Better temperature uniformity allows for better control of PCR reactions
Michelle Cayouette Jane Moores Jason Capodanno
Stratagenes RoboCycler temperature cycler # produces consistent PCR results in every well. The RoboCycler systems unique design uses thermal blocks and heating techniques that are optimized for block temperature uniformity. In this study, fluid temperature profiles and PCR results were determined using both a RoboCycler 96 temperature cycler and a typical Peltier single-block thermal cycler, the MJ Research PTC-100. The RoboCycler system showed better fluid temperature well-to-well uniformity, faster fluid temperature ramping rates, and more consistent PCR-product banding patterns.
When performing PCR amplifications, reaction components and PCR parameters are not the only criteria that affect the quality of results; differences may also exist in the ability of thermal cyclers to maintain a consistent temperature both within and between experiments. If the well-to-well temperature across the thermal block is not uniform, it can have a significant impact on the PCR. This is an especially important consideration for researchers performing quantitative or diagnostic PCR assays using multiple samples.
The four-block design common to all members of Stratagenes RoboCycler line of thermal cyclers provides a higher degree of well-to-well uniformity than the single-block design of many other thermal cyclers. Each of the three RoboCycler hot blocks is heated using a microprocessor-controlled resistive element heater, which covers 95% of the bottom surface of each block. Other thermal cyclers, such as the MJ Research thermal cyclers, use Peltier thermal electric modules attached to the bottom of a single block for heating and cooling. However, the Peltier modul es cover only approximately 60% of the bottom surface of the block; this design creates a temperature gradient from the center to the edges of the block. Moreover, to ramp temperatures up and down quickly, use of the Peltier modules requires that the blocks mass be limited.
Maintaining a consistent temperature across a thermal block is easier when the exposed surface area is kept to a minimum, thereby limiting heat loss due to convection. Each hot block of the RoboCycler temperature cycler maintains its programmed temperature throughout the course of a PCR amplification experiment. A robotic arm moves PCR samples from one preset hot block to another at user-specified times. This eliminates the need to ramp blocks up and down in temperature and permits the use of blocks with minimal surface area. Consequently, RoboCycler temperature cyclers maintain a more consistent temperature throughout all wells of the block and, hence, provide improved PCR results.
To compare well-to-well temperature consistency and sample ramping rate, fluid temperature profiles were determined during thermal cycling using both a RoboCycler 96 temperature cycler fitted with Stratagenes Hot Top Assembly and an MJ Research PTC-100 with heated lid enabled. In each case, fluid temperature measurements were taken in six tubes positioned in the four corner wells and two center wells as shown in Figure 1. Similar testing parameters were used for each thermal cycler. T-type thermocouple temperature probes were inserted into six, 200-l thin-walled tubes containing 50 l of 1X Taq DNA polymerase buffer and cycled through three cycles of 95C for 1 minute, 55C for 2 minutes, and 72C for 1 minute. The temp erature profile for the MJ Research PTC-100 shows that the sample temperature in tubes in the center of the block is up to 1.1C higher than the sample temperature in tubes positioned in the edges of the block (Figure 2). The fluid temperature during the denaturation step varied from 95.3 to 96.4C, with center wells 1.2 to 1.4C higher than the set temperature. The annealing temperature varied from 54.4 to 54.8C, and the extension temperature varied from 72.4 to 73.2C. On average, the PTC-100 changed fluid temperatures at a rate of approximately 0.67C/second, with a coefficient of variation (the standard deviation divided by the mean) of 1.12% between tubes. Therefore, samples placed in different well positions are not only exposed to different temperatures once ramping is complete but also change temperature at different rates. Both of these components combined provide vastly dissimilar PCR conditions between wells of the Peltier-controlled PTC-100.
The temperature profile of the RoboCycler 96 temperature cycler shows a much higher degree of well-to-well temperature uniformity (Figure 2). The fluid temperature during the denaturation step only varied from 95.0 to 95.4C; the annealing temperature varied from 54.4 to 54.6C, and the extension temperature varied from 72.1 to 72.3C. On average, the fluid temperature rate of change for the RoboCycler system was approximately 0.85C/second with a 0.22% coefficient of variation between tubes. This translates to a 27% increase in the average rate of fluid temperature change compared to the PTC-100. Also, unlike the PTC-100, the ramping rates were more consistent from well to well for the RoboCycler system. These fluid temperature results show that PCR cycling conditions using the RoboCycler 96 cycler are virtually identical from well to well.
Experiments were performed to determine if, and to what extent, the temperature variability observed across the block of the MJ Research PTC-100 during fluid temperature testing affected PCR product formation. A fragment of the trkA gene was PCR-amplified from a cDNA library made from human brain frontal cortex. A single PCR master mix was prepared large enough to accommodate six reactions for each of the two thermal cyclers tested. The 50-l aliquots from this master mix were placed into 12, 200-l thin-walled tubes. Six of these tubes were positioned in a RoboCycler 96 temperature cycler fitted with Stratagenes Hot Top Assembly, and six were positioned in an MJ Research PTC-100 with hot top enabled. As described for the fluid temperature measurements, all four corners and two center wells were included. The annealing temperature chosen was known to produce several PCR products that could be used for comparison. Following thermal cycling, aliquots from each reaction were run on a vertical 6% polyacrylamide/TBE minigel, stained with ethidium bromide, and visualized using the Eagle Eye II Still Video System (Figure 3).
The yield and specificity of PCR products obtained using the RoboCycler 96 were similar for all wells tested. However, PCR results obtained using the MJ Research PTC-100 showed a significant degree of well-to-well variability. Similar results were observed when using the RoboCycler standard paddle and the MJ Research PTC-100 with the hot top disabled (data not shown). These results correlate well with the temperature profile obtained from the PTC-100, which showed a difference of up to 1.1 C from the middle to the edges of the block. These PCR results corroborate the block temperature profiles and indicate that researchers can obtain more consistent PCR results from well to well using the RoboCycler 96 thermal cycler.
The RoboCycler temperature cycler provides more consistent fluid temperature profiles between center and edge well positions during PCR thermal cycling than the MJ Research PTC-100. When comparing PCR results from both cyclers, the RoboCycler 96 also shows a greater well-to-well uniformity of amplified products. This temperature uniformity results from the RoboCycler systems unique four-block design that uses thermal blocks and heating techniques optimized for block temperature uniformity.
Cayouette, M. et. al. (1997) Strategies 10: 75-76.
* US Patent number 5,525,300 and patents pending