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Thermal Cyclers and PCR Plasticware - The Most Undervalued PCR Components
Rita Weispfenning, Promega
During the 22 years that I have been working in Technical Support at Promega, I have had plenty of opportunities to interact with many international scientists. Through the investigative processes that served to find and correct possible application or handling errors and numerous scientific discussions, I have gained detailed insights into daily laboratory routines and the variety of equipment and lab consumables used for short tandem repeat (STR) analysis at different customer sites. To my surprise, I found a striking contradiction in customers’ attitudes regarding PCR amplification.
How is it possible that people who show the utmost conscientiousness in the preparation, processing, and documentation of their laboratory procedures, often seem to completely underestimate two important variables that impact the amplification success to the highest degree? The selection of thermal cyclers and PCR plasticware for STR amplification, is often done without giving it much thought.
While at customer sites, I have repeatedly come across a wide variety of thermal cyclers in the customers’ PCR rooms. Typically, I have seen different models of all ages from various manufacturers, with profoundly different performance capabilities, line up one after another on the benches. It is rare to find more than two cyclers of the same model series at one place. Usually, two or three somewhat modern cyclers are dedicated to the PCR of a particular new generation STR kit. This in itself is a problem if the cyclers are of quite different model series; it is even common for some laboratories to use additional cyclers, often antiquated models when funding gets tight. As far as the choice of PCR plates goes, quite often I have noticed boxes of generic yet diverse PCR plates stacking up on the shelf above the thermal cyclers. Observing this made me realize that it is a common belief among forensic scientists that both thermal cyclers and PCR plasticware are completely interchangeable.
Yet, the selection of the individual thermal cycler models and PCR plasticware—and the combination—is absolutely critical for the success of amplification with fast STR kits. Their interaction regulates the temperature of the reactions in the wells at any time during PCR. This regulation determines whether or not reactions reach the required temperatures quickly enough and maintains those temperatures for a sufficiently long time – which is key for a successful PCR. Accordingly, thermal cyclers and PCR plates, strips, or tubes for STR analysis must be chosen with the utmost care.
In the example of PCR laboratory equipment described above, it can be assumed that the PCR results will vary greatly depending on the cycler and PCR plate used. In any case, one cannot hope for reliably good results when using the incorrect thermal cyclers and plate combination, because it seems to be a matter of luck whether a perfect PCR result is obtained.
However, I admit that it is incredibly challenging right now to get the PCR plastic consumables you want. The pandemic, container shortages, factory fires, the blockade of the Suez Canal by a container ship and other logistical problems have caused production delays in many plastics industries. Constraints on the supply of their raw materials—especially polyethylene (PE) and polypropylene (PP)—are leading to factory shutdowns, sharp price increases, and production delays across a range of industries. Consequently, some suppliers of PCR plastics must resort to other subcontractors or risk delivery delays. In this situation, many scientists see themselves forced to change their routine plasticware just so they can continue working at all. There is light at the end of the tunnel, though: market analysts see the world's supply disruptions as temporary, with things heading back toward normal in 2022.
Tips for choosing thermal cyclers and PCR plasticware for STR amplification
Of course, there are many high-quality thermal cyclers and PCR plasticware products on the market. Still, it is by no means sufficient just to select a superior product for each of them. Not all thermal cyclers and PCR plastic items are optimal in combination, i.e., some do not offer an optimal heat transfer from the heating block to the samples in the wells.
Considerations for buying a thermal cycler suited for fast STR kits
Pay attention to the material of the heating block
For successful PCR to occur, it is essential that the thermal cycler provides high temperature stability and accuracy, as well as temperature homogeneity, over the entire heating block. Cyclers with blocks made of silver, gold or their alloys fulfill these criteria better than cyclers with aluminum heating blocks. The latter often do not reach the desired temperatures at all, or only after a delay; moreover, they are unable to spread the temperature evenly over the heating block.
Don’t only rely on the maximum block ramp rates
New generation STR kits require fast cycling programs, so how quickly a thermal cycler can switch between different temperatures is essential for successful PCR.
For this reason, many thermal cycler manufacturers only specify the maximum block ramp rates of their models. However, this value describes the highest achievable block performance, which can only be maintained during a very short period during the ramp. When selecting a thermal cycler, it is crucial to inquire about the average block ramp rate, which reveals the rate of temperature change over a longer section of the ramp and is more representative of the performance and speed of the thermal cycler.
Ask about the sample ramp rates
Just because the heating block has achieved the desired temperature does not mean that the samples in the wells have reached the same temperature value. It takes some time and varies depending on the PCR plasticware used for the heat to be transferred from the thermal cycler block to the sample. Some manufacturers therefore specify average and maximum sample ramp rates for their thermal cyclers, in addition to the ramp rates of the heating blocks. These decisive variables should help you in the selection of thermal cycler models. Unfortunately, the manufacturer's specifications for the sample ramp rates of various cycler models are usually lacking any information about which PCR plasticware was used in the tests. In the cases where the manufacturer offers both thermal cyclers and consumables, one may be able to guess which PCR plates were used for the experiments.
