The time taken to complete a standard PCR protocol is becoming increasingly important for researchers today. Whilst many manufacturers of thermal cyclers focus on pushing the limits of ramp rates, in this article we highlight how Thermo's approach of focussing on achieving accurate sample temperatures can yield PCR results in less than 1 hour.
The Active Tube Control feature of Thermo's thermal cyclers measures the actual temperature of the reaction mix during PCR. This enables reduced dwell time PCR programmes to be run. A one second dwell time PCR programme was successfully run on Thermo's MBS and Px2 thermal cyclers. Yield of PCR product was comparable to that obtained from a standard PCR programme with a 30 second dwell time, but total run time was reduced by up to 50%.
A standard 3 temperature PCR programme can take in excess of 1 hour 30 minutes to complete. For modern researchers it is desirable to reduce these experimental times to speed up laboratory workflow. The use of Active Tube Temperature Control provides accurate monitoring and adjustment of in-sample reaction conditions, significantly reducing reaction dwell times and, therefore, experimental times without affecting PCR product yield.
Thermo thermal cyclers can use up to five control modes for monitoring temperature during PCR reactions. Block Control is the sole control method used on some thermal cyclers. As the name implies, the block temperature is monitored during the PCR reaction, irrespective of the in-sample temperatures achieved. This approach is adequate for prolonged static incubations. However, if Block Control is used during short incubation periods, such as during a PCR reaction, the reaction mix only achieves the set temperature for a time considerably shorter than that programmed or in extreme cases, will not reach the target temperature at all (figure 1).
The method of choice for monitoring any thermal cycling reaction is to measure the actual temperature the reaction mix achieves during PCR and adjust the heating or cooling accordingly. In this way, the researcher can be assured that the temperatures and dwell times they have set are actually achieved by the sample and not just the block. This control mode, which all Thermo thermal cyclers can use, is termed Active Tube Control (figure 2).
The improved accuracy in Active Tube Controlled protocols allows reaction conditions to be more stringent and subsequently overall run time can be significantly reduced.
Successful PCR using Block Control
While Block Control is less than ideal for performing PCRs, protocols can be written to compensate for inaccuracies in the PCR machine and it is ultimately possible to successfully amplify a gene fragment (figure 3).
Comparison of Block Control and Active Tube Control
The results of a PCR using the standard 30 second dwell time PCR with Block Control can be directly compared with those of a PCR performed using Active Tube Control (figure 4). Using Active Tube Control in this way caused an increase in the run time on both the MBS and the Px2 (table 1). In these equivalent protocols, the faster run time of the block controlled reaction occurs because the dwell time clock starts counting down as soon as the block achieves the set temperature, irrespective of the sample temperature. In the case of the Active Tube Controlled protocol, the unit more accurately monitors the in-sample temperature and the dwell time clock will not count down until it has achieved the set temperature.
Superior accuracy of PCR protocols run under Active Tube Control allows reduction of dwell times.
Due to the results obtained in the standard block controlled protocol (figure 3), we can confidently assume that it would be possible to successfully amplify these samples with a much shorter dwell time by using the more accurate Active Tube Control (figure 5).
As can be seen in figure 5, the Active Tube Control successfully allowed the reduction in dwell times and, hence, total run time, without a comprise on yield quality for both the Px2 and MBS instruments.
The accuracy and yield of a PCR is dependant on the accuracy of hitting and maintaining a precise target temperature. In Block Control, the only method available to ensure that the sample has reached the target temperature is to lengthen dwell times. In contrast, Active Tube Control measures the actual sample temperature allowing the use of shortened dwell times. Using these short dwell times on the Px2 and MBS allowed completion of a 3 temperature PCR protocol in less than 1 hour.
*The Polymerase Chain Reaction (PCR) is covered by U.S. patents 4683195, 4683202, and 4965188, which are owned by Hoffman-La Roche Ltd.