GB2399776A

GB2399776A - Cyclical heating and cooling device and associated methods

Info

Publication number: GB2399776A
Application number: GB0306768A
Authority: GB
Inventors: Thomas Neudeck; Duncan James Westland; Richard Harley Grenville Owen
Original assignee: PA Knowledge Ltd
Current assignee: PA Knowledge Ltd
Priority date: 2003-03-24
Filing date: 2003-03-24
Publication date: 2004-09-29
Also published as: GB0306768D0; WO2004085065A1

Abstract

A cyclical heating and cooling apparatus comprises a heating cycle of denaturing t1 annealing t2 and extension t3 phases, a detector 12 able to detect the amplification of a polymerase chain reaction process at a plurality of times during each heating cycle, and a monitor, which may monitor 18 the detector 12, at a plurality of times. The detector 12 may be a light detector 12, and the monitor 18 may be a data acquisition unit 18. A fluorescent marker may be placed within the polymerase chain reaction, and may provide differing levels of fluorescence during the heating cycle, which changes may be detected 12, and monitored 18. Also disclosed are two associated methods, consistent with normal usage of the above described device in which DNA may be amplified by a polymerase chain reaction.

Description

METHOD OF POLYMERASE CHAIN REACTION AND APPARATUS FOR

CONDUCTING THE SAME

The present invention relates to a method of polymerase chain reaction for amplifying a target template of DNA and an apparatus for conducting the same. It also relates to a method of controlling such an apparatus.

The polymerase chain reaction (PCR) is a well known technique for amplifying a target fragment or sequence of double stranded DNA (template) . The process is described for instance in M. J. McPherson, S. G. Moller (2000) The Basics PCR, Understanding PCR pp 1-18 BIOS Scientific Publishers, Oxford. It generally involves a plurality of cycles, each cycle including a denaturing phase in which the double strands are separated, an annealing phase in which primers bind to specific sequences of DNA and an extension phase in which, from the primers, second strands of DNA are synthesized. À À . The construction of a suitable apparatus for conducting this process is well known. Àe

and is described for instance in M. J. McPherson, S. G. Muller (2000) The Basics..

PCR, Reagents and Instrumentation pp 23-59 BIOS Scientific Publishers, Oxford. À.. .

Often, commercial quantative-PCR systems use a fluorescent dye, such as Sypr À A Green 1, in order to monitor the amplification progress. Free in solution, the dye molecule has poor fluorescence upon excitation. However, once the dye binds to the DNA, fluorescence is greatly enhanced. Other fluorescent dyes are known which specifically bind to double stranded DNA so that the dye binds and a signal can be generated. The increase m fluorescence is a measure of the progressive amphfication of the target template and allows cycle-to-cycle monitoring of the reaction. In particular, the fluorescence signal is measured at the end of the extension phase, such that, together with a meltcurve analysis, this data can be used to calculate the mitial template concentration and the product quantification.

Although the known PCR process is very effective, in practice, the PCR efficiency is disappointing. In theory, each cycle of the process should double the amount of target DNA, giving the process exponential increases. However, in practice, PCR efficiencies of around 60 to 70% are more realistic.

It is an object of the present invention to provide a method and apparatus by which performance may be increased according to the needs of the user.

According to the present invention there is provided a method of polyrnerase chain reaction for amplifying a target template of DNA in a reaction mix, the method including: at least one cycle having a denaturing phase, an annealing phase and an extension phase; and for each cycle, monitoring the amplification of the target template at a plurality of times. .; According to the present invention there is also provided an apparatus for conducting.. e a polymerase chain reaction process on a reaction mix to amplify a target template of A. DNA in the reaction mix, the apparatus being configured to operate at least one cycle having a denaturing phase, an annealing phase and an extension phase, the apparatus À.

including: À . . À a detector for detecting the amplification of the target template; wherein the detector is able to detect the amplification at a plurality of times during each cycle; and the apparatus further includes: a monitor for monitoring the amphficaton as detected by the detector at a plurality of times during each cycle.

In this way, unlike previous systems, where the amplification is only measured at the end of each extension phase, by monitoring the amplification at a plurality of times during each cycle, it is possible to construct a profile for the amplification occurring during that cycle. This allows much better control of the process, since, from the l shape of the profile, it is possible to determine not only how much amplification has occurred, but also how the process is progressing on an ongoing basis. In this way, the overall process may be fully analysed or, indeed, adjusted on a continuous basis.

