Quantitative real-time PCR (qPCR) is one of the most widely used laboratory techniques in biotech, pharmaceutical, and academic research labs. Researchers have long relied on qPCR to quickly and accurately detect and quantify target DNA. Today, digital PCR (dPCR) is beginning to stake its claim in the laboratory due to its increased sensitivity, precision, and reproducibility for applications such as copy number variation and rare event detection. A survey conducted jointly by The Scientist and research firm Frost & Sullivan revealed that 10% of its readers were using dPCR in 2012 and another 30% intended to use the technology in 2013.1
Finding uses for both qPCR and dPCR
Biogazelle, a PCR data analysis company, has used real-time PCR for more than a decade. The company considers real-time PCR the gold standard for gene expression analysis due to its high-throughput and low costs.
According to Biogazelle’s project manager, Ariane DeGanck, the workflows are mature and the MIQE guidelines help them to set-up, analyze, and report on their qPCR studies. In addition, Biogazelle also utilizes qPCR for SNP and mutation detection, as well as gene copy number analysis.
However, a rising demand from researchers interested in copy number variation analysis and rare event detection led the company to purchase the QX100 Droplet Digital PCR (ddPCR) system from Bio-Rad Laboratories.
“Digital PCR offers higher resolution and sensitivity that gives researchers the ability to measure smaller differences and accurately quantify minority variants in the background of a wild type sequence,” said De Ganck.
As a company based on the principles of real-time PCR, Biogazelle sees a place for both qPCR and dPCR. According to De Ganck, deciding which system to use depends on the results their clients are hoping to obtain.
Case in point, Biogazelle was contracted by a biotech firm to design a PCR-based experiment for transgene copy number variation. The experiment using qPCR, which is standard practice for biopharmaceutical companies, did not allow detection of fold-changes lower than 50%, leaving researchers with poor-quality data that could not be reproduced. Biogazelle proposed using droplet digital PCR, which detected the smaller differences in copy number, providing them with more precise and reliable data.
Even as companies like Biogazelle adopt dPCR for specific applications, qPCR will remain a valuable tool for certain applications. Thus, the question becomes, “Which technology is right for me?”
qPCR: A well-established technology
qPCR has been in use since the 1990s and is established as a credible and capable technology. Basic research and clinical microbiology labs have relied on it for its speed, sensitivity, specificity, and ease-of-use. The technology can be used for a broad range of applications including gene expression analysis, genotyping, pathogen detection, viral quantification, DNA methylation analysis, and high resolution melting (HRM) analysis, among others. One advantage of qPCR is that because it has been around for so long, there is a large body of literature available for reference. Researchers can also rely on the continuity of their own historical data for designing and interpreting their experiments.
In addition, qPCR is considered the gold standard for nucleic acid quantification. A set of PCR best practices called MIQE—Minimum Information for the Publication of Quantitative Real-time PCR Experiments—has been established to help ensure the integrity of the scientific literature, promote consistency among laboratories, and increase experimental transparency.
The power of qPCR
As the name suggests, real-time PCR measures PCR amplification as it occurs. The relative nature of qPCR, where concentration or relative expression of a target is determined from comparison to a sample of known concentration or control sample, makes it particularly well suited to gene expression analysis. Commonly in these experiments, changes in target(s) expression results are most meaningful when compared between experimental conditions, such as the relative expression in diseased versus healthy tissue.
qPCR offers the greatest flexibility in the choice of detection chemistry. Researchers can select from inexpensive intercalating dyes (such as SYBR green) to a variety of target-specific probes (TaqMan, molecular beacons, FRET, etc.). A highly beneficial attribute of qPCR is the per sample pricing flexibility that comes from being able to easily change reaction volume, throughput, and detection method to meet experimental needs.
Well-designed qPCR assays can detect as few as several to as many as millions of copies of a target sequence per reaction giving it a considerable dynamic range. This attribute enables detection of targets with very low and very high copy number in the same run, well-suited for screening or downstream validation experiments. The high sample throughput capabilities due to qPCR instruments’ varying block capacity (96 and 384-well, for example) and automation compatibility also make qPCR a good choice for experiments with either high sample or high target number screening requirements.
qPCR is useful for relative gene expression experiments, as a mid-level discovery approach, and as a validation tool supporting other genomic methods including DNA microarrays and some next generation sequencing (NGS) applications. In addition, because of its widespread adoption, most researchers have easy access to the technology.
