Part 2: Speed up your Reaction – Reduction of Preparation Time and Avoiding Runs Entirely

Reducing the time spent on PCR runs is doubtlessly beneficial, as results will be available more quickly and the thermocycler will be free to be used for additional experiments. The entire PCR-protocol, however, encompasses much more than just the actual run on the instrument; the time required for preparation, as well as subsequent analysis, must not be underestimated. In addition, it is worth considering whether each individual PCR run is indeed necessary. In summary, further to a reduction in total run time, optimization of preparation and analysis must be taken into consideration and, wherever possible, runs should be avoided altogether. These factors have a substantial impact on the productivity as well as on the efficiency of a laboratory – especially if many PCR experiments are carried out in a routine environment.

Prior to the run, reagents must be prepared and the instrument must be programmed. Preparing the reaction demands great care and attention, as multiple dispensing steps are involved which rely on accuracy as well as strict avoidance of contamination. This scenario thus results in higher complexity and expenditure of effort, combined with a high risk of error. Handling optimization starts with the selection of the right type of vessel which, in turn, is dependent on the number of samples to be processed as well as the sample volume. The more samples there are, the more comfortable it will be to process them in the plate format. The plate format allows for transport of the samples using just one hand, and it provides (better) compatibility with multichannel dispensing systems or automated dispensing stations. In addition, the correct dispensing technique, as well as suitable tools, should be employed to handle the tasks at hand.

The technique of dispensing is superior to simple pipetting: the solution (e.g. the PCR Master Mix) needs to be aspirated into the tip only once, followed by subsequent dispensing of defined portions of the liquid into multiple vessels. Practical tools to help fill plates efficiently include multichannel pipettes – even 16 and 24-channel pipettes are now commercially available. The tips, which are tailored to work with the 384-well plate format, also allow the loading of agarose gels (figure 1).

In the case of higher plate throughput, automated processing saves considerable time and relieves the operator of repetitive routine tasks.

Figure 1: Loading of an agarose gel using a 24-channel pipette

These days, electronic instruments are all around us – at home and at work – and it takes time to learn to operate and subsequently master all their different features. Operating manuals always provide critical support; it is, however, even more helpful if they do not need to be consulted all the time. As such, intuitive operation of an instrument is a basic hallmark of user-friendliness and thus plays an important role in saving time on an ongoing basis during routine applications. A touch screen enables direct interaction with the instrument, thus facilitating quick operation, as long as information is organized in an intuitive manner and the user interface is optimized for this purpose. In the case of a thermocycler, new protocols must often be generated quickly, and it will definitely be beneficial to have different PCR methods handy for use as possible templates. At the same time, it is necessary to adapt protocols for more specific applications through flexible programming. Easy copying of methods and transfer between cyclers (for example, via USB) adds to overall convenience.  

One further point on the list of ways to reduce expenditure is the elimination of entire PCR runs. How is this possible? Consider the following two scenarios: on the one hand, routine runs of established protocols are carried out, and on the other hand, new experiments are developed. The latter often require optimization in order to achieve accurate and reliable results – a process that can easily necessitate a large number of different reactions. It is the aim of PCR to obtain large yields of the desired band(s) without generating unwanted products. To achieve this goal, the optimal annealing temperature must be applied, which, in turn, depends on a number of factors including the primer sequence, the buffer and the PCR reagent – as well as the heating properties of the cycler. For this reason, the annealing temperature must be determined empirically by sequentially testing different temperatures. While there are formulas to help calculate the annealing temperature based on the primer sequences and their melting temperatures, respectively, these methods are generally considered to be merely a first approximation and the basis upon which to build optimization runs. This procedure was considerably simplified by the first thermocycler with gradient technology (Mastercycler® gradient), launched by Eppendorf in 1997. This allows the design of a temperature gradient (across 12 columns) during one temperature step, through separate control of the Peltier elements present within the block. In this way, different annealing temperatures can be tested simultaneously in one run (figure 2).

Figure 2: Screenshot of the Mastercycler® X50 with programmed gradient for the annealing temperature and display of the resulting temperatures in each column.

At this time, this technique has long been standard practice. There are, however, experiments that call not only for optimization of the annealing temperature, but which also require adjustment of the denaturation temperature. In the case of GC-rich templates, for example, the temperature must be high enough to effect denaturation of the DNA-strands while at the same time enzyme activity must be preserved. Frequently, gradient cyclers also allow programming of a temperature gradient in the denaturation step; however, until now, this particular type of optimization needed to be carried out in an independent experiment, separate from the process of optimizing the annealing temperature.  

With the new 2D-gradient technology, which allows programming a vertical as well as a horizontal gradient within a single cycler block, both optimization reactions can now be combined in one experiment (figure 3)

Using this approach, a number of experiments may now become redundant: while separate testing of the denaturation temperature and the annealing temperature is of course possible (using single gradients), the combination of the resulting temperatures may not necessarily bring the desired result. In Application Note 423, a respective experiment is described which shows that the simultaneous optimization of denaturation temperature and annealing temperature may increase yield and/or specificity, and that false-negative results can thus be prevented [1].

Figure 3: Screenshot of the Mastercycler X50 with a programmed 2D-gradient, where the vertical gradient was applied during the denaturation step and the horizontal gradient was applied during the annealing step.

Further to the expedited establishment of new experiments, there are also ways to reduce the total number of routine runs. The technique of Multiplex-PCR is especially well suited to this task since multiple individual reactions are pooled into one reaction in order to amplify different target sequences simultaneously. Optimizing Multiplex-PCR proves more complex than optimizing standard PCR as appropriate conditions must be defined for several primer pairs. For these reasons, this application, too, benefits considerably from 2D gradient technology [2].

The speed of the workflow is only one aspect of productivity in the laboratory; another factor that can influence productivity is throughput – which is the topic of our next article …

Eppendorf®, the Eppendorf Brand Design, Eppendorf twin.tec® and Mastercycler® are registered trademarks of Eppendorf AG, Germany. All rights reserved, including graphics and images · Copyright © 2020 by Eppendorf AG.