February 15, 2013 (Vol. 33, No. 4)

PCR has allowed scientists to amplify scarce amounts of DNA and engage in the type of research that will help us understand the genetics of who we are, why people are prone to developing certain diseases, and how to develop diagnostics and therapeutics for better health.

Advances in PCR technologies have transformed the nonquantitative methods of traditional PCR to real-time measurement of PCR amplification, and most recently, to the absolute quantitation of DNA.

Recently, scientists at CHI’s “Digital PCR: Application and Advances” conference shared their findings and excitement about emerging PCR technologies and new tools that improve existing PCR processes. These emerging methods are making a pivotal impact in life science research and all share a set of common features, which include independence from controls and standards, an increase in sample throughput, and the absolute measurement of DNA concentration to standardize assays and to improve the quality of data shared between investigators.

One option for scientists that can help improve sample throughput without the need for new equipment is a cloud-based, scalable system developed by DNA Software. John SantaLucia Jr., Ph.D., the firm’s president and CEO, discussed the qPCR CopyCount™ cloud-based service. CopyCount can fit raw qPCR data using mechanism-based modeling to obtain relative and absolute DNA concentrations.

The service implements a method known as Counting PCR (cPCR) and provides scientists the ability to upload their raw qPCR data for analysis. For each qPCR well, users receive the absolute copy number of DNA at cycle zero without the need for a standard curve. Highlights of the service are instrument independence, compatibility with commonly used fluorophores, and the ability to dedicate more well space for samples rather than for controls.

“The high-throughput of qPCR CopyCount will enable a variety of applications including determination of viral load, copy number variations, and quantification of fragment libraries for next-generation sequencing,” said Dr. SantaLucia.

Alternatively, absolute DNA quantitation may be performed using digital PCR. In its simplest form, digital PCR is a single-molecule counting method that gives a direct quantitative measurement of absolute DNA concentration and thus does not require a standard curve. It has potential impact in gene expression and mutation detection as well as the detection and diagnosis of infectious and hereditary diseases. Digital PCR can also be used to standardize qPCR assays and reduce the number of false positives.

The basic workflow with digital PCR is to partition a DNA sample, perform PCR in each partition, score a positive or negative for the presence of the target sequence, and return to the user an absolute value of DNA concentration (e.g., copies per microliter). Platforms utilizing digital PCR can partition a DNA sample either by using fixed arrays or by creating individual nanoliter or picoliter droplets, a strategy utilized by the Bio-Rad QX100 Droplet Digital PCR and the RainDance RainDrop Digital PCR systems.

No Drop in the Bucket

Bio-Rad believes that the digital PCR space is experiencing an inflection point at which implementing digital PCR daily has become affordable and practical. George Karlin-Neumann, Ph.D., director of scientific affairs at Bio-Rad’s Digital Biology Center, explained that “implementing digital PCR with past technology was very expensive and did not provide enough throughput. People used to use 96-well plates. Performing limiting dilution in a 96-well plate is painful and extremely tedious to have a sufficient number of partitions.”

He talked about the performance and applications of Bio-Rad’s QX100 Droplet Digital PCR system. “The QX100 system is an affordable, turnkey system that enables one person to run 300 samples in an 8 hour day,” said Dr. Karlin-Neumann.

The system is versatile as it can operate in a high-throughput mode where analysis of each sample requires only a single well, or it can achieve ultra-high sensitivity and precision by utilizing multiple wells, he explained. By partitioning each well into approximately 20,000 nanoliter droplets, one can obtain absolute counts of target molecules.

Because the QX100 system is able to interrogate droplets with up to 5 or even 25 target copies per droplet, it can screen through as many as 100,000 to 500,000 target copies in 20,000 droplets in the search for a few rare mutants or foreign sequences. This would require 1 million to 5 million droplets if one operated under the limiting dilution regime of conventional digital PCR, according to Dr. Karlin-Neumann.

As a consequence, this allows for the QX100 to operate with a dynamic range of nearly 5 logs. In addition, Bio-Rad’s digital PCR system can achieve high reproducibility in part because of the system’s “highly uniform droplet size, which allows us to use Poisson statistics to obtain precise concentrations even with multiple target occupancy per droplet,” says Dr. Karlin-Neumann.

Smaller Droplets Yet

RainDance Technologies has pushed the idea of digital PCR further by developing a platform that creates picoliter droplets, allowing for single molecule encapsulation and billions of reactions per day. Darren Link, Ph.D., co-founder and vp of R&D, spoke of the company’s RainDrop Digital PCR system.

“The power of any digital PCR system, droplet-based or otherwise, is in having a sufficient number of reactions to do single molecule counting across a range of starting concentrations. It is essential to be able to simultaneously count high and low levels of expression.”

The advantage of picoliter partitions is being able to generate millions of partitions from a sample size of less than 100 µL. For example, “With just 50 µL, 10 million 5 picoliter droplets can be generated. While it is possible to count 10 million with 1 nanoliter droplets, it requires 10 mL of starting volume, which is impractical and costly for most applications,” said Dr. Link.