As a result, parameters for the process may be optimised, for instance when new enzymes and primer reaction mixes are used.

Preferably, the detector is able to detect continuously the amplification.

In this way, the amplification may be monitored continuously or at a predetermined sample rate. This allows a more complete and accurate profile for the amplification to be determined. As a result, an analysis or control of the polymerase chain reaction may be improved.

It will be appreciated that, where the amplification is merely monitored at a predetermined sample rate, it is sufficient for the detector to be able to detect the À amplification at that sample rate. À.

Preferably, the detector is able to detect the amplification at least once during each. . phase of each cycle and the monitor is arranged to monitor the amplification as detected by the detector at least once during each phase of each cycle. À À:-e This can be used to give an Indication of how each of the denaturing, annealing and extension phases are progressing for each cycle. As a result, the user can choose alternative parameters andlor knows where a process is not working efficiently.

In order to allow for more accurate control of the process during individual phases, the detector is preferably able to detect the amplification at a plurality of times during at least one phase of each cycle and the monitor is arranged to momtor the amplification as detected by the detector at a plurality of times during the at least one phase.

g - '-

-

In this way, a profile is provided of the amplification during the at least one phase such that improved analysis and control of that phase can be achieved.

Preferably, a controller is attached to the monitor for controlling, on the basis of the monitored amplification, at least one parameter for the polymerase chain reaction process.

In this way, the apparatus is able to conduct an adaptive process in which parameters for the polymerase chain reaction process can be controlled in real time according to the monitored amplification.

Preferably, the at least one parameter includes one or more of a number of cycles, the temperature of the reaction mix and the period of one or more of the denaturing phase, annealing phase and extension phase. a.- À .

Hence, as will be clear from the following description, the temperatures and periods À.

used for the various phases of the cycles can be varied and controlled according to A. the monitored reaction process. In this way, the process can be optimised according ^ . to the requirements of the user. Indeed, the overall number of cycles need not be determined in advance, but can be judged by the apparatus on the basis of the À ..

monitored amplification. À.

The apparatus may further include a memory, the controller being arranged to store in the memory one or more profiles of previous monitored processes.

In this way, it is possible to compare the profile of the monitored amplification with a stored profile of the amplification for a previous polymerase chain reaction process.

This may enable diagnosis of problems with a particular process run. The profile of that particular process run may be similar to the profile of a previous process run for which error conditions are already known.

Preferably, the detector is able to detect the amplification at a plurality of times during at least one of the denaturing, annealing and extension phases of each cycle, the monitor is arranged to monitor the amplification as detected by the detector at a plurality of times during said at least one of the denaturing, annealing and extension phases and the controller is arranged to determine when the process of said at least one of the denaturing, annealing and extension phases is complete and to control the period of said at least one of the denaturing, annealing and extension phases accordingly.

Hence, one or more of the phases can be controlled in length according to the progress of the polymerase chain reaction during that phase as monitored.

The controller may additionally or alternatively be arranged to adj ust the temperature of the reaction mix according to the rate of change of amplification monitored for the at least one of denaturing, annealing and extension phases. À À Be. : Hence, in a similar way, the temperature used for individual phases can be controlled and varied according to the monitored progress of the polymerase chain reaction. In..

particular, it can be varied between consecutive cycles and even be varied during an individual phase of a cycle according to progress of the process so as to optimise the À process according to the requirements of the user. À ...

Thus, a feed-back loop may be provided, thereby allowing optimisation of the parameters for the phases of each cycle. Because the optimum periods during the process can be automatically adjusted for every individual cycle, the overall yield may be increased. Similarly, the overall runmng time may be reduced as well as the number of required cycles.

Previous systems lengthen the extension phase so as to maximise the number of target templates which are fully duplicated. However, with these longer periods, non-specific amplification, i.e. duplication of unwanted sequences, Is more likely to occur. Hence, by monitoring the amplification as proposed with the present invention and controlling the period of the extension phase so as to optimise the extension phase, non- specific amplification is less likely to happen, resulting in a cleaner PCR product. s

It is also possible to provide more accurate quantification of start template DNA, because higher yield and cleaner product throughout a PCR run reduces the need for post data processing currently used for DNA quantification, for instance using proprietary software algorithms.

