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Technical Briefs |
1 Institute for Milk Hygiene, Milk Technology and Food Science, Veterinärplatz 1, 1210 Vienna, Austria
2 Institute for Medical Physics and Biostatistics, Veterinärplatz 1, 1210 Vienna, Austria
3 Congen Biotechnologie, Robert-Roessle Strasse 10, 13125 Berlin, Germany
4 Danish Veterinary Institute, Bülowsvej 27, DK-1790 Copenhagen, Denmark
aauthor for correspondence: fax 43-1-25077-3590, e-mail dagmar.schoder{at}vu-wien.ac.at
Since the publication of the first article describing PCR, thermocyclers have become a staple in academic and industrial laboratories (1). The thermocycler is a programmable cycling incubator that performs repeated PCR steps of DNA denaturation, primer annealing, and primer elongation at defined intervals. Rapid heat transfer from the heating block to the in-tube sample liquid ensures a high efficiency of amplicon multiplication; therefore, a thermal processor should guarantee temperature uniformity for all samples within an individual run as well as run-to-run repeatability.
PCR-based protocols can give unsatisfying results (2)(3). Several collaborative studies have shown weak reproducibility with random amplified polymorphic DNA (RAPD) protocols (4)(5). One reason might be the influence of the thermocycler on amplification efficiency.
Despite its striking importance for PCR, the literature on thermocyclers is scarce. Some studies were published on the first generation of cyclers (6)(7)(8). Others determined the amplification efficiency but did not evaluate the physical characteristics (9)(10). The impact on PCR of the variation within thermocyclers, with regard to their thermocycling settings, has not been fully determined. The goal of this study was to define the physical characteristics of performance of the latest generation of thermocyclers and to discuss the influence of the physical properties on amplification efficiency.
Six new thermocyclers were selected for this performance study: (A) Gene Amp 9700 (Applied Biosystems), (B) Multicycler PTC 200 (MJ Research, Inc.), (C) Tgradient (Whatman Biometra GmbH), (D) Mastercycler gradient (Eppendorf Netheler-Hinz GmbH), (E) Touchgene (Techne, Inc.), and (F) Primus 96 (MWG AG Biotech). All experiments were performed with the lid temperature set to 105 °C and with the maximum ramp rate available.
The temperature was measured in 0.2-mL PCR tubes (MicroAmp; Applied Biosystems) containing 50 µL of distilled water. Fast-response type T microthermocouples (copper/constantan; i.d., 0.6 mm; isolated with polytetrafluorethyl; RS Components GmbH) and a 263A Data Bucket data logger (Fluke Cooperation) were used. The data logger was connected to a personal computer via the RS232 with use of IEEE-488 as communication language. A special software package (ScanScape; Fluke Coop.) enabled data collection and data storage. The setup of the experiments allowed the measurement of the in-tube temperature at 13 block positions (A1, H1, B2, G2, D4, A6, F6, C7, E10, B11, G11, A12, and H12).
The measurement unit was calibrated in accordance with specifications established for legal metrology, which documents traceability to national standards. The calibration was carried out by an officially accredited calibration authority (TESTO Comp.) at the following three temperature set points: 39, 72, and 97 °C. The certified temperature accuracy and reproducibility were ± 0.3 °C and ± 0.1 °C, respectively.
Each thermocycler was programmed to perform a dynamic temperature protocol (PCR RAPD short) that corresponded to a typical RAPD assay: 30 cycles of 97 °C for 30 s, 39 °C for 30 s, and 72 °C for 30 s (11). Evaluation of the temperature profiles allowed us to group the six test cyclers into three categories. Cycler A and B were assigned to category 1 (Fig. 1A
), cyclers D and E were assigned to category 2 (Fig. 1B
), and cyclers C and F were assigned to category 3 (Fig. 1C
).
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The performance of category 1 cyclers resembled an idealized chart (Fig. 1A
). The in-tube temperatures precisely followed the programmed temperatures. The mean PCR step duration (Stepmean) was calculated to be 30 s for cycler B and 31 s for cycler A (Table 1
).