The power of digital PCR
What distinguishes digital PCR from qPCR is the partitioning of the sample and reaction components into hundreds or thousands of reaction chambers so that it can then count the presence or absence of target molecules in each partition after endpoint PCR amplification. dPCR provides an absolute measurement of copies present per sample volume assayed (i.e. concentration) and does not require the user to compare an unknown to a standard, thus eliminating the need for a standard curve. The precision achievable with dPCR is an essential property both for making reliable CNV measurements for greater than 3 copies and for excellent day-to-day and lab-to-lab reproducibility. Furthermore, partitioning the sample decreases the amount of background DNA in each partition, giving greater specificity and sensitivity in amplifying the target when present. This yields improved sensitivity in the detection of rare mutations and sequences.
Analyzing copy number variations
CNVs include deletions, insertions, duplications, and complex amplifications ranging from tens to hundreds of thousands of base pairs long and underlie diseases of both germline and somatic origins. The accelerated discovery of CNVs has increased the need for high-throughput, low-cost options, such as droplet digital PCR, for validation and follow-up studies.
Steve McCarroll’s research group from Harvard Medical School has utilized ddPCR in this way.2 The lab has been studying the differences in human genomes to identify the genes underlying biological processes and human diseases. This research requires the ability to measure the precise copy number of genome segments in hundreds to thousands of individuals. By using droplet digital PCR, the McCarroll lab discriminated copy number states of three and higher—a challenge for qPCR—in close agreement with next-generation sequencing results, enabling the use of ddPCR technology to quantitate copy numbers in large populations.
Identifying low-frequency CNVs
Reprogramming somatic cells into induced pluripotent stem cells has been suspected of causing de novo copy number variation. A paper published in Nature used ddPCR to show that a significant portion of CNVs had not simply arisen de novo during reprogramming.3 Instead, approximately half of the CNVs originated as low-frequency variants present in skin cells prior to reprogramming.
Findings like these are an important step in understanding the extent to which cells of the human body normally acquire structural alterations in their DNA post-zygotically. This understanding could shed light on the challenges in identifying the genetic contribution of such alterations particularly in neurodevelopmental diseases, for which determining the exact loci for genetic predisposition has proven difficult.
Detecting rare mutants
Generally, the detection of somatic mutations poses an analytical challenge due to the heterogeneous nature of most samples. A gene carrying a mutation may differ from the highly abundant wild type sequence by only a single nucleotide. Most conventional methods have poor selectivity and fail to detect mutant sequences below 1 in 100 wild type sequences.
A presentation at the American Association for Cancer Research Annual Meeting in 20124 showed how the enrichment of mutant sequences upon partitioning in the ddPCR system enabled the detection of somatic mutations with high selectivity and sensitivity, promising earlier and less invasive diagnosis of disease. The ddPCR system allowed for the quantification of the JAK2 V617F mutation in peripheral blood mononuclear cells with several logs greater sensitivity than pyrosequencing, noting the presence of mutants in patient samples classified as normal by the latter method. Similarly, common mutations in clinically relevant cancer genes such as KRAS, EGFR, and BRAF were readily detectable in FFPE patient samples. These findings suggest ddPCR technology may have a role to play in the earlier detection of cancer, as well as in monitoring the progress of disease and patient response to therapeutics.
Developing new diagnostic tests
Private companies, as well as academic and clinical laboratories around the world, are turning to digital PCR. Monoquant, a molecular diagnostics firm founded by researchers at Flinders University and Medical Center in South Australia, is in the process of conducting final refinements before rolling out a new clinical test for chronic myeloid leukemia (CML). Initially, the company used qPCR to develop a highly sensitive method for isolating and quantifying the abundance of the translocation breakpoint in CML DNA, but qPCR proved challenging because this breakpoint is different in each patient and qPCR primers and conditions may vary. Monoquant eventually turned to droplet digital PCR to overcome variations in qPCR amplification efficiency.
Outlook for qPCR and dPCR applications
Real-time PCR will continue to remain the gold standard technique for target DNA quantitation and gene expression analysis. However, droplet digital PCR provides new levels of sensitivity, precision, and reproducibility, with the ability to extend nucleic acid quantification beyond previous limits.
1. Frost &Sullivan. Market penetration leadership award quantitative and digital PCR instrumentation North American, 2012. Frost & Sullivan Best Practices Research, November 2012.
2. Boettger LM; et al. Structural haplotypes and recent evolution of the human 17q21.31 region. Nature Genetics. 2012, 1–5.
3. Abyzov A; et al. Somatic copy number mosaicism in human skin revealed by induced pluripotent stem cells. Nature. 2012, 1-7.
4. Hindson B; et al. Ultra-sensitive detection of rare mutants by droplet digital PCR with conventional TaqMan assays. April 15, 2012. AACR, Chicago, IL.