The CONSTELLATION Digital PCR Priming Module primes your PCR reaction into microfluidic channels where partitioning can occur. The priming module, with a 20 second cycle time, helps to remove sample preparation as a bottleneck in your high-throughput digital PCR workflow.
The CONSTELLATION Digital PCR Rolling Module seals 5 µL of each reaction into 8,000 or 12,000 partitions depending on the plate type used. Since partitioning has a 6 minute cycle time per plate, many rolling modules can be integrated in a single workflow to facilitate higher throughput sample quantification.
The CONSTELLATION Digital PCR Imaging module reads partition fluorescence to calculate the number of target DNA molecules in each sample. The module reads up to 8 different wavelengths providing researchers with a high degree of flexibility in highly multiplexed digital PCR.
1. What is Digital PCR?
Digital PCR is a new alternative to qPCR. It uses microfluidics to provide absolute quantification of the target nucleic acid sequence. In digital PCR, the sample, master mix and primers are mixed, then split into many individual partitions of equal volume. When the partitions are thermally cycled, only the partitions containing the target DNA will amplify. This results in a mixture of “positive” fluorescent partitions and “negative” dark partitions, hence the name “digital” PCR. By counting the number of positive and negative partitions, the original concentration of the target sequence can be determined.
2. How is this different from standard qPCR?
qPCR and dPCR use similar reagents, and the amplification process is the same, but the readout is very different. In qPCR, the fluorescence of the reaction mixture is monitored every cycle, and the cycle that exceeds a threshold fluorescence value is recorded. This threshold cycle is compared to a standard curve, created with samples of known concentration, to calculate a target concentration. With digital PCR, the partitions are thermally cycled to completion (typically 40 cycles). The reactions do not need to be monitored during amplification. After cycling is complete, the number of positive and negative partitions are counted with a fluorescence imaging system. This gives the original concentration directly, without having to compare to a standard curve.
3. Why is dPCR better than qPCR?
Although digital PCR is not always better than qPCR, there are many cases where it gives a more accurate and precise answer. Because the quantification is absolute and binary, as opposed to relative, digital PCR is not dependent on small changes in the amplification efficiency. It is easy to directly compare multiple targets without having to create standard curves or match amplification efficiency.
4. Is amplification efficiency really not important for dPCR?
With digital PCR, each partition is cycled to completion. As long as the amplification efficiency is enough to produce a signal distinguishable from background fluorescence, it will not affect the quantification.
5. What if there is more than one copy of the target in a partition?
Digital PCR can’t detect the number of copies of a template in a single partition, but using Poisson statistics, this can be accounted for. The random distribution of a high concentration of templates in a known number of partitions follows the Poisson distribution. While CVs will increase at very high concentrations, concentrations of up to an average of four templates per partition can still obtain CVs under 10%.
1. How does the CONSTELLATION Digital PCR System work?
The CONSTELLATION microplate has 96 or 24 sample input wells on the top surface, and microfluidic channels and chambers on the bottom surface. Samples are partitioned, thermal cycled, and read on the CONSTELLATION instrument.
2. How many partitions are there per well?
The CONSTELLATION digital PCR system offers distinct plate types designed to accommodate a wide range of throughput and dynamic range requirements of applications from gene expression analysis to rare mutation detection. For higher throughput applications such as gene expression analysis, plates that accommodate 96 samples will provide partitioning into 8000 microfluidic chambers per sample. For more sensitive applications, such as rare mutation detection, plates that divide 24 samples into 36,000 partitions per sample will be available.
3. How much volume do I need per sample?
The minimum per sample reaction volume is 10ul. This includes master mix, primers and DNA samples.
4. How long does it take to run a dPCR experiment on the CONSTELLATION?
The CONSTELLATION integrates partitioning, thermal cycling, and imaging for full walkaway automation of digital PCR. Following simple sample preparation, the instrument takes usersfrom sample to answer in under 1.5 hours for a single plate.
5. What detection chemistry does the CONSTELLATION support?
The CONSTELLATION is designed to work with probe based chemistries (for example, Taqman or IDT probe assays). The CONSTELLATION is also compatible with EvaGreen.
6. Can the CONSTELLATION do multiplexing?
Yes, the CONSTELLATION can multiplex up to 5 probe wavelengths per sample. The default wavelengths are FAM and VIC/HEX. The third, fourth, and fifth can be configured by the user.
Please Contact Us if you have any other questions. We will be happy to answer any additional questions or discuss whether your application would benefit from digital PCR.