29 Proteomics, Genomics & Microarrays
Figure 1: 2D-gradient function can be used in a matrix-style optimisation of both denaturation and annealing temperatures concurrently to fi nd the optimal condition for highest PCR yield.
Optimal PCR result is defi ned by maximum yield of the specifi c amplicon of interest. Therefore, the aim of PCR is always fi rst and foremost specifi city followed closely by yield. While this can be primarily achieved through optimising 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-specifi c product be reduced.
Figure 2 shows the result of the matrix-style optimisation technique of the 2D-gradient in amplifying the human ß-actin gene. This PCR system was chosen because of its temperature sensitive nature. Specifi c amplifi cation will yield 484 bp fragments while sub-optimal condition will give rise to non-specifi c amplifi cation visible as a 350 bp artefact in the gel.
Ordinarily, gradient optimisation is only performed for the annealing step at a fi xed 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-specifi c product and at 70.5°C, only specifi c product will be obtained. Depending on the objective of the PCR, both of these temperatures can be considered ‘optimal’ conditions that are usually suffi cient 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 specifi c bands almost doubled in intensity. In addition, the results in this study showed that increasing
TD leads to decreasing non-specifi c amplifi cation. For PCR systems where non-specifi c amplifi cation is a problem, especially those with multiple bands, running a gradient at denaturation step would be especially benefi cial. Hence the 2D gradient allows users to easily obtain a rich amount of information about the characteristic of their PCR system,
Figure 2: PCR optimisation of ß-actin gene with 2D gradient technique.
which in turn is benefi cial for various application objectives such as increasing yield or resolving non-specifi c amplifi cation problems.
Conclusion
The 2D-gradient function of the Mastercycler X50 allows users to simultaneously optimise both denaturation and annealing temperatures to determine the conditions for combined optimal yield and specifi city for best PCR result. Not only does the convenience of this function allow users to save much time and effort in their optimisation 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-specifi c amplifi cation 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 fl exible operation on the dual block thermocycler Mastercycler® nexus GX2. Eppendorf Application Note 289.
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