STABILITY OF TAQ DNA POLYMERASE AT ELEVATED TEMPERATURES
Ampliqon Taq DNA Polymerase is a very stable enzyme with a half-life time of 40 minutes at 95°C. The half-life time rapidly increases with only a few degrees of temperature drop. To evaluate the thermostability we tested the activity of Taq polymerase at three different temperatures.
Experimental set-up:
Ampliqon Taq DNA Polymerase of the same batch was distributed into several tubes and incubated at the temperatures 40 °C ± 2 °C, 25 °C ± 2 °C and 5 °C ± 3 °C. At specified time points (table 1) samples were collected in duplicates and transferred to -20 °C until analysis. One sample (in duplicates) was stored at -20 °C from the start to serve as T0 sample which never was exposed to elevated temperatures. The T0 sample serves as reference for 100 % activity and is used to prepare the standard series.
Table 1: Time points of Taq Stability Test.
Analysis was done in two ways. Firstly, real-time PCR was applied to monitor relative changes in enzyme activity. This was done by diluting the samples to a concentration, where the amount of Taq DNA Polymerase was limiting, thereby allowing to monitor changes in enzyme activity. Starting with the same dilution, a standard curve for Taq polymerase activity was prepared from the sample without incubation at elevated temperatures for relative quantitation. Secondly, an end-point PCR was performed to show the influence of enzyme activity to a standard PCR reaction. To challenge the system, the PCR was done using 3 dilutions of gDNA.
Ampliqon Taq Polymerase incubated at 40 °C:
Real-time PCR using 100 mU/reaction of Ampliqon Taq polymerase which was incubated at 40 °C for the indicated times. Sample amplification plots are shown in red. The amplification plot of 100 % activity is shown in yellow and the rest of the standard series is shown in black.
Conclusion: A decrease in enzyme activity is represented by the change of the slope of the curves as demonstrated with the standard series. After 2 weeks at 40 °C, there is no loss of activity seen. After 4 weeks still 70 % of activity is retained. After 6 weeks, the activity has decreased to about 50 % and after 8 weeks to about 10 %
The end-point PCR in figure 2 shows the same amount of yield for enzyme incubated at 40 °C for up to 6 weeks. The 8 week sample still provides an amplicon but a reduction in yield is seen. In a standard PCR reaction, the polymerase is present in vast excess. Therefore, a loss of 50 % enzyme activity is not visible.
Conclusion: Standard end-point PCR using 1 U/reaction of Ampliqon Taq polymerase which was incubated at 40 °C for the indicated times and 50 ng, 10 ng or 0,6 ng of gDNA in triplicates. M = marker, ntc = no template control.
Ampliqon Taq Polymerase incubated at 25 °C:
Real-time PCR using 100 mU/reaction of Ampliqon Taq polymerase which was incubated at 25 °C for the indicated times. Sample amplification plots are shown in green. The amplification plot of 100 % activity is shown in yellow and the rest of the standard series is shown in black.
Conclusion: There is no loss of activity after 2 months. After 4 months, 95 % of activity is retained. After 6 months at RT, the activity still lies between 60 % and 70 % and after 8 months, the activity has decreased to about 60 %.
Standard end-point PCR using 1 U/reaction of Ampliqon Taq polymerase which was incubated at 25 °C for the indicated times and 50 ng, 10 ng or 0,6 ng of gDNA in triplicates. M = marker, ntc = no template control.
Conclusion: The end-point PCR displayed in figure 4 shows no loss of yield for enzyme incubated at 25 °C for up to 4 months and only a slight loss of yield for enzyme incubated at 25 °C for 6 and 8 months. Again, the excess polymerase present in a standard PCR reaction compensates for the loss of up to 40 % of enzyme activity.
Ampliqon Taq Polymerase incubated at 5 °C:
Real-time PCR using 100 mU/reaction of Ampliqon Taq polymerase which was incubated at 5 °C for the indicated times. Sample amplification plots are shown in blue. The amplification plot of 100 % activity is shown in yellow and the rest of the standard series is shown in black.
Conclusion: The amplification plots for Taq polymerase incubated at 5 °C for 18 months and 24 months both lay close to the amplification plot of 100 % activity indicating, that there is no loss of activity after storage of Taq polymerase at 5 °C for 18 months and only a slight decrease of activity after 24 months.
Standard end-point PCR using 1 U/reaction of Ampliqon Taq polymerase which was incubated at 5 °C for the indicated times and 50 ng, 10 ng or 0,6 ng of gDNA in triplicates. M = marker, ntc = no template control.
Conclusion: End-point PCR showed that Taq polymerase stored at 5 °C for up to 24 months show no changes in yield compared to the enzyme kept at -20 °C for the whole period of time.
AMPLIQON TAQ POLYMERASE IN THE FREEZE-THAW STRESS TEST
To test the resistance of Ampliqon Taq polymerase to freezing and thawing, a freeze-thaw test was performed applying 50 freeze-thaw cycles. To make the test even more challenging, Ampliqon Taq polymerase was kept in a buffer without glycerol. Glycerol is a cryoprotectant, and are normally part of the storage buffer where it serves to protect Taq polymerase during freezing conditions.
Experimental set-up:
Samples were thawed at 30 °C for 7 minutes, shortly vortexed and spun down at room temperature (RT) and then placed on ice. 30 minutes after the start of thawing, samples were placed at -20 °C for at least 1 h before the cycle was started again. At defined numbers of cycles, the according samples were collected and stored at -20 °C until analysis. One sample was kept at -20 °C without any thawing to serve as reference for 100 % activity and to prepare the standard series.
Analysis:
Analysis was done using real-time PCR. Samples were diluted to a concentration, where the amount of Taq polymerase was limiting, thereby allowing to monitor changes in enzyme activity. Starting with the same dilution, a standard curve for Ampliqon Taq polymerase activity was prepared from the sample without freeze-thaw cycles. The standard curve amplification plots are shown in yellow for 100 % activity and in black for the rest of the standard series.
Freeze-thaw test. The amplification plots show the sample curves in purple, 100 % activity in yellow and the rest of the standard series in black. The rectangle depicts the area where the curves are in the linear range. The right part of the figure shows an enlargement of the depicted area of the amplification plots.
Conclusion: A decrease in enzyme activity is represented by the change of the slope of the curves as demonstrated with the standard series. The amplification curve of the sample with 40 freeze-thaw cycles lies close to the standard amplification curve with 100 % activity, indicating only a slight decrease in Taq polymerase activity. After 50 freeze-thaw cycles, still 90 % of the full activity for Taq polymerase is retained.
CONCLUDING REMARKS:
Ampliqon Taq DNA polymerase is a remarkable thermostable enzyme. Our results show that it is tolerant to incubation at elevated temperatures for long periods without losing activity: at 40 °C for 2 weeks; at 25 °C for 2 months and at 4 °C for 18 months.
Ampliqon Taq DNA polymerase can withstand a high number (> 40) of freeze-thaw cycles with only slight decrease in activity. At lower temperatures, Taq polymerase is stable for even longer time. Storage at -20 °C is therefore recommended in order to guarantee maximal shelf life. However, if you forget your enzyme on your lab bench even for several days, no harm is done due to the high stability of Taq polymerase at room temperature.
Taq DNA polymerase is mainly used to perform end-point PCR. In standard PCR setups, Taq DNA polymerase is present in vast excess, which makes the method tolerant to up to 50 % decrease of enzyme activity.
Ampliqon Taq DNA polymerase offers a perfect combination of heat resistance, robustness, specificity, sensitivity and yield and is commonly agreed to be one of the best polymerases available.
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