Digital PCR – replacement or addition?

A digital version of the gold standard for nucleic acid quantification is allowing new levels of precision, but the analogue equivalent still has its advantages. Can you find a place in your lab for both techniques? 

Researchers in biotech, pharmaceutical, and academic labs have long relied on quantitative real-time (qPCR) to quickly and accurately detect and quantify target DNA. Recently, digital PCR (dPCR) is beginning to stake its claim in the laboratory for applications where qPCR meets its technology limits. Digital PCR’s increased sensitivity, precision, and reproducibility is particularly useful for applications such as higher order 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.

 Companies that have long relied on qPCR are turning to digital PCR when left with unanswered questions. Monoquant, a molecular diagnostics firm founded by researchers at Flinders University and Medical Center in South Australia, is a perfect example of this. Initially, the company used qPCR to develop a highly sensitive method for isolating and quantifying the abundance of the translocation breakpoint in chronic myeloid leukemia (CML) DNA to develop a new clinical test. This method extends the sensitivity of qPCR and allows researchers to routinely detect down to about 1 in 10 million cells. However, one potential challenge with the qPCR-based test is that the FDA might take issue with the fact that amplification efficiency may vary by patient, due to the use of patient-specific PCR primers.

Monoquant is looking at droplet digital PCR to overcome variations in qPCR amplification efficiency which could affect quantitation accuracy. They hope the results they are seeing from their digital PCR system will fast-track the FDA approval process for the test, which will offer patients a better degree of monitoring and better disease management by tracking the progression or remission of CML.

“Advancements in digital PCR have given us the ability to overcome variations in real-time PCR amplification efficiency and have also enabled us to do away with using a standard curve,” said Professor Alec Morley, head of Monoquant and a digital PCR pioneer.

 Even as companies like Monoquant turn to dPCR for answers, qPCR will remain a valuable tool for certain applications. Thus, the question becomes: “Which technology is right for me?”

 In use since the 1990s, qPCR has been established as a credible and capable technology in basic research and clinical labs. It 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. Researchers rely on qPCR mainly for its speed, sensitivity, specificity, and ease-of-use and is considered the gold standard for nucleic acid quantification, and a set of PCR best practices has been established. MIQE (Minimum Information for the Publication of Quantitative Real-time PCR Experiments) helps ensure the integrity of the scientific literature, promote consistency among laboratories, and increase experimental transparency. In addition, since qPCR 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.

 As the name suggests, real-time PCR measures PCR amplification as it occurs. It is particularly well-suited to gene expression analysis due to 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. Changes in target expression results are most meaningful in these experiments when compared between experimental conditions, such as the relative expression in diseased versus healthy tissue.

Well-designed qPCR assays have a considerable dynamic range as they can detect several to millions of copies of a target sequence per reaction. This enables detection of targets with very low and very high numbers of copies in the same run, which is well-suited for screening or downstream validation experiments. qPCR instruments’ varying block capacity (96- and 384-well, for example) and automation compatibility make qPCR a good choice for experiments with either high sample or high target number screening requirements. qPCR also offers the greatest flexibility in the choice of detection chemistry as researchers have the option to select from inexpensive intercalating dyes (such as SYBR green) to a variety of target-specific probes (TaqMan, molecular beacons, FRET, etc.). Researchers will also find pricing flexibility that comes from being able to easily change reaction volume, throughput, and detection method to meet experimental needs.  Because of the widespread adoption of qPCR, most researchers have easy access to the technology. qPCR is most 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.

After using qPCR for more than a decade, Biogazelle, a PCR data analysis company, considers it the gold standard for gene expression analysis due to its high throughput and low costs.  Biogazelle relies on the mature workflows and the MIQE guidelines to help them set up, analyse, and report on their qPCR studies. In addition, the company also utilises the technology for SNP and mutation detection, as well as gene copy number analysis. However, Biogazelle has seen a rising demand from researchers interested in copy number variation analysis and rare event detection that led the company to try out digital PCR. Case in point, Biogazelle was contracted by a biotech firm to design a PCR-based experiment for transgene copy number variation between 8-10 copies. The approach 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. As a company based on the principles of real-time PCR, Biogazelle sees a place for both qPCR and dPCR. Deciding which system to use depends on the results their clients are hoping to obtain.

