利用Mastercycler X50系列PCR独有的2D梯度实现高效的PCR条件...
利用Mastercycler X50系列PCR独有的2D梯度实现高效的PCR条件优化
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.