16 overshot ct free sample

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Soon after the introduction of the first commercial instrument in 1987 (Perkin-Elmer Cetus DNA thermal cycler), other suppliers offered similar devices. Today the thermocycler market is dominated by a few major manufacturers. Thermocyclers of the previous generations differ widely from the contemporary models. Developments of heating/cooling systems (circulating water versus Peltier technology), internal temperature control options (block control versus in-sample probe control and calculated temperature control), and evaporation control concepts (oil overlay versus heated lid) have been changing the technical architecture of the cyclers completely (15, 16). Accurate thermocyclers are a prerequisite for advanced applications such as cycling sequencing, in situ PCR, and real-time quantitative PCR in sample formats of up to 384 block positions. All these techniques rely on (i) an efficient heating and cooling system, (ii) an efficient heat transfer from the block to the sample liquid, and (iii) a sophisticated steering algorithm which allows the reproducibility of a given set temperature profile as stringent as possible.

The performance studies on thermal cyclers published so far lack relevance (5, 8, 14, 21). On the one hand, the studies refer to the older generation of thermocyclers, or processors from only one or two manufacturers were tested (5, 8, 21). Interlaboratory trials, on the other hand, compared only the amplification efficiency of used cyclers of various makes and models (16). The physical functionality of the machines was not determined.

This study focused on brand-new cyclers of the last generation. By embedding a fast-response microthermocouple inside the PCR tube, it was possible to determine the temperature uniformity within the sample block and to study the thermodynamic process from the block via the tube to the PCR sample in more detail. If available, the calculated temperature control mode, which is known to bring the samples more easily to the programmed temperatures (16), and each maximum heating rate had been chosen. By performing a PCR assay (PCR ListStand), the amplification efficiency of each model was determined.

Both physical testing using a static protocol and PCR testing allowed us to distinguish accurate (A, B, D, and E) from less-accurate (C and F) cyclers. This was also shown using a dynamic three-level protocol in a recent study (19). The most obvious irregularities of the machines were premature timing, followed by under- and overshooting and spatial irregularities of heat transfer. These results indicated that a difference in the cycler performance is not only a matter of abrasion after years of use, as suggested by previous studies (7, 16). It is an outcome of the variable technical architecture of the cyclers, which differs in the number, size, and quality of the Peltier elements, the location and number of the thermosensors steering the Peltier elements, and the underlying algorithm. A difference in the ITT from the block temperature might also result from poor heat transfer from the block to the PCR sample. In particular, the models which were notable for high heating and cooling rates seemed to be “susceptible” to premature timing.

The short denaturation step of PCR ListStand was seen to be the critical point. The cyclers C and F failed to amplify the target DNA. The physical testing of cyclers C and F showed a significant tendency for undershooting and premature timing. Usually the optimum denaturation temperature is between 91 and 94°C (11). Referring to the PCR ListStand protocol (94°C/15 s, 68°C/30 s, and 72°C/60 s), cylers C and F performed the effective temperature level only for 5 s (33%) or 7 s (47%), respectively (data not shown). Consequently, we concluded that the lack of amplification of cyclers C and F was most likely caused by the insufficient melting of the template DNA. This hypothesis could be proved by modifying the original denaturation step by increasing the temperature (PCR Listmod1), time (PCR Listmod2) or both (PCR Listmod3). The amplification efficiencies of cyclers C and F could be improved significantly. The best results could be obtained by expanding the denaturation time from 15 to 60 s (PCR Listmod2). This was seen as a proof that premature timing was the main reason for the lack of amplification of cyclers C and F.

By increasing and expanding the denaturation step up to 96°C and 60 s (PCR Listmod3), the tendency of cycler E for overshooting became most evident. Its amplification efficiency decreased dramatically, perhaps due to polymerase inactivation or deoxynucleoside triphosphate breakdowns (3, 22, 23, 24).

Each PCR user should have an effective and simple tool to test the performance of in-house cyclers. Several cycler companies developed end-user-friendly temperature validation systems, such as the Temperature Verification system (Applied Biosystems, Foster City, CA) or the Temperature Validation system (Eppendorf Netheler-Hinz GmbH, Hamburg, Germany). These single-sensor test systems allow static temperature measurement only within the block wells. Our study could show that static temperature procedures, on condition that they are done in-tube, are appropriate to detect some of the main weak points of thermocyclers (19). Another approach could be a cycler performance test using a standardized temperature-sensitive PCR system, such as SureCycle (Congen, Berlin, Germany), which was recently developed and introduced into the market (9). Cycler performance testing should regularly be carried out and become a part of any accreditation of an expert or an end-user lab devoted to PCR diagnostics. New PCR strategies should demonstrate the robustness of a temperature protocol, and validation of PCR protocols must include the testing of a PCR assay on several thermocyclers.