The process allows instant quality control for batch-to-batch variation of the enzymes, i.e. Taq enzymes, and primers. Hence, it also allows evaluation of PCR reagents from different manufactures.

In the preferred embodiment, a fluorescent dye is added to the reaction mix. A light sensor may be used to measure the fluorescence in order to monitor the amplification. À.

However, the present invention is also applicable to other systems of measurement, .. . for instance by detecting heat, impedance and such like. A.: It will be appreciated that, knowing the reaction mix and the process parameters, it is possible to predict the approximate profile for the amplification over time. À À.

Preferably, the reaction process may be determined as being in error when the monitored amphficahon rises either one of substantially more quickly and more slowly than expected.

In this way, any problems with the reaction mix, for instance by virtue of the primers or Taq enzyme will immediately be detected during the polymerase chain reaction.

According to the present invention, there is also provided a method of controlling a polymerase chain reaction apparatus configured to operate at least one cycle having a l, denaturing phase, an annealing phase and an extension phase, the apparatus having a detector for detecting the amplification of a target template of DNA, the method including: monitoring the amplification as detected by the detector at a plurality of times during each cycle.

There may also be provided a computer program and a computer program product for causing such an apparatus to operate in this way.

The invention will be more clearly understood from the following description, given by way of example only, with reference to the accompanying drawings in which: Figure 1 illustrates schematically an apparatus embodying the present invention; Figures 2(a) and (b) illustrate schematically the temperature profile of a single PCR cycle and the resulting fluorescence signal; Figures 3(a) and (b) illustrate schematically the temperature profile for 3 consecutive À cycles; and. . Figure 4 illustrates the expected fluorescence signal during the extension phase. . . À - . As illustrated in Figure 1, in order to carry out the PCR process, a PCR reaction mix À ..

is provided as a sample 2 in a container 4. A heater 6 is provided to adjust the ' . À temperature of the sample 2 under the control of a controller 8.

The apparatus, under the control of the controller 8, is configured to conduct at least one cycle of DNA amplification including the steps of denaturing, annealing and extending.

Figure 2(a) illustrates a suitable temperature profile for the sample 2 dunug one cycle.

The controller 8 controls the heater 6 to carry out the denaturing process during period to. The DNA molecule is heated to approximately 94 C to break the relatively weak hydrogen bonds between the bases and to separate the double strands. This process is conducted for a period in the order of 5 seconds.

The controller 8 then reduces the temperature of the heater 6 and sample 2 so as to carry out the annealing process over the period t2. The denaturing process is reversible and bonds between complementary bases can be formed when the temperature is decreased. The PCR mix in the sample 2 contains an excessive amount of the short single stranded DNA called primer. At the lower temperature provided during the annealing period, the primers bind to specific sequences of DNA.

The annealing period is ofthe order of 10 seconds.

Finally, once the primers are bound to the target DNA, the controller 8 causes the heater 6 to raise the temperature of the sample 2. During this extension period t3, the Taq enzyme in the PCR mix synthesises a new double stranded DNA so as to À duplicate the target DNA. . . The sample 2 may be provided with a fluorescent dye, such as Sypr Green 1, in order

- -

to monitor the amplification progress. Free in solution, the dye molecule has poor À ..

fluorescence upon excitation. However, once the dye binds to the DNA, fluorescence À . À is greatly enhanced. Other dyes specifically bind to double stranded DNA so that the dye binds and becomes visible.

Figure 2(b) illustrates a representation of the fluorescence produced during the cycle of Figure 2(a).

In order to allow monitoring of the fluorescence, the apparatus is provided with a light source 10 and an appropriate photo detector or light sensor 12. In this respect, in the preferred embodiment, a light filter 14 may also be provided to improve the signal provided to the light sensor 12. -9 -

The apparatus differs from previous arrangements in that the light sensor 12 is able to detect fluorescence in the sample 2 at a plurality of times during the each cycle.

Depending on the control process used, it may be sufficient for the light sensor 12 to detect fluorescence at a plurality of times during one of the phases of each cycle or at least once for each phase of each cycle. However, in a preferred embodiment, the light sensor 12 is able to detect fluorescence at a plurality of times during each phase of each cycle. The light sensor 12 could monitor the fluorescence on a continuous basis or at a plurality of discrete times.

