Proteomics, Genomics & Microarrays
How to Achieve Ultimate PCR Optimisation Authors: Arora Phang, Tim Schommartz, Eppendorf AG, Hamburg, Germany
The new Eppendorf Mastercycler X50 is the only thermal cycler in the market equipped with the innovative 2Dgradient function. PCR optimisation, typically of the annealing temperature using gradient function is an established technique. Optimisation of the denaturation temperature is less commonly done and typically limited to applications dealing with complex or GC-rich DNA templates.
This is mainly due to the high amount of effort required to obtain useful optimal result from the combination of denaturation and annealing conditions. The 2D-gradient function reported herein allows optimisation of both denaturation and annealing temperatures in just one PCR run. This provides users with rich amount of information in the least amount of time and effort, thus greatly shortening the scientifi c research process.
Introduction
Since the inception of PCR, the technique has gone through numerous evolution steps. Similarly, the thermal cycler, a device designed to carry out PCR, has evolved from a simple heating device to one with numerous functions that allows PCR to be performed more efficiently. Perhaps one of the most powerful innovations in the thermal cycler is the gradient function. This function directly targets the fundamental principle of PCR, that the annealing step in PCR is primer-dependent and the correct temperature for this step is very ambiguous and hard to predict. Determination of the correct annealing temperature generally involves much trial and error and this fine-tuning can be very time-consuming. Thermal cyclers with gradient function are able to simultaneously provide multiple different temperatures at a certain step. When used at the annealing step, this function can thus reduce the time and effort needed in optimising the annealing temperature of a primer [1, 2].
On the other hand, the denaturation step in PCR has less ambiguous working temperature, generally only deviating slightly from the temperature specified by the manufacturer. This is because most DNA will be completely denatured at 95°C and most enzymes have a maximum temperature tolerance around that temperature. However, while not as variable as primers, each DNA template has its own characteristic and hence a certain degree of variation is unavoidable. Complex DNA or DNA templates with rich GC content naturally require higher denaturation temperature. Thus, while PCR might be successful without optimising the denaturation step, the quality and yield of the PCR might not be optimal. An optimal PCR is thus to a smaller or larger degree also affected by the denaturation temperature used [2].
To date, it is possible to optimise the denaturation and annealing steps of a PCR system by doing two separate runs (by keeping either the denaturation or the annealing temperature constant while changing the other). To find the best combination of optimal denaturation and annealing temperatures, one would have to first run a gradient for the annealing temperature. Subsequently, for each of the annealing temperatures tested, a gradient is then repeated for the denaturation step. This would result in multiple PCR runs that is both time- and resource-consuming. With the introduction of the new Eppendorf Mastercycler X50 however, this difficulty can now be solved. This article will present a new innovative technique called the 2Dgradient that allows for the ultimate PCR optimisation with utmost ease and speed.
Materials and Methods
PCRBio Taq DNA polymerase (Nippon Genetics) and Human Genomic DNA (Roche®) were used for the following amplifi cation. PCR reaction master mix containing 1X reaction buffer, 0.25U of enzyme, 0.2 µM of each primer and 20ng DNA template was prepared. 10 µl of the master mix was dispensed into each respective 96 wells of Eppendorf twin. tec® skirted PCR plates. Dispensing was carried out by Eppendorf epMotion® 5073. Plates were sealed with adhesive PCR fi lm and PCR was carried out on Mastercycler X50s.
The following primers were used for amplifi cation of the human ß-actin gene:
Cycling conditions are listed in Table 1. The PCR products were detected using GelRedTM (Biotium) following agarose gel electrophoresis and visualised using the Gel Doc XR+ (BioRad®).
Table 1: PCR condition with two concurrent gradient setting at denaturation and annealing steps.
Results and Discussion
The new 2D-gradient function of the Mastercycler X50 enables optimisation of both the denaturation and annealing temperatures in one PCR run. This was achieved through a matrix-style temperature set-up whereby the fi rst gradient at denaturation step is set vertically while the second gradient at annealing step is set horizontally. This means that each of the eight rows of the thermal block has a different temperature at the denaturation step while each of the 12 columns of the thermal block has a different temperature at the annealing step.
Hence, for each denaturation temperature (TD), 12 samples would be amplifi ed at that temperature (e.g. wells A1–A12 would be subjected to 99°C TD while B1–B12 would be subjected to 98.5°C TD). After the denaturation step, samples under the same column would be subjected to the same annealing temperature (TA), thus giving rise to 12 different TA across the block (e.g. A–H1 would be subjected to 51.9°C TA and A–H2 would be subjected to 52.3°C TA). At the end of the completed PCR, the best combination of denaturation+annealing temperatures can then be determined (Figure 1).
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