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Feb 15, 2014 (Vol. 34, No. 4)

Digital PCR Comes of Age

  • The digital polymerase chain reaction (dPCR) consists of many discrete, small-volume reactions to represent genetic information from a single sample.

    If a DNA template is sufficiently diluted so that some of these reactions contain no target molecules, while others contain discrete amounts, statistical analysis can then be used to quantify the exact number of target molecules in the sample. Therefore, dPCR can be used to accurately determine the number of nucleic acid molecules in a sample without comparison to a reference standard, provided that many reactions are performed in parallel.

    Because the reaction volumes for dPCR are generally very small, and the template must be sufficiently dilute to achieve single-molecule resolution, nucleic acids can be isolated from minimal quantities of specimen. The dilution of template also removes many of the inhibitors of PCR that are present in the original sample, further improving accuracy and efficiency.

    These properties make dPCR a superior diagnostic tool for the clinic, as well as any application where extreme sensitivity or precise quantification is essential, such as identifying mutations or copy number variations in tumor cells, or examining gene expression at the single-cell level.

    While dPCR uses established thermal cycling technologies and enzymes, and fluorescent probes are used to read the amplification signal, as they are in qPCR, its essence lies in the partitioning of the sample into many small reactions. The number of reactions that can be run depends on the equipment and the process it uses for partitioning.

    There are two types of dPCR machines currently available: chip-based and digital droplet PCR (ddPCR). For chip-based dPCR systems, such as the LifeTechnologies QuantStudio™, the sample is mixed with reagents and loaded into individual reaction chambers in plates that are about the size of a microscope slide. The reaction mixture is loaded into these small partitions by hydrophobic/hydrophilic interactions and capillary forces and the machine monitors the reactions as they occur.

    In ddPCR, employed by Bio-Rad and RainDance machines, the samples are first mixed with the reagents and dispersed into nanoliter-sized droplets. The droplets for each sample are then placed in a tube and PCR is performed in a thermocycler. A droplet reader then detects a fluorescent signal to determine whether or not reactions have occurred. Unlike chip-based systems, ddPCR does not use physical partitions to separate the reactions, but the properties of the droplets themselves. For further discussion on dPCR technologies, please see Nature Methods 9, 541-544 (2012).

  • A Chip-Based System

    Click Image To Enlarge +
    The Life Technologies™ QuantStudio™ 3D Digital PCR System uses a microfluidic chip that partitions samples based on hydrophobic/hydrophilic interactions and partitioning valves.

    The QuantStudio™ 3D dPCR system became available from Life Technologies in June 2013. According to Stephen Jackson, Ph.D., associate director of product applications at Life Technologies, approximately 20,000 reactions can be run on a single, dime-sized, silicon-wafer chip in this system. All of the partitions are of identical size. This system offers the additional advantages of a single-step workflow, where a 15–20 µL sample of the template DNA and reaction mix is loaded and processed by the system, which quantifies it as copies of nucleic acid per µL.

    The QuantStudio™ 3D dPCR system is compact; the thermal cycler fits easily on the benchtop; and the reader instrument is “the size of a shoebox.” It is also priced to be within the budget of most research scientists, with a system that includes the QuantStudio™ 3D Digital PCR Instrument, Applied Biosystems® GeneAmp® 9700 dual flat-block thermal cycler, chip loader, and 384 digital PCR chips for under $50,000.

    While there is a risk of cross contamination for any PCR system that processes a large number of reactions, Dr. Jackson believes that the risk will be reduced in this system, since each sample is loaded on a separate chip and the reactions are physically separated by partitions. With an eye toward the certification of dPCR as an in vitro clinical diagnostic, Dr. Jackson adds that “no mixing of patient samples is something the FDA will pay attention to.”

  • Detection of Rare Noncoding RNAs

    Click Image To Enlarge +
    The distribution of lncRNAs within the cell can be determined by cellular fractionation followed by dPCR. These results can be verified by FISH, as in this image, where it is shown that many lncRNAs permanently reside in the nucleus. Moreover, the absolute number of transcripts per cell can be determined by dPCR, and this number gives clues as to their mechanism of action. Low-abundance ncRNAs are found to be cis acting while high-abundance ncRNAs are likely to act in trans. [K. Gagnon and L. Li, Corey Lab, UT Southwestern Medical Center]

    The dilution of template into a very large number of reactions makes dPCR ideal for identifying targets that would otherwise be undetectable in a sample containing many thousands of copies of a genome, such as preoncogenic mutations. David Dodd, Ph.D., a postdoctoral fellow in Dr. David Corey’s laboratory at the University of Texas Southwestern Medical Center in Dallas, uses a Bio-Rad QX100 ddPCR System to detect long noncoding RNAs (lncRNAs) in the nucleus that regulate gene activity. Differences in the expression of lncRNAs have been associated with several diseases.

    According to Dr. Dodd, dPCR is essential for this area of research, where knowledge of the exact number of a particular lncRNA can reveal whether it acts on a gene on the same chromosome (cis), or a different one (trans). “You get real numbers really quickly,” Dr. Dodd says, “and you don’t need to rely on a standard curve, as in real-time PCR, or quantitative PCR (qPCR), which can introduce problems due to structural variations in the RNA targets.”

    Still, he does not think that the ddPCR system he uses will replace qPCR; although dPCR is important for his work, he mentions that few of his colleagues in academia use it because of the high cost of the equipment. Because of this, he sees it more as a service that would be offered by core facilities than something each lab would purchase. There are also “lots of hands-on steps” to prepare the droplets, he says, further adding to the inconvenience.

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