by Michelle Racey, Applications and New Product Support, Digital Biology Group, Bio-Rad Laboratories
Measurement is the torch of scientific exploration. It illuminates all natural processes and directs researchers to the best next steps toward evidence-driven discovery. For this reason, technology that produces accurate, quantifiable data drives modern research from experimental lab procedures to results that inform biological discoveries and healthcare solutions. In molecular biology, the analysis of nucleic acid components within samples, especially those in limited quantities, requires advanced and sensitive tools. One such method is Droplet Digital PCR (ddPCR) technology, which offers a versatile approach with applications in clinical settings and various research and development pursuits. Harnessing the power of ddPCR methods elevates life science research to a whole new level of precision insights.
What is ddPCR Technology?
The ddPCR technique begins by dividing a sample into 20,000 emulsified droplets, each containing a single copy of a specific target nucleic acid sequence. Traditional PCR methods are then employed to amplify the contents of each droplet, and any amplified target DNA can be identified using a fluorescent probe. This tagging mechanism allows for the differentiation of "positive droplets" containing the target sequence from droplets lacking it. By examining the statistical distribution of positive and negative droplets, a definitive and quantitative measurement of the relative abundance of the nucleic acid sequence in the original sample is obtained.
The ddPCR technique possesses numerous advantages that set it apart as a sensitive, statistically reliable, and cost-effective option for nucleic acid quantification. Firstly, its ability to deliver absolute quantification of target DNA eliminates the need for standard curves, reducing hands-on time and the risk of human error and ensuring robust analysis. The exceptional accuracy and unparalleled precision of ddPCR data offer detailed and dependable insights crucial for life science research. Moreover, the superior partitioning of the sample into droplets mitigates PCR bias by ensuring equal amplification opportunities for each DNA sequence. The smaller sample volume also reduces background noise, producing a clearer signal-to-noise ratio. Alongside its technical excellence, the streamlined cost-per-run and reduced consumable costs further establish ddPCR technology as an economical and uncompromising solution.
Despite its simplified workflow, ddPCR design enables a wide range of applications involving quantifying biomarkers, pathogenic DNA strands, target genes in rare diseases, and other molecules of interest to clinicians and basic researchers.
Wide-ranging Applications of ddPCR Methods
The versatility of ddPCR technology has transformed numerous fields of life science research, with particularly fruitful results in molecular diagnostics. Among these, oncology has witnessed remarkable advancements driven by ddPCR innovation. The ability to detect trace amounts of tumor DNA in liquid biopsy samples has distinguished ddPCR analysis as an effective method for monitoring cancer progression, evaluating treatment efficacy, and identifying patients at high risk for relapse. Additionally, genetic testing dovetails with ddPCR methods for detecting and quantifying genetic variations within clinical samples. Employed in this manner, ddPCR assays can help evaluate an individual's predisposition to specific diseases and turbo-charge research that helps inform diagnostics and treatment selection to improve patient outcomes. Furthermore, ddPCR assays enable researchers to assess biological indicators of diseases in clinical samples. By accurately measuring biomarker levels, ddPCR technology facilitates early disease detection, allows effective monitoring of treatment response, and paves the way for the development of targeted therapies.
Wastewater testing also benefits immensely from ddPCR technology. Its capacity to detect and quantify pathogens in environmental samples provides invaluable information for evaluating circulating pollutants and pathogens and characterizing microbial communities. The same ddPCR assays can equip life science researchers with a detailed look at environmental ecosystems and serve as an early warning system for disease outbreaks.
Biopharmaceutical development and manufacturing can also benefit from ddPCR implementation. The high-resolution detection capacity of ddPCR assays aids quality control and regulatory compliance procedures, such as evaluating biopharmaceutical products for viral, bacterial, or fungal contaminants. It can also directly quantify the concentration of viral vectors and transgene copies to aid in proper dosing for patient treatment. Improvements in the quality, safety, and efficacy of biopharmaceutical products and manufacturing practices have motivated the growing adoption of ddPCR methods across the industry.
The exceptional ability of ddPCR technology to provide accurate, sensitive, and quantitative measurements across a wide range of clinical and environmental samples holds immense potential for advancing personalized medicine and biological discovery. As dedicated and ambitious scientists continue to explore novel applications and refine ddPCR techniques, public health initiatives aimed at managing complex diseases and life science research zeroed in on advanced ecological discovery will reflect the ongoing transformation within the biotechnological landscape.
Case Studies: ddPCR Technology in Action
To highlight significant progress across these wide-ranging applications, Bio-Rad Laboratories awarded four distinguished scientists as 2022 Positive Droplet Award recipients, all of whom have driven positive changes in their field of research using ddPCR methods.
