims, wherein at least a partial volume of the sample fluid is being withdrawn of the reactor chamber by passing the outlet port (9).

45. A method of claim 34or anyone of the above claims, wherein the withdrawn at least partial volume is introduced into an analysis device (15) to undergo analysis.

46. A method of claim 34 or anyone of the above claims, wherein the introduction of the sample fluid into the reactor chamber (1 ) is performed via injection of sample fluid.

47. A method of claim 34 or anyone of the above claims, wherein the introduction of the sample fluid is performed via fluid flow, deriving from at least one fluid support source.

48. A method of claim 34 or anyone of the above claims, wherein the fluid flow derives from a first, a second and a third fluid support source.

49. A method of claim 34 or anyone of the above claims, wherein the first fluid support source is a sample well containing fluid sample (20), the second fluid source is a gel- or other chemical reagents containing well (21 ) and a third fluid source is a well containing a DNA standard fluid (22), wherein the reactor system is operatively attached with at least one of said fluid support sources.