Explore the Future of Quantification With dPCR

Digital PCR applications Introduction Digital PCR (dPCR) is a powerful technology for detecting and quantifying nucleic acids. In dPCR, nucleic acids within a sample are randomly distributed into numerous smallvolume micro-reactions and amplified in parallel. dPCR does not rely on references or standards to obtain target concentrations, which helps reduce variability caused by relying on these standards for quantitative measurement. It is