Polymerase chain reaction (PCR) is a foundational technique for medical and biological research. While quantitative PCR is used for applications such as expression profiling, traditional end-point PCR is most commonly used to amplify DNA and RNA for downstream applications. These include DNA cloning for sequencing or functional analysis of genes, the diagnosis of hereditary diseases, the identification of a genetic fingerprint, and the detection and diagnosis of infectious diseases.
PCR relies on specific primers to amplify defined DNA or RNA sequences of interest, through the use of multiple cycles of denaturation, primer annealing, and synthesis of double-stranded copies by specialized DNA polymerases.
Consistent and uniform amplification is highly dependent on the accuracy and precision of the thermal cycler used to carry out the cycles of amplification.
A thermal cycler for end-point PCR must have several important characteristics to provide specific, efficient, and easily accessible amplification for a variety of applications. Temperature accuracy and uniformity are essential, as annealing temperatures are very precise. Ramp rate is also important, as it determines how fast the thermal cycler can change temperatures, and therefore how fast it can complete a given number of cycles. The thermal cycler must also work efficiently for a wide range of sample volumes. Most importantly, a thermal cycler must allow rapid optimization of cycling parameters, in order to accelerate protocol development and deliver optimal amplification.
Optimization is essential to successful end-point PCR. Unfortunately, it can be a major bottleneck in the amplification workflow, requiring time-consuming and tedious setup and amplification of samples at a series of annealing temperatures. Optimization must be repeated when any of the components of the reaction, including its volume, change.
The SureCycler 8800 Thermal Cycler from Agilent Technologies allows rapid and accurate optimization and affords the ability to generate a temperature gradient across a 96-well or 384-well plate, allowing 12 different annealing temperatures to be tested simultaneously. When combined with a very fast (6 C/sec) ramp rate, this feature enables the rapid, accurate, and efficient optimization required to speed up protocol development.
This thermal cycler also has the ability to change the annealing temperature (Tm) for a given sample location in small downward increments to enable touchdown PCR. Any difference in Tm between correct and incorrect annealing will produce an exponential advantage of twofold per cycle, thus assuring accurate and efficient amplification. Protocol creation is quick and easy with the protocol wizard.
This thermal cycler offers A high ramp speed (6 C/sec) even for reactions as large as 100 µL, enabling rapid optimization and amplification. Throughput is also flexible, due to the ability to interchange 96- and 384-well blocks. Remote access allows users to control the thermal cycler from any location.
Optimization of PCR reactions can be a major roadblock to rapid assay development and high throughput in the laboratory. Temperature gradient capability, fast ramp rate for reactions as large as 100 µL, and the ability to perform touchdown PCR, are keys to overcoming this obstacle.