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Category 2 cyclers (D and E) had short over-/undershooting phases before performing each PCR step (Fig. 1B
). The Stepmean was 38 s for cycler D, which means that the time for one cycle of denaturation, annealing, and elongation was extended by up to 30%. In cycler E, on the other hand, the annealing and elongation phases were reduced by up to 10%, whereas the denaturation was extended by 7% (Table 1
).
The temperature profile of cyclers C and F (category 3) did not correspond to the programmed temperature protocol. Both failed to achieve a temperature plateau for the denaturation, annealing, and elongation steps (Fig. 1C
). As a consequence, the Stepmean was shorter than with the other thermocyclers: 12 s for cycler C and 15 s for cycler F.
The performance of a thermocycler has a critical influence on PCR efficiency. Interlaboratory trials comparing up to 33 thermocyclers of various makes and models reported a failure to obtain reproducible banding patterns by RAPD assays (5)(9). Use of inappropriate cyclers was mentioned as one of the main reasons for these results; this was perhaps attributable to differences in age, temperature control options, and calibration status (9). The physical characteristics of performance, however, were never examined in detail.
To circumvent these shortcomings, we exclusively compared new thermocyclers of the latest generation. By embedding a fast-response microthermocouple inside a PCR reaction tube, we were able to study the thermodynamic process from the block via the tube to the PCR sample in more detail. This physical evaluation allowed us to distinguish accurate performers (category 1 and 2 cyclers) from less accurate performers (category 3 cyclers).
At the denaturation step, during the first 15 s the thermal inhomogeneities became most evident. Mai et al. (12) showed that the minimum denaturation temperatures that enabled amplification were 86–88 °C. The best results were obtained with denaturation temperatures between 91 and 94 °C (12). We found that category 3 cyclers (C and F) failed to reach the critical temperature of 90 °C for 8 and 10 s, respectively, after the start of timing of this step (data not shown). Consequently, these less-accurate performers may not reach adequate denaturation temperatures during short denaturation holds. We concluded that false-negative PCR results would most likely be caused by insufficient melting of the template DNA. Because of the less stringent annealing temperatures and shorter primers used, RAPD assays might be even more sensitive to temperature inhomogeneities than conventional PCR.
Acknowledgments
We thank Kurt Wimmer for assistance with designing and constructing the temperature recording unit; we also thank the companies for loan of the cyclers. We thank Drs. Martin D’Agostino and Nigel Cook (Central Science Laboratory, York, United Kingdom) for careful reading of this manuscript. This project was supported by EC Grant QLK1-CT-1999-00226.
References
The following articles in journals at HighWire Press have cited this article:
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  M. Fricker, U. Messelhausser, U. Busch, S. Scherer, and M. Ehling-Schulz Diagnostic Real-Time PCR Assays for the Detection of Emetic Bacillus cereus Strains in Foods and Recent Food-Borne Outbreaks Appl. Envir. Microbiol., March 15, 2007; 73(6): 1892 - 1898. [Abstract] [Full Text] [PDF]
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 D. Schoder, A. Schmalwieser, G. Schauberger, J. Hoorfar, M. Kuhn, and M. Wagner Novel Approach for Assessing Performance of PCR Cyclers Used for Diagnostic Testing J. Clin. Microbiol., June 1, 2005; 43(6): 2724 - 2728. [Abstract] [Full Text] [PDF]
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B. Malorny, E. Paccassoni, P. Fach, C. Bunge, A. Martin, and R. Helmuth Diagnostic Real-Time PCR for Detection of Salmonella in Food Appl. Envir. Microbiol., December 1, 2004; 70(12): 7046 - 7052. [Abstract] [Full Text] [PDF]
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J. Hoorfar, N. Cook, B. Malorny, M. Wagner, D. De Medici, A. Abdulmawjood, and P. Fach Making Internal Amplification Control Mandatory for Diagnostic PCR J. Clin. Microbiol., December 1, 2003; 41(12): 5835 - 5835. [Full Text] [PDF]
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