Eppendorf Mastercycler X50系列PCR儀是當前市售唯一具有2D梯度功能的熱循環(huán)儀。利用梯度功能設定退火溫度的變化是當前PCR優(yōu)化中最為成熟的技術。然而，對于變性溫度的優(yōu)化卻并不常見，通常只限于處理復雜或是富含GC的DNA模板。
為了從變性與退火的組合條件中獲得有用的優(yōu)化結(jié)果，需要進行大量的工作，這往往會讓人望而生畏。本篇應用旨在介紹如何在一次PCR運行中同時優(yōu)化2D（變性和退火）梯度參數(shù)，這可以使得用戶在有限的付出與時間下獲得豐富足量的信息，極大地縮短了科研進程。

Ultimate PCR Optimization with Eppendorf Mastercycler® X50 2D-gradient
Arora Phang, Tim Schommartz,
Eppendorf AG, Hamburg, Germany
 

Abstract

The new Eppendorf Mastercycler X50 is the only thermal cycler in the market equipped with the innovative 2Dgradient function. PCR optimization, typically of the annealing temperature using gradient function is an established technique. Optimization 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 optimization 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 scientifc 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 al lows PCR to be performed more efciently. Perhaps one of the most powerful innovations in the thermal cycler is the gradient function. This function directly targets the funda mental 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 fne-tuning can be very time-consuming. Thermal cyclers with gradient function are able to simulta neously 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 optimizing the annealing temperature of a primer1, 2.
On the other hand, the denaturation step in PCR has less ambiguous working temperature, generally only deviating slightly from the temperature specifed 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 charac teristic and hence a certain degree of variation is unavoid able. Complex DNA or DNA templates with rich GC content naturally require higher denaturation temperature. Thus, while PCR might be successful without optimizing the dena turation 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 used2.
To date, it is possible to optimize 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 fnd the best combination of optimal denaturation and anneal ing temperatures, one would have to frst 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 difculty can now be solved. This Application Note will present a new innovative technique called the 2D gradient that allows for the ultimate PCR optimization with utmost ease and speed.

Materials and Methods

PCRBio Taq DNA polymerase (NIPPON Genetics) and Hu man Genomic DNA (Roche®) were used for the following amplifcation. PCR reaction master mix containing 1X reac tion buffer, 0.25U of enzyme, 0.2 µM of each primer and 20 ng 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 Eppen dorf epMotion® 5073. Plates were sealed with adhesive PCR flm and PCR was carried out on Mastercycler X50s.
The following primers were used for amplifcation of the human ß-actin gene:
  Forward primer: 5’- ATCGCCGCGCTCGTCGTC-3’
  Reverse primer: 5’- TGGGTCATCTTCTCGCGGTTGG-3’
Cycling conditions are listed in Table 1. The PCR products were detected using GelRedTM (Biotium) following agarose gel electrophoresis and visualized 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 optimization of both the denaturation and annealing temperatures in one PCR run. This was achieved through a matrix-style temperature set-up whereby the frst 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 temper ature at the denaturation step while each of the 12 columns of the thermal block has a different temperature at the an nealing step.

Figure 1: 2D-gradient function can be used in a matrix-style optimization of both denaturation and annealing temperatures concurrently to fnd the optimal condition for highest PCR yield.

Hence, for each denaturation temperature (TD), 12 samples would be amplifed 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 T A across the block (e.g. A–H1 would be subjected to 51.9 °C T A 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 deter mined (Figure 1).
Optimal PCR result is defned by maximum yield of the specifc amplicon of interest. Therefore, the aim of PCR is always frst and foremost specifcity followed closely by yield. While this can be primarily achieved through optimiz ing the annealing temperature, there is no guarantee that the result obtained is the true “optimal” result. It is always possible that the yield could be increased or the amount of non-specifc product be reduced.
Figure 2 shows the result of the matrix-style optimization technique of the 2D-gradient in amplifying the human ß-actin gene. This PCR system was chosen because of its temperature sensitive nature. Specifc amplifcation will yield 484 bp fragments while sub-optimal condition will give rise to non-specifc amplifcation visible as a 350 bp artefact in the gel.
Ordinarily, gradient optimization is only performed for the annealing step at a fxed denaturation temperature at ca. 95 °C. Taking the example from Figure 2, when 95.6 °C is used, gradient result for annealing step showed that 65.9 °C gives the best yield with small amount of non-specifc prod uct and at 70.5 °C, only specifc product will be obtained. Depending on the objective of the PCR, both of these tem peratures can be considered “optimal” conditions that are usually sufcient for most applications.
However, in certain cases such as low target copy number, a small difference in yield can be crucial to the application. In the example above, it can be clearly seen that 95.6 °C is not an optimal TD for this PCR system. By lowering the TD to 93.4 °C, the specifc bands almost doubled in intensity. In addition, the results in this study showed that increas ing TD leads to decreasing non-specifc amplifcation. For PCR systems where non-specifc amplifcation is a problem, especially those with multiple bands, running a gradient at denaturation step would be especially benefcial. Hence the 2D gradient allows users to easily obtain a rich amount of information about the characteristic of their PCR system, which in turn is benefcial for various application objectives such as increasing yield or resolving non-specifc amplifca tion problems.

Figure 2: PCR optimization of ß-actin gene with 2D gradient technique.

Conclusion

The 2D-gradient function of the Mastercycler X50 allows users to simultaneously optimize both denaturation and annealing temperatures to determine the conditions for combined optimal yield and specifcity for best PCR result. Not only does the convenience of this function allow users to save much time and effort in their optimization work, it also has important implications for applications relating to low target copy number and GC-rich targets. In addition, this function is highly useful in troubleshooting non-specifc amplifcation issues.

References

[1] Ong, W.K. (2010) Using the gradient technology of the Mastercycler® pro to generate a single universal PCR protocol for multiple primer sets. Eppendorf Application Note 220.
[2] Gerke, N. (2013) Straightforward PCR optimization and highly flexible operation on the dual block thermocycler Mastercycler® nexus GX2. Eppendorf Application Note 289.