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The introduction of quantitative real-time PCR (qPCR) methodologies has greatly improved the analysis of nucleic acids. While traditional PCR techniques1 typically rely on end-point, and sometimes semi-quantitative analysis of ampliﬁed DNA targets via agarose gel electrophoresis, qPCR methods use fluorescence-based detection to allow the measurement of accumulated amplified product as the reaction progresses.
Today, scientists are able to use Digital PCR (dPCR), an absolute quantitation method in which samples are diluted and divided into many separate reactions. This can be accomplished through the physical partitioning of samples in separate chambers or droplets. Each partition contains either one copy, or zero copies of the target. PCR is run and the end point fluorescence is measured to determine if the partition is positive or negative. The exact amount of copies of a DNA molecule in the original sample can then be determined by counting the number of 'positive' partitions (sequence detected) versus 'negative' partitions (sequence not detected). The distribution of target DNA molecules among the reactions follows Poisson statistics, where at limiting dilution, the vast majority of reactions contain either one or zero target DNA molecules.1–3 Ideally, the number of PCR positive reactions equals the number of template molecules originally present. Increasing the number of partitions offers the potential to increase sensitivity for the detection of very small amounts of targets. The RainDrop Plus™ Digital PCR System is an ultra-sensitive droplet-based platform that is capable of generating millions of droplets.
With the increased sensitivity, dPCR is being used for important applications such as copy number variation, expression analysis, and the detection of rare mutants that require high sensitivity and have restricted sample availability.
dPCR is an ultra-sensitive method for absolute quantification of nucleic acids. It uses the same primers and probes as qPCR, but is capable of higher sensitivity and precision. The concept of dPCR was first described in 1992 by Sykes et al.1 However, it was Kinzler and Vogelstein who coined the term “digital PCR” in a paper in which they described the quantitation of ras mutations in a sample by partitioning the sample in order to perform a series of PCRs in 384 well microplates.3
dPCR provides a highly sensitive approach to the accurate and reproducible quantitation of DNA or RNA present in a sample. This method is similar to qPCR in the reaction assembly components and amplification reaction, but differs in the way the sample target is measured. dPCR provides absolute quantiﬁcation by avoiding any reliance on a calibration curve or endogenous controls. A major advantage of dPCR over qPCR is the ability to resolve rare events and assess targets at ultra-low template concentrations in a high background. In addition, detection using dPCR is more tolerant to PCR inhibitors because it is based on the detection of presence or absence of a reaction end-point. Samples which amplify slowly due to inhibition give late CT values in qPCR, appearing as lower than true concentration. In dPCR, this endpoint is still captured and the true concentration is determined. The initial reaction is assembled using the same reaction components as those used in qPCR. The reaction is then separated into thousands or millions of partitions where each partition behaves as an individual PCR reaction. The method relies on the assumption that sample partitioning will follow a Poisson distribution resulting in either zero or one target per well. Upon completion of sample partitioning, PCR amplification is performed to endpoint. The presence or absence of fluorescence in each partition is then used to calculate the absolute number of targets present in the original sample. Partitions with fluorescent signal are positive and scored as “1”; partitions with background signal are negative and scored as “0”. The number of positive vs negative reactions is determined and the absolute quantification of target is calculated.
Increasing the number of partitions offers the potential to increase sensitivity for the detection of very small amounts of targets, particularly when rare targets are present in a high background. This can be accomplished through the droplet-based RainDrop Plus Digital PCR System.
dPCR relies on the generation of two criteria: partitions with (positive) and without (negative) a fluorescent signal from the amplified partitions, and data that fits a Poisson distribution model. It is possible for a positive partition to contain two or more copies of your target at high concentrations of target relative to the number of partitions. This can be addressed using Poisson distribution calculations. Multiple copies of targets per positive partition do not become an issue until more than 10% of the partitions are positive. The RainDrop Plus Digital PCR System can generate up to 10,000,000 droplets, meaning Poisson statistics aren’t necessary until the number of target molecules exceeds 1,000,000. For human genomic DNA the total amount added to a reaction would need to exceed 3 µg before Poisson statistics became necessary. However, the RainDrop Analyst II Software provides the option of Poisson statistics if very large amounts of target material is needed when quantitating low levels of virus in a high host background.. The RainDrop Digital PCR System is ideally suited for the analysis of rare events. Large samples can be efficiently analyzed in a cost effective manner in a single 50 µl reaction.
Data can be reproducibly obtained across different instruments, different end users in different laboratories since Poisson distribution is not dependent on target concentrations derived from a calibration curve. The RainDrop Analyst II Software reliably analyzes samples containing multiple target molecules per droplet.
The generation of 10 million droplets by the RainDrop Plus dPCR System enables robust multiplexing capabilities. Probe-based dPCR can be multiplexed, through the use of different spectrally resolvable fluorophores for each target. Multiplexing is achieved by adjusting the probe concentrations to alter the intensity of fluorescence. Alternatively, two different probes can be blended together at different concentrations to further increase the multiplexing capability. Compared to multiplexing using qPCR, dPCR may perform better when quantifying multiple targets with very disparate expression levels. In qPCR, the stronger reaction can suppress the weaker reaction in the same tube. By contrast, dPCR works at limiting dilutions where each partitioned reaction is generally only positive for one assay template and is therefore free of reagent competition
Thanks to its high sensitivity, precision, and absolute quantification, dPCR extends the range of nucleic acid analysis beyond the reach of other methods in a number of applications:
dPCR is a robust PCR method that enables the detection and accurate absolute quantitation of nucleic acid molecules through the diluting and partitioning of biological samples. Applications requiring high sensitivity, accurate quantification, reproducibility, dynamic range, and improved multiplexing capability have benefitted from dPCR compared to other PCR technologies. Click here to learn more about our dPCR applications.
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