|Absolute Quantification for:||Gene Expression Analysis – Rare Mutation Detection – Copy Number Variation|
In digital PCR (dPCR), the sample is divided into many separate partitions, with each partition containing either a single template molecule or none. After thermal cycling, the number of partitions containing the amplicon are counted, resulting in absolute quantitation of the target gene. A major advantage of dPCR is that it is not sensitive to reaction efficiency, nor does it require the preparation of standard curves.
Because partitions containing templates are cycled to completion and counted as positive or negative, digital PCR (dPCR) doesn’t require perfect amplification. Quantification using standard qPCR is sensitive to small changes in amplification efficiency, so each experiment needs to be well-optimized to obtain accurate data. Efficiency can be affected by: inhibitors in the sample, changes in annealing temperature, different master mixes, and/or different primers. With the CONSTELLATION® digital PCR system, each partition either contains the target sequence or it doesn’t, so the quantification is unaffected by small differences in amplification efficiency. This allows dPCR experiments to maintain accuracy in the presence of inhibitors.
Flexible Experimental Setup.
dPCR allows direct comparison between different targets. If you are testing several different targets that vary in melting temperature or other factors, dPCR can still directly compare quantities without the need to optimize amplification conditions.
Direct and Absolute.
You don’t have to create standards with known quantities of target genes and pipette them to each plate. As the number of templates in the sample can be extrapolated and quantified, absolute quantification is straight-forward, which allows you to compare results obtained by different labs, at different times, and under different experimental conditions.
The CONSTELLATION dPCR microplate provides increased detection specificity. In cases where the target is relatively rare compared to the amount of non-target DNA, the background DNA can compete for reagents and cause non-specific amplification. Partitioning the sample into many small chambers on the dPCR microplate increases the effective concentration of rare targets in the partitions.
The CONSTELLATION facilitates high throughput without high capital cost. Standard qPCR requires a real-time thermal cycler for each plate for the full cycling process, and scaling-up throughput requires purchasing multiple real-time thermal cyclers. The CONSTELLATION dPCR microplate can be thermally cycled on non-real-time thermal cyclers and then analyzed with the CONSTELLATION. Thermal cyclers can be added to increase throughput.
Digital PCR has been widely adopted for sensitive applications such as copy number variation and rare gene detection for which the lower limit of detection excludes qPCR as a viable solution. However, due to limitations in the platforms currently available for digital PCR, such as inadequate throughput, high cost per sample, and extensive hands-on time, researchers cannot justify the time and cost associated with transferring more mainstream applications over from qPCR. Even though digital PCR provides absolute quantification of target nucleic acid sequences, meaning that in practice, it provides more accurate data, quantitative PCR has provided, until now, the only feasible way to quantitate at the throughput and cost demanded by mainstream applications, such as gene expression analysis. The CONSTELLATION, however, mimics contemporary qPCR with a plate-based and high-throughput workflow capable of processing up to four plates per run.
Flexible Sample Formats.
The CONSTELLATION 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.
The CONSTELLATION integrates partitioning, thermal cycling, and imaging into a single fully automated instrument that takes users from sample to answer in under one and a half hours. With up to five channel filters, the CONSTELLATION is the first ever digital PCR system to offer five color multiplexing making it a high-throughput platform capable of displacing qPCR as the method of choice for high-throughput quantification of nucleic acid targets.
In addition to the throughput and analysis capabilities of the system, the CONSTELLATION also provides genomics researchers with a more familiar workflow than other existing digital PCR systems. Since the system is plate-based, the addition of sample, master mix, and primers can be performed just like in qPCR workflows, enabling researchers to more easily transfer their current multiplex qPCR assays to digital PCR. Additionally, a plate based system is more amenable to automated sample preparation, and FORMULATRIX intends to couple the capabilities of the MANTIS® Liquid Handler, a reagent dispenser designed to dispense primers and enzymes, enabling high-throughput, reproducible, and efficient sample preparation, with the new digital PCR platform.
Sample Preparation Workflow.
The CONSTELLATION® software can be used to easily view digital PCR results generated on the instrument. In addition to controlling the instrument, the software can run on desktop PCs for experiment setup and analysis at your desk.
After pipetting the PCR reaction mixture into the proprietary 96- or 24- well CONSTELLATION® Digital PCR System microplate and sealing the plate, the plate is placed into the CONSTELLATION drawer. Each sample-containing-well is separated into 8,000 or 36,000 reaction chambers depending on the plate format. The instrument then thermal cycles the samples based on your method and images each partition. Based on the number of positive partitions, the CONSTELLATION calculates the absolute concentration of each sample.
The microplate is compatible with most liquid handlers.
Five Reporter Multiplex Detection using the Constellation Digital PCR (dPCR) System
The ability to detect and quantitate multiple nucleic acid targets within the same reaction is advantageous in a number of biological applications, including copy number variation, pathogen detection, detection of allelic variants, and simultaneous detection of multiple transcripts. Researchers attempting to increase the number of assay targets that can be detected in both realtime and digital PCR systems have adopted several strategies for both detection using intercalating dye assays as well as conventional exonuclease probe assays. For intercalating dyes, where the detection of the reporter dye is limited to a single optical channel, the use of multiplexed assays with different amplicon lengths – hence different endpoint fluorescence – has demonstrated successful.
Detection of Rare Nucleic Acid Targets using the Constellation Digital PCR (dPCR) System
Detecting low levels of specific nucleic acid target sequences is of crucial importance to a variety of applications. For example, accurate and reliable measurement of molecules making up a small fraction of the total population is vital when judging the efficacy of eradication treatment1 by identifying pathogen reservoirs in host tissue. Similarly, detecting viral or bacterial load to measure host response is helpful when predicting the future course of a virus- or bacteria-borne disease.
Determination of Copy Number Variation using the Constellation Digital PCR System
Copy number variation, or the structural variation of a DNA genome, is an important source of genetic variation for an array of medically- and agriculturally- relevant traits. In order to understand the biological basis for a number of important variations, it is critical to discriminate between the different number of genomic copies of specific DNA sequences. Digital PCR is a highly accurate technique for the absolute quantification of nucleic acid targets, and can be used to determine copy number variations between individuals in a population, between normal and tumor cell populations, and within somatic mosaics.
Constellation Twofold Dilution Series
A twofold dilution series was performed with a synthetic target and a probe based assay. Representative images of each concentration show the number of partitions containing the target. The software automatically calculates concentration from the images. Fine differences in target levels can be determined using the Constellation. (The error bars are one standard deviation)
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. Please see this "Advantages" for the benefits of dPCR over qPCR.
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. See the “How it works” and “Interactive Demo” page for a more detailed explanation.
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.