Depending on the nature of the detector 12, a signal amplifier 16 can be provided to pass a signal from the detector 12 to a data acquisition unit 18. The data acquisition unit 18 acts as a monitor for monitoring the amplification as detected by the detector at a plurality of times during each extension phase. It may be embodied as software, for instance, running on a PC. À À. I-. :

The fluorescence can be monitored continuously or with a particular sampling rate, À.

for instance five samples per second. A. À . : À Figures 3(a) and (b) are provided to illustrate the relationship between consecutive cycles of the process and, by way of example, illustrate the 20th, 21st and 22nd cycles of a particular process run. Since the quantity of required DNA should..

approximately double for each cycle, then, as illustrated in Figure 3(b), the peak fluorescence at the end of each extension phase should similarly double for consecutive cycles. As Illustrated, during the denaturing phase of each cycle, all of 2: the double strands should be separated, such that the fluorescence should return to zero. However, m practice, as illustrated, the fluorescence will always remain at a minimum base hne level resulting from background noise.

By monitoring the fluorescence in this way, the controller 8 can monitor the level of required DNA in the reaction mix. As a result, the controller is able to control adaptively various parameters of the PCR process so as to optimise the process as required. This adaptive process takes the form of a closed loop whereby the controller detects the results of the process on an on-going basis and varies accordingly the parameters influencing that process.

As will be apparent from the description given above, the relevant parameters include at least the temperature for each phase, the length or period of each phase and the overall number of cycles. By monitoring the amplification process, for instance by means of the fluorescence, the controller can control one or more of these parameters so as to vary adaptively the PCR process in real time. This is in contrast to earlier arrangements where a fluorescence signal is measured only at the end of the extension phase so as to calculate the initial template concentration and the product quantification. According to these earlier arrangements, for each PCR process that is conducted, the temperatures and periods for the denaturing, annealing and extension phases are set in advance of each process run. ..

15. Hi.

As explained above, at the end of the denaturing phase, i.e. at the end of period to, the fluorescence signal should have returned to the base line or background noise level. ..

By using the detector 12 to measure the fluorescence of the reaction mix and À'. . determine how much target DNA remains, it is possible for the controller 8 to ensure that the reaction mix does return to the base line. '''' .' The controller 8 can vary the period of the denaturing phase. In particular, it can maintain the denaturing phase until the base line is reached. In addition, if this period is longer than expected, the controller can also increase the temperature of the reaction mix during the denatunng phase. In a preferred embodiment, the controller can judge from the rate of fall of fluorescence (and hence rate of fall of double stranded DNA) whether or not the reaction mix will reach the base line in the expected period. On this basis, it can increase the temperature of the reaction mix so as to achieve the desired rate of change. Of course, there may also be some circumstances where the controller 8 decreases the temperature of the reaction mix.

Having brought the reaction mix to the base line, the controller 8 then drops the temperature of the reaction mix so as to start the annealing phase. During this phase, when the primers bind to the specific sequences of DNA, the fluorescence level should rise again to the peak level of fluorescence for the previous cycle of the PCR process.

The controller 8 can extend the period of the annealing phase until the required fluorescence is achieved. Furthermore, if the time for this is beyond that expected, the controller 8 can also decrease the temperature of the reaction mix. In a preferred embodiment, the controller 8 is sensitive to the rate of change of fluorescence and can vary the temperature of the reaction mix so as to achieve a desired rate of change and period for the annealing phase. Similarly, again, there may be circumstances where the controller 8 judges that the temperature should be increased rather than decreased. À. a A- .

Figure 4 illustrates a typical profile for the rise in fluorescence during the extension À . phase. As explained above, this is indicative of the extension process itself. In particular, it will be seen that the process slows down as all of the target DNA to..

which primers have been bound is synthesised into new double stranded DNA. ', À

If the period t3 of the extension phase is too small, then the PCR efficiency is À .'.

unnecessarily reduced. On the other hand, if the period t3 is too long, primers may bind to the wrong target and incorrect synthesis may occur.

The period of the extension phase is generally of the order of lO seconds and, in previous arrangements, the period is fixed according to various process parameters.