Digital PCR Techniques: Alec Morley, MD
Dr. Alec Morley, a pioneer in digital PCR, sought to discover a more sensitive detection method for leukemia in 1987. He recognized the limitations of conventional microscopy-based assessment in identifying leukemic cells when their proportion fell below 1-2% in bone marrow samples. To overcome this challenge, Morley turned to PCR and identified three potential targets for applying gene amplification techniques to achieve higher-resolution analysis. He and his team developed patient-specific markers for acute lymphoblastic leukemia (ALL) and adapted a quantification method to PCR to measure and detect gene rearrangements. This groundbreaking research gave birth to a novel digital PCR method known as "limiting dilution PCR," which offered a more precise and sensitive approach to leukemia monitoring.
However, the early technique faced challenges due to its labor-intensive nature and lack of modern automation, limiting its widespread adoption. Undeterred, Morley continues to engineer new methods for measuring and monitoring leukemia. He now employs ddPCR technology to detect BCR-ABL translocations characteristic of chronic myeloid leukemia (CML). This innovative approach driven by translocations necessitates patient-specific primers, enhancing the method's selectivity and distinguishing it from traditional practices. Dr. Morley emphasizes the advantages of this new technique, stating, "Instead of setting up 25 laborious electrophoresis gels, you can set up 20,000 droplets within minutes and obtain results in a short time." His commitment to advancing leukemia detection and monitoring through cutting-edge technologies demonstrates an inspiring dedication to the field.
Wastewater-based Epidemiology: Raul Gonzalez
Hampton Roads Sanitation District (HRSD) adopted ddPCR for its remarkable precision and sensitivity in 2015. Raul Gonzalez, an environmental scientist, led his team in transitioning from qPCR to ddPCR assays to optimize their practices in wastewater-based disease surveillance, performance evaluations of water reuse systems, and pathogenic aerosol detection, among other applications. Amid the COVID-19 pandemic, HRSD implemented ddPCR approaches to enhance the research and laboratory capacity of the utility, uncovering the most effective methodological approaches for detecting viral compounds in wastewater.
For Gonzalez and HRSD, ddPCR approaches have expanded their sample capacity from a modest ten to over a thousand samples processed per month. Gonzalez emphasizes that this expanded capability, coupled with HRSD's increased confidence in the accuracy of the data, "has made our data more actionable, consequently instilling confidence in our collaborators and downstream data users."
Oncology: Remco de Kock
Remco de Kock from Catharina Hospital Eindhoven has played a pivotal role in adapting multiplex ddPCR methods to analyze circulating tumor DNA (ctDNA) in lung cancer treatment and monitoring. His research focuses on assessing the clinical value of ctDNA analysis in diagnosing, selecting therapy, and monitoring treatment response in patients. Its capacity to amplify low concentrations of ctDNA and deliver reliable and absolute quantification distinguished ddPCR technology as the best tool for the task. Furthermore, de Kock's research tracks changes in ctDNA concentration over time, enabling a correlative evaluation of how concentrations evolve in response to therapy. This analysis can facilitate the early detection of progressive diseases through therapy response monitoring.
Additionally, de Kock's team proposes an innovative diagnostic strategy that employs ddPCR assays to screen patient ctDNA, allowing for the optimal selection of candidates for mutation analysis. Implementing an initial screening step could reduce the overall costs of tumor profiling, enabling resources and efforts to be redirected toward diagnostics and targeted therapies for lung cancer patients.
Respiratory Disease: Raphael Nyaruaba
Raphael Nyaruaba, conducting research at the Wuhan Institute of Virology, investigates the potential applications of digital PCR in developing rapid molecular diagnostic tools for infectious diseases such as SARS-CoV-2 and tuberculosis. Nyaruaba's research demonstrates how ddPCR technology surpasses other commonly used qualitative molecular diagnostic methods by rapidly detecting changes in bacterial concentration.
Leveraging the heightened sensitivity of ddPCR techniques to detect even minute bacterial amounts, Nyaruaba's team showcased, in a proof-of-concept article, how ddPCR methodology can reduce the time required for drug sensitivity detection from weeks to just four days. Additionally, the research group adapted ddPCR multiplexing approach to create an assay capable of detecting two biomarkers of Mycobacterium tuberculosis within a single channel.
Empowering Tomorrow's Life Science Research
Droplet Digital PCR technology has emerged as a powerful and versatile tool that drives advancements in research and clinical diagnostics. Its accurate, sensitive, and quantitative measurements have enhanced many fields, including oncology, infectious disease testing, molecular diagnostics, and biopharmaceutical development and manufacturing.
By delivering real-time molecular information, ddPCR technology has the potential to transform the landscape of biological research. The remarkable achievements of scientists like Morley, Gonzalez, de Kock, and Nyaruaba, who harness ddPCR capabilities in their respective fields, demonstrate the profound impact of this methodology.
Researchers continue to innovate and expand the applications of ddPCR technology and, in doing so, bring essential quantification and precision to life science and clinical research.