He noted that early users of RainDance’s RainDrop Digital PCR system have been pleased with the ease at which samples are processed and the data they can generate. “There is only a single pipetting step throughout the entire workflow, and having millions of droplets eliminates the need for titrations and allows for robust multiplexing,” explained Dr. Link.

Dr. Karlin-Neumann shared a few noteworthy applications of droplet digital PCR that include the measurement of rare HIV DNA in patients for effective combination antiretroviral therapy (cART) and the monitoring of chronic myeloid leukemia patients to determine response to therapy, presence of residual disease, and the emergence of relapse or resistance mutations.

“Droplet digital PCR can provide an order of magnitude more precise, accurate, and reproducible measurements versus real-time PCR. In regard to rare HIV DNA, the use of droplet digital PCR as a duplex assay can yield a fivefold decrease in the coefficient of variation of pol copy number and a 10-fold accuracy improvement for 2-LTR circles.”

Droplet digital PCR has also been used for the detection of cancer biomarkers in peripheral blood. For example, one of RainDance Technologies’ early users was able to detect a single mutated copy of KRAS in a background of 200,000 wild-type KRAS copies, claimed Dr. Link.

“When it comes to cancer detection, this kind of result is a real game-changer,” he said. The RainDance technology is also being used for the detection of cancer mutations in cell-free circulating DNA and the characterization of heterogeneity of tumor biopsies. The benefit of this technology is the noninvasive approach toward personalized treatment plans for cancer patients.

RainDance Technologies has developed a platform that, it says, creates picoliter droplets, allowing for single molecule encapsulation and billions of reactions per day.

Digital Microfluidics

Digital microfluidics is not limited to PCR and has inspired researchers in other areas of genomic research. One researcher has extended the use of digital microfluidics to the application of whole-genome haplotyping. Christina Fan, Ph.D., formerly at Stanford University, discussed her work with digital microfluidics on determining the fetal genome noninvasively from maternal blood.

“To achieve whole-genome haplotyping, one needs to look at many loci across the chromosome, and in our case, we focused on digital amplification of metaphase chromosomes and looked at 1 million loci using digital MDA.” Unlike digital PCR, which amplifies specific targets in the genome present in the digital compartment, digital MDA (multiple-strand displacement amplification) amplifies all DNA within the compartment.

The current limitation in digital amplification of a single metaphase chromosome has been “the amplification bias, yielding biased representation across the chromosome,” said Dr. Fan. However, she added, “we can pool together amplified materials from replicates of single chromosomes with our solution” to overcome this.

“Noninvasive determination of fetal genome is one application enabled by whole-genome haplotyping, which may ultimately facilitate the diagnosis of all inherited diseases,” says Dr. Fan. She highlighted one example of the importance of haplotyping.

“Given that a person has two mutations at two different loci with the same gene, either mutation would abolish function of the gene. In one scenario, the two mutations can be on the same strand, yielding still one functional copy of the gene. In another scenario, the two mutations can be on different strands, yielding no functional copy. The two scenarios can only be differentiated by haplotyping while genotyping alone would only inform that the person is heterozygous at the two loci,” explained Dr. Fan.

As droplet digital microfluidics becomes more accessible to researchers, the technology is expected to deliver on answering questions that previously could hardly have been imagined.

“One of the things scientists are discovering is that the more they interrogate genomes, the more complexity they find. At this point, we are still at the tip of the iceberg in fully appreciating the biological complexity of our cells. But, technologies such as droplet-based digital PCR are opening up new possibilities for this research and will help scientists piece together these biological puzzles,” said Dr. Link.

The next wave of droplet microfluidic technology can be found at the Micro/Nano Fluidics Fundamentals Focus (MF3) Center at the University of California at Irvine. There, Abraham Lee, Ph.D., oversees the development of a 1 million droplet array platform that simultaneously improves the dynamic range of digital PCR detection and integrates digital PCR with qPCR.

His lab has developed a platform that enables real-time PCR that not only gives absolute count of gene copies but also has the potential to determine starting copy numbers in each droplet when there is more than one DNA molecule. “This should further increase the dynamic range of digital PCR and may potentially detect the subtle variations in gene sequences that influence the extension efficiency of the enzymes,” Dr. Lee added.

“While digital PCR is an exciting technology that provides a new tool to identify absolute copies of target DNA, there is so much more that droplet microfluidics can provide in conjunction with this platform,” said Dr. Lee.

“Droplet microfluidics can provide solutions to sample processing and sample preparation, multiplexing, and in-line or parallel sample detection. Moreover, droplet manipulations that provide pre- and postprocessing steps allow for more automation and enable applications such as point-of-care diagnostics.”

Currently, his students are working on label-less detection schemes (nonoptical, nonfluorescence), some multiplex gene detection techniques, and some next-generation sequencing related applications using digital PCR.

For more on digital PCR, be sure to check out the Expert Tips “8 Reasons Labs Rely on Digital and Quantitative PCR“.

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