For successful PCR of new-generation PowerPlex® systems, the cyclers should be able to offer maximum sample ramp rates as fast as 4.4°C/sec for heating up and 3.4°C/sec for cooling down. The respective required maximum block ramp rates are, of course higher: 6.0 °C/sec for up ramping and 4.4°C/sec for down ramping (1).
What should be considered when selecting PCR plates, strips or single tubes suited for fast STR kits?
PCR plasticware can be found in countless designs. There are single tubes, strip tubes or 96-well plates. Plates are further divided into non-skirted, semi-skirted or full-skirted products. There are items made of polypropylene or polystyrene, with and without barcodes, and of course you can choose between different well sizes and colors. Who’s not getting lost here?
As a rule, non-skirted plates fit most cycler models, while semi- or full-skirted plates are easier to handle due to their higher stability and are therefore more suitable for automated laboratory processes.
With this wide range of choices, however, keep in mind that even slight differences between the various products (in terms of shape, size and wall thickness) can significantly affect the heat transfer rate between the heating block and the sample. To ensure that the interaction between thermal cycler and PCR plasticware is optimal, there are no shortcuts, and it is essential to test both in combination.
Test, test, test
Whether PCR plasticware is suitable for a particular cycler model can only be determined by performing side-by-side amplifications on this cycler between the specific plasticware and any of our recommended PCR plasticware (see below) and comparing both results. When comparing plates, distribute the reaction mixture from the same preparation on both plates, using the same areas in each case with at least one complete column. Following PCR, make sure to load the amplicons from both PCR batches onto the CE plate; it is vital that these amplicons are separated by the same capillaries. Only then can a fair comparison between both PCR plastic items be guaranteed.
Our recommendations
If you want to save yourself tedious testing and evaluation time, you can make things easier by simply choosing the combination of thermal cyclers and plasticware that Promega has tested already for optimal results. We used these combinations during the development and for the developmental validations of our PowerPlex® systems. Both groups of instruments and consumables are completely synchronized with each other and promise optimal PCR results with our PowerPlex® Systems.
Once you have identified the ideal combination of one particular thermal cycler model and one specific PCR plasticware for your needs, stick with it and, if possible, equip your entire PCR room with it. If you need to purchase additional cyclers later, buy the same model over and over again. If it becomes unavailable at some point, opt for its successor.
Following this approach, you will only have to optimize the PCR for specific STR kits once and you will always get reliably good results, no matter which of your cyclers you use.
Troubleshooting
Suppose you are in the unfortunate position of not having an optimal thermal cycler, or your combination of cycler and PCR plasticware is not ideal for the amplification of fast STR kits. If you further lack the resources to get the optimal instrument or combination of instrument and consumables, you may find the following explanations and tips helpful.
What are the consequences of inefficient heat transfer?
When heat transfer from the block to the samples is very slow, it can have a drastic effect on modern STR systems, which require fast cycling programs. The denaturation and annealing steps appear to be affected most under these conditions.
Impairment of DNA denaturation
As can be seen from the illustration of the PowerPlex® ESX and ESI Fast PCR cycling program (Fig. 1), a duration of just 5 seconds is allocated for DNA strand separation. As soon as the heating block reaches the temperature of 96°C, the 5-second countdown begins, which again is immediately followed by the cooling phase.
So, it is easy to imagine what happens when heat transfer is slowed down. The cooling phase of the heating block is initiated before the samples have even reached the target temperature, causing the double-stranded DNA to be improperly denatured in this case. As a result, some loci (such as TH01 or D2S1338) amplify poorly, which may be related to their tendency to develop secondary structures (Fig 2.).
As mentioned previously, this problem occurs regularly in thermal cyclers with aluminum heating blocks, because the blocks have difficulties reliably reaching the high temperatures. Further, the heat transfer rate can be handicapped by the use of unsuitable PCR plasticware.
Fig. 1: Thermal Cycling Protocol for PowerPlex® ESX and ESI Fast Systems
Fig 2.: Peak height comparison of samples amplified using the PowerPlex® ESI 17 Fast System and the Applied Biosystems® 2720 thermal cyclers with an aluminium heating block or GeneAmp® PCR System 9700. Amplifications were performed as described in the PowerPlex® ESI 17 Fast System Technical Manual (TMD041). Panel A: the blue dye channel, Panel B: the yellow dye channel, Panel C: the green dye channel, Panel D: The red dye channel.
What can you do if you have to rely on a cycler with an aluminum heating block?
Thermal cyclers equipped with an aluminum heating block can be countered with a trick:
Increase the denaturation temperature
If the temperature of the denaturation step is increased by 2°C to 98°C (2) it is more likely that at least some areas of the heating block will reach the 96°C set point (Fig. 3).