This leads to a practical PCR etficency of around 60 to 70 %. Possible reasons for this are primers binding to the wrong target (mispriming), primers binding to each other (primer dimers) reducing resource, incorrect bases being m-cooperated into the growing chain, contamination (particularly of the starting template), wrong annealing and extension temperahre, too short dwell times and exhaustion of primer or enzymes before reaction completion.

By monitoring the fluorescence signal thoughout the entire extension dwell time for each cycle, it becomes possible to assess the actual extension process and vary the extension dwell time accordingly. In particular, the controller 8 can provide automatic dwell time optimization.

In a preferred embodiment, the controller 8 can take account of the rate of change of fluorescence so as to judge how the process of extension is proceeding and to vary the temperature of the reaction mix accordingly.

It should be appreciated that the controller 8 can be configured to optimise the PCR process in a number of different ways. In particular, it need not be the case that the..

controller 8 always optimises the process for the highest yield. Alternatively, the controller 8 could optimize the process to achieve high specificity. Thus, with an expected possible increase of 100% as compared with a previous cycle, the controller.'.

8 could control the process to produce an increase of only 95% by maintaining the À'. . . temperature during the extension phase at a value slightly higher than that required.

for optimum yield. In this way, non-specific development is less likely to occur and.., a reaction mix may be created having a substantially 100% pure intended target with '' substantially no undesired artifacts.

The controller 8 can cause the PCR process to be Nan according to the requirements of the user For instance, where detection is required, the process could be run as quickly as possible so as to provide a high yield. However, in contrast, for cloning applications, specificity is very important.

It should be appreciated that the controller 8 can consider not only individual cycles of the process, but also the overall process having the plurality of cycles. On the one hand, the controller 8 can vary parameters on a cycle by cycle basis, such that, as ! conditions change, for instance concentrations change, the parameters can be changed accordingly. This may be of particular use where the initial reaction mix contains contaminants. Contaminants, such as biological molecules, act as inhibitors.

During early cycles of the process, the controller 8 might provide long periods for the phases so as to overcome these inhibitors. However, for later cycles, the periods may be reduced to more normal lengths.

The controller 8 may also vary the overall number of cycles. In particular, by monitoring the fluorescence and, hence, the amount of target DNA, it is possible for the controller 8 to end the process once the desired amplification has been achieved.

Since the controller 8 is able to optimise the process at each cycle, the overall number of cycles is likely to be reduced as compared to previous systems. Indeed, according to previous systems, it was necessary to preset the overall number of cycles to a number high enough to ensure that the required amplification takes place. Ale.:

In a preferred embodiment, it is envisaged that the user inputs to the controller 8 suggested parameters for the process, for instance the periods and temperatures for À.

the venous phases, together with the overall number of cycles. The controller 8 can then start the overall process on this basis, but then vary the parameters according to....

the detected fluorescence. On the other hand, it is possible for the apparatus, effectively to run itself. By giving the controller the sequence of the primers and the expected length of the target sequence, it will be possible for the controller 8 to estimate the required temperatures and periods for the various phases of the process.

However, of course, the controller 8 could still apply the adaptive process described above, varying the parameters according to the actual detected fluorescence.

As illustrated in Figure l, the apparatus can also be provided with a memory 9.

Using the memory 9, the controller 8 is able to store profiles of previous PCR processes. Indeed, it would also be possible to transfer into the memory 9 profiles known or obtained by other users of similar systems.

Using these earlier profiles, the controller 8 is able to conduct a diagnostic process.

For example, where a particular run of the PCR process fails, the controller 8 can compare the profile to earlier profiles to find any which are similar. This may provide useful information to the user regarding the factors surrounding the failure.

In one embodiment, the user could name earlier profiles according to the circumstances of the particular respective runs. It is envisaged that the process runs could be conducted intentionally with too much enzyme, too tattle enzyme etc and the profiles stored with appropriate names. In this way, when the controller 8 finds a similarity between the profile of a failed process run and an earlier process run, the l O user may be provided with an indication of the reason for that failure.

It may be possible for a user to determine whether a process run has been successful merely by analysing the end product. However, where the controller 8 determines that the profile of the process differs significantly from the expected response, the l 5 user can be warned that an error has occurred. In particular, the monitored À À . . amplification indicated by the fluorescence might rise substantially more quickly or À substantially more slowly than expected. ... :

Figure l illustrates schematically an error detector 20 as part of the unit l 8.

-

2 0. . A It will be appreciate that the general method described with reference to Figure l À ' .: could be used with other similar apparatuses. Indeed, for an apparatus having detection equipment (with light or otherwise) monitoring the amphfication at a plurality of times during a cycle, it would be possible to provide an appropriate control process for use with that apparatus, for instance as a software routine.

By using the present invention, it is possible to increase the overall yield and/or produce a cleaner PCR product since non-specfic amplification is less likely to happen. Similarly, more accurate quantification of the start template DNA is possible, because the higher yield and cleaner product throughout a PCR run reduces the need for post data processing as is currently required for DNA quantification (for Al instance proprietary software algorithms). By virtue of the present invention, it is also caster to cope with new enzyme and primer reaction mixes, since the optimization of the PCR parameters is caster and faster, with the periods and/or temperatures being determined automatically. Similarly, instant qtrality control is provided for batch-to-batch variations of Taq enzymes and primers, together with evaluation of PCR reagents from different manufacturers. The overall running time will also be reduced, since the number of required cycles will decrease. Beet I: À^ À 4.e r

Claims

- CLAIMS

1. An apparatus for conducting a polymerase chain reaction process on a reaction mix to amplify a target template of DNA in the reaction mix, the apparatus being configured to operate at least one cycle having a denaturing phase, an annealing phase and an extension phase, the apparatus including: a detector for detecting the amplification of the target template; wherein the detector is able to detect the amplification at a plurality of times during each cycle; and the apparatus further includes: a monitor for monitoring the amplification as detected by the detector at a plurality of times during each cycle.

2. An apparatus according to claim 1 wherein the detector is able to detect continuously the amplification.

I À'..:

3. An apparatus according to claim 1 or 2 wherein the monitor is configured toÀ monitor the amplification at a predetermined sample rate.. . À.

4. An apparatus according to claim 1, 2 or 3 wherein the detector is able to detect the amplification at least once during each phase of each cycle and the monitor À...

is arranged to monitor the amplification as detected by the detector at least once À '.

during each phase of each cycle.

5. An apparatus according to any preceding claim wherein the detector is able to detect the amplification a plurality of times during at least one phase of each cycle and the monitor is arranged to monitor the amplification as detected by the detector a plurality of times during said at least one phase.

6. An apparatus according to any preceding claim further including: l a controller connected to the monitor for controlling, on the basis of the monitored amplification, at least one parameter for the polymerase chain reaction process.

7. An apparatus according to claim 6 wherein said at least one parameter includes one or more of: the number of cycles; the temperature of the reaction mix; and the period of one or more of the denaturing phase, annealing phase and extension phase.

8. An apparatus according to claim 6 or 7 further including: Àe a memory, the controller being arranged to store in the memory one or more À . profiles of previous monitored processes. À.

9 An apparatus according to claim 6, 7 or 8 wherein: ... Àe

the detector is able to detect the amplification at a plurality of times during at least one of the denaturing, annealing and extension phases of each cycle; À the monitor is arranged to monitor the amplification as detected by the À A detector at a plurality of times during said at least one of the denaturing, annealing and extension phases; and the controller is arranged to determine when the process of said at least one of the denaturing, annealing and extension phases is complete and to control the period of said at least one of the denaturing, annealing and extension phases accordingly.

10. An apparatus according to any one of claims 6 to 9 wherein: the detector is able to detect the amplification at a plurality of times during at least one of the denaturing, annealing and extension phases of each cycle; the monitor is arranged to monitor the amplification as detected by the detector at a plurality of times during said at least one of the denaturing, annealing and extension phases; and f the controller is arranged to adjust the temperature of the reaction mix according to the rate of change of amplification monitored for said at least one of the denaturing, annealing and extension phases.

S

11. An apparatus according to any preceding claim further including: an error detector connected to the monitor, the error detector being responsive to the momtored amplification rising either one of substantially more quickly and more slowly than expected to indicate an error in the reaction process.

12. An apparatus according to any preceding claim when the detector is a light sensor for sensing fluorescence of a fluorescent dye added to the reaction mix.

13. A method of polymerase chain reaction for amplifying a target template of. . DNA in a reaction mix, the method including: I. . at least one cycle having a denaturing phase, an annealing phase and an À:.

extension phase; and À.

for each cycle, monitoring the amplification of the target template at a À plurality of times. À :- À À À. e.

14. A method according to claim 13 including: monitoring the amplification continuously.

1S. A method according to claim 13 including: momtoring the amplification at a predetermined sample rate.

16. A method according to claim 13, 14 or 15 including: monitoring the amphfication at least once during each phase of each cycle.

17. A method according to any one of claims 13 to 16 including: monitoring the amplification at a plurality of times during at least one phase of each cycle.

18. A method according to any one of claims 13 to 17 including: adaptively controlling at least one parameter of the polymerase chain reaction on the basis of the monitored amplification.

19. A method according to claim 18 wherein said at least one parameter includes one or more of the number of cycles; the temperature of the reaction mix; and the period of one of more of the denaturing phase, annealing phase and extension phase

20. A method according to any one of claims 13 to 19 including: monitoring the amplification at a plurality of times during at least one of the À denaturing, annealing and extension phases of each cycle; and À controlhng the length of said at least one of the denaturing, annealing and extension phases on the basis of the monitored amplification. ..

21. A method according to any one of claims 13 to 20 including: À monitoring the amplification at a plurality of times during at least one of the. . denaturing, annealing and extension phases of each cycle; and controlling the temperature of the reaction mix according to the rate of change of monitored amplification.

22. A method according to any one of claims l3 to 21 including: comparing the profile of the monitored amplification with a stored profile of the amplification for a previous polymerase chain reaction process.

23. A method according to any one of claims 13 to 22 further including: determining that the reaction process is in error when the monitored amplification rises either one of substantially more quickly and more slowly than expected.

24. A method according to any one of claims 13 to 23 further including: adding a fluorescent dye to the reaction mix; and measuring the fluorescence to monitor the amplification.

25. A method of controlling a polymerase chain reaction apparatus configured to operate at least one cycle having a denatunug phase, an annealing phase and an extension phase, the apparatus having a detector for detecting the amplification of a target template of DNA, the method including: momtoring the amplification as detected by the detector at a plurality of times during each cycle.

26. A method according to claim 25 further including: monitoring the amplification continuously. .. À Àe

27. A method according to claim 25 further including: À.

monitoring the amplification at a predetermined sample rate. À

28. A method according to claim 25, 26 or 27 including: . . monitoring the amplification at least once during each phase of each cycle. . .

29. A method according to any one of claims 25 to 28 including: monitoring the amplification at a plurality of times during at least one phase of each cycle.

30. A method according to any one of claims 25 to 29 including: adaptively controlling at least one parameter of the polymerase chain reaction on the basis of the monitored amplification.

31. A method according to claim 30 wherein said at least one parameter includes one or more of: the number of cycles; ( the temperature of the reaction mix; and the period of one of more of the denaturing phase, annealing phase and extension phase

32. A method according to any one of claims 25 to 31 including: monitoring the amplification at a plurality of times during at least one of the denaturing, annealing and extension phases of each cycle; and controlling the length of said at least one of the denaturing, annealing and extension phases on the basis of the monitored amplification.

33. A method according to any one of claims 25 to 32 including: monitoring the amplification at a plurality of times during at least one of the denaturing, annealing and extension phases of each cycle; and.... À.-

controlling the temperature of the reaction mix according to the rate of change... À.

of monitored amplification. À -

34. A method according to any one of claims 25 to 33 including:À.: comparing the profile of the monitored amplification with a stored profile of the amplification for a previous polymerase chain reaction process. .

35. A method according to any one of claims 25 to 34 further including: indicating an error in the reaction process when the monitored amplification rises either one of more quickly or more slowly than expected.

36. A computer program comprising program code means for perforrmng all the steps of any one of the claims 25 to 35 when said program Is run on a computer.

37. A computer program product comprising program code means stored on a completer readable medium for performing the method of any one of the claims 25 to 35 when said program product is run on a computer. l

38. An apparatus constructed and arranged substantially as hereinbefore described with reference to and as illustrated by the accompanying drawings.

39. A method of polymerase chain reaction substantially as hereinbefore described with reference to and as illustrated by the accompanying drawings.

40. A method of controlling a polymerase chain reaction apparatus substantially as hereinbefore described with reference to and as illustrated by the accompanying drawings. À

I- À.. À- Àe À À.

À - o. À Àe À À À À .e I,