Unlike qPCR, dPCR partitions the sample and reaction components into hundreds or thousands of reaction chambers to count the presence or absence of target molecules in each partition after endpoint PCR amplification. As a result, quantitation using dPCR is less sensitive to factors affecting PCR amplification efficiency, including the presence of inhibitors in the sample.  Digital PCR provides an absolute measurement of copies present per sample volume assayed (i.e. concentration). It thus does not require the user to compare an unknown to a standard, eliminating the need for a standard curve. Excellent day-to-day reproducibility and reliable CNV measurements for even greater than three copies is readily achievable due to the precision of dPCR. In addition, partitioning samples decreases the amount of background DNA in each partition, which gives greater specificity and sensitivity in amplifying the target when present. This leads to greatly improved sensitivity in the detection of rare mutations and sequences.

Over the years there has been an increased interest in research towards the eradication of HIV. This has emphasised the need for new assays that will monitor the disease burden in infected patients on combination antiretroviral therapy.  For effective monitoring in clinical trials, assays should be cost-effective, and should provide high sensitivity, specificity, and reproducibility. Currently, there is not an assay that meets all of these criteria. Real-time PCR has been used most frequently, but digital PCR has recently been proposed as an alternative to qPCR due to its improved accuracy and precision. A new study by Matthew Strain and Douglas Richman of the University of California San Diego found that digital PCR improves the precision of real-time assays by providing an increased precision of four-fold to over 20-fold versus qPCR, when using identical quantities of clinical samples from peripheral blood.

An early adopter of droplet digital PCR, Sangamo BioSciences has been using the technique to develop therapies designed to provide a complete cure for HIV.  One of the major challenges in Sangamo’s research is measuring residual levels of HIV DNA in trial subjects to evaluate the effectiveness of their zinc-finger nuclease-based therapy. Initially, they used qPCR to detect and quantify HIV DNA in patient samples. However, this technology was not sufficiently sensitive or reproducible to accurately quantify the low residual levels seen after treatment (? 1 copy per 10,000 cells). The Sangamo team turned to droplet digital PCR for a solution. In comparison, qPCR was ~10-fold less sensitive than ddPCR—due to imperfect amplification efficiencies and the necessity of establishing a standard curve—preventing qPCR from delivering the precision and sensitivity needed to detect these rare target DNA sequences.  Due to the digital nature of the assay and the fact that no standard curve is required, ddPCR technology can discriminate differences in target DNA concentrations as low as 10% with a single well -- and much lower with multiple wells – and can detect as few as several target copies per well.

Sangamo was able to easily measure low copy events in a sample of genomic DNA. Droplet Digital PCR now plays a central role in the company’s research, providing highly precise, reproducible data in support of complete cure development.

Droplet digital PCR has also recently been adopted by the Ji Research Group at Stanford University to accurately and precisely measure cancer genome amplifications in archival cancer tissue samples. Detecting amplifications in cancer tissue is technically challenging due to the fact that genomic amplifications are diluted both by the presence of normal tissue and cancer cell heterogeneity whereby some or many cells within the tumor may lack a later acquired mutation. In addition, clinical samples are of poor quality because they are traditionally processed as formalin fixed paraffin embedded (FFPE) tissues that lead to irreversible damage to the genomic DNA. Traditionally, microarray assays or qPCR have been used for this application. However, they often lack the sensitivity and precision to detect copy number alterations present in the small amounts of amplifiable tumor DNA found in degraded FFPE samples.  ddPCR’s reduced sensitivity to factors affecting amplification efficiency, use of a duplexed reference assay in the same reaction and lack of reliance on a standard curve offer advantages that enable fine discrimination of copy number changes in these samples. When researchers in the Ji group compared qPCR and ddPCR for measuring FGFR2 gene amplifications, they were able to demonstrate the superiority of ddPCR for copy number analysis of archival material in DNA due to its precision, reproducibility, and sensitivity. Targeting these amplified oncogenes could move us closer to long sought after personalised therapies for cancer treatment.

Real-time PCR will continue to remain the gold standard technique for target DNA quantitation and gene expression analysis. However, for particular challenging applications digital PCR provides new levels of sensitivity, precision, robustness and reproducibility, with the ability to extend nucleic acid quantification beyond previous limits.

Authors:

George Karlin-Neumann, Director of Scientific Affairs, Bio-Rad’s Digital Biology Center and Rachel Scott, Marketing Manager, Gene Expression Division, Bio-Rad Laboratories.

References

1. Frost &Sullivan. Market penetration leadership award quantitative and digital PCR instrumentation North American, 2012. Frost & Sullivan Best Practices Research, November 2012.
2. Strain, M.C.; Richman, D.D. New assays for monitoring residual HIV burden in effectively treated individuals. Lippincott Williams & Wilkins 2013.