This "block overshoot thermal energy" is actually a method as applied by thermal cyclers equipped with modern temperature control mechanisms. They use an algorithm that drives the block temperature above the set point to allow the sample to reach the required temperature faster and more reliably. Moreover, these “super cyclers” have the advantage that the thermal overshoot energy is calculated based on the sample volume, and that the time for this PCR step is not counted down until the samples have reached the desired temperature. Of course, the emergency solution for cyclers with aluminum heating blocks cannot offer all these advantages, but it will enable the heating blocks to reach the desired temperature.
Fig. 3: Peak height comparison of samples amplified using an Applied Biosystems® 2720 thermal cycler with aluminum heat block with the PowerPlex® ESI 17 Fast System and 96°C and 98°C denaturation temperatures. Panel A: the blue dye channel, Panel B: the yellow dye channel, Panel C: the green dye channel, Panel D: the red dye channel.
Difficulties with primer annealing
If there is a substantial delay for samples to adopt the temperature of the heating block, this will negatively impact primer annealing.
As the annealing phase follows the denaturation step, the heating block must cool down by 36°C from the highest temperature in the PCR cycle to 60°C. Reaching this set point is necessary because it represents the temperature at which primers anneal to their complementary regions on the DNA under optimal conditions Once the block reaches this perfect annealing temperature, it holds at that temperature for 35 seconds, as seen in the example of the PowerPlex® ESI and ESX Fast PCR. We know, of course, that even under ideal conditions, there is always a short delay in the temperature transfer from block to sample. It takes a few seconds for the samples to reach 60°C, which does not matter because the short delay was taken into account when developing the PCR program. Nonetheless, it does become critical if the plastic materials further hinder the temperature transfer to the samples. Then, in the worst case, the samples may never reach the desired temperature of 60°C in the short time allotted for primer annealing. Instead, they remain at higher temperatures throughout the entire annealing phase, which is detrimental to the amplification of loci that are particularly sensitive to elevated annealing temperatures, such as Amelogenin, and SE33 (Fig. 4). Their amplification success is already reduced by more than half at 62°C.
Fig. 4: PowerPlex® ESI 17 Fast System with low peak heights at Amelogenin and SE33 due to inadequate heat transfer from heating block to sample.
What can you do if Amelogenin and SE33 are poorly amplified due to insufficient heat transfer from heating block to sample?
Two measures can help here. Before applying the first one, you must be entirely sure that the PCR is affected by insufficient heat transfer and not for other reasons, which do exist. PCR inhibition should be mentioned here which, when it occurs, exhibits very similar symptoms. Therefore, be sure to confirm that Amelogenin and SE33 have amplified poorly in the PCR of the positive amplification control, the 2800M DNA.
Other loci—such as FGA, D8S1178 or D2S441—may also be weak in the profile, but it is always Amelogenin or SE33 whose amplification is affected first or most severely.
Reduce the annealing temperature by 1–2˚C
Contrary to the thermal overshoot method, this measure is applied in the other direction and thus portrays a thermal undershoot of the setpoint. Reduced annealing temperatures of 59°C or 58°C ensure that the samples adopt the desired annealing temperature reliably despite the insufficient heat transfer (Fig. 5).
Fig. 5: PowerPlex® ESI 17 loci average peak heights at two different annealing temperatures using a thermalcycler-PCR Plate combination with insufficient heat transfer.
Warning: If you, however, observe poor amplification of Amelogenin and SE33 only in DNA samples but not in the positive DNA control, it is not inefficient heat transfer rate that impairs PCR. Do not decrease the annealing temperature This measure would be an attempt to correct a problem that is essentially due to issues elsewhere in the analysis workflow, such as in the extraction or purification of the samples. In addition, you would risk more artefacts being generated.
Alternatively, you can counteract the reduced heat transfer with a second measure that allows more time for the PCR annealing process.
Extend the duration of the annealing step
By extending the length of the annealing phase, the sample is given more time to reach the desired temperature. This action naturally leads to a longer PCR time. For example, if you extend the annealing step by 15 seconds, the total PCR time of a 30-cycle program will increase by additional 7.5 minutes. However, this is a reasonable tradeoff, given the improved results that would be achieved when doing so.
If you have additional questions that are not addressed in this article or need assistance with optimizing Promega STR chemistries on thermal cycler models that are not mentioned in this article please don't hesitate to contact the author or another member of the Promega Technical Support team for assistance.
References
- Thermo Fisher Scientific Inc. (2015) Thermal cyclers: key thermal cycling concepts and ramp rates. (Application note)
- McLaren, R.S. et al. (2014) Developmental validation of the PowerPlex1 ESI 16/17 Fast and PowerPlex1 ESX 16/17 Fast Systems. Forensic Sci Int. Genet. 13, 195–205
PowerPlex is a registered trademark of Promega Corporation.
Applied Biosystems, GeneAmp, Veriti, ProFlex and MicroAmp, are registered trademarks of Applied Biosystems.