Why validate cyclers?

It would be a waste of time if the reason a temperature validation was carried out is only to conform to quality norms. However, the majority of all laboratory engineers have the experience of unexplicable problems with their PCR experiment, with no relation to the composition of the reaction mix. Just like the fact that there is often an undefined preference for a specific instrument at which “my reaction always works”. From our experience, this seems to be really the case; not a pair of thermal cyclers, even identical types of the same brand, shows the same temperature profile. And it is exactly this profile that is crucial for the success of the reaction. The more reason to study this subject seriously.

As explained before, there is not a pair of cyclers with the same temperature behaviour. The accuracy of the block temperature can deviate either positively or negatively from the set temperature, and the different wells in a block sometimes show a spread (non-uniformity) in temperature up to several °C. The lack of control in some cyclers causes an occurence of over- and undershoots, while trying to reach a plateau.

A first explanation of these differences might be found in the differences in technology. There are many ways to regulate the temperature in a thermal cycler (Joule heating, Peltier elements, air or liquid compression) and to bring a block to, and to keep it at a certain temperature. Every technology has its pros and cons. Secondly, the level to which these technologies are controlled is essential for the level of performance, but differs greatly. Most cyclers have only a poor level of control. Differences between single and multi-sensor control mechanisms are obvious causes for temperature inaccuracies.

Although a PCR instrument normally does not have any mechanical or moving parts, this does not imply that it cannot be subject to wear and tear. The performance of electronic components depreciates in time, and an intensive use can reinforce this effect. Failure and wear of Peltier elements can give rise to hot or cold spots in a block. In general, cyclers do not recognise this loss of performance caused by wear and tear, block extension and shrinking. Therefore it is crucial to monitor the temperature performance in time,in order to be aware of these effects. By the same token, a bad thermal performance is often a strong indicator for upcoming failures and breakdowns of a cycler.

Last, but not least, several external factors also influence the temperature performance of the cycler. Probably the most important external factor is the way in which the tubes are loaded into the block. As a result of the sensor positioning of a certain instrument, a specific way of loading the tubes (e.g. a so-called left load) can lead to substantial temperature over-/undershoots, and also inaccuracies.

The mechanism of a PCR reaction is presented quite oversimplified, often because of didactic reasons. There are three temperature plateaus (denaturation, annealing and elongation) at which a defined reaction occurs. Nothing less is true. During the courses between plateaus, reactions are still taking place. E.g. annealing, the hybridisation of the primers to the DNA strands, is not limited to a specific temperature, but can happen over a range of temperatures. Lower temperatures lead to non-specific binding, while higher temperatures obstruct a good annealing. Extension, the step at which the thermostable enzyme elongates the DNA strands, is not restricted to 72 °C, but occurs at a non-optimal level, also at higher and lower temperatures. Denaturation is the most dualistic one of the three steps. A good denaturation is of great importance for the course of the PCR reaction. When the strands are not separated, there will not be enough attachment possibilities for the primers, and the final yield will be reduced. On the other hand, heat inactivation of the enzyme caused by elevated temperatures is a very underestimated factor. Although the thermostable enzyme is resistant to higher temperatures, sooner or later, and depending on the half-life, it will decrease in activity and finally stop working. The higher the denaturation temperature is, the sooner this will occur. A decrease in the activity of the thermostable enzyme is especially fatal in the last cycles of the PCR reaction. These are the cycles where relatively the most substrate is offered to the enzyme, and where the largest amount of DNA is amplified. Investigations into the effects of increasing the denaturation temperature, or an extension of the denaturation time, show dramatic effects on the yield.

Denaturation is the most dualistic one of the three steps. A good denaturation is of great importance for the course of the PCR reaction. When the strands are not separated sufficiently, there will not be enough attachment possibilities for the primers, and the final yield will be reduced. On the other hand, the inactivation of the enzyme caused by elevated temperatures is a very underestimated factor. Although the thermostable enzyme is resistant to higher temperatures, sooner or later, and depending on the half-life, it will stop working. The higher the denaturation temperature is, the sooner this will occur. A decrease in the activity of the thermostable enzyme is especially fatal in the last cycles of the PCR reaction. These are the cycles where relatively the most substrate is offered to the enzyme, and where the largest amount of DNA is amplified. Research into the effects of increasing the denaturation temperature, or an extension of the denaturation time, show dramatic effects on the yield.

It is obvious that the differences in temperature performance between brands and types of cyclers directly influence the accuracy and non-uniformity at the three temperature plateaus, and thus the results of the overall PCR reaction. Besides that, differences in speed, non-uniformity during ramps, and overshoots and undershoots, are also substantial factors. In the worst-case scenario, this could lead to a negative result, which should have been positive. For Real Time PCR, where the results of fluorescence, signal,yield and melting curves are in direct relation with these temperatures, the effect is even more dramatic.

In the light of the above mentioned arguments, the arguments mentioned above, many laboratories have the obligation to comply with a certification of some kind. With ISO, GMP, GLP norms, one needs to proof that all the lab proceedings are performed according to standardised procedures. Some laboratories may call themselves accredited, when they comply with tested and accepted procedures from their coordinating organisation or professional association. Others are held to compelling governmental stipulations for quality surveillance regarding certain tests (e.g. food or water quality). Moreover it is recognised that the PCR reaction, when used in these kind of laboratories should be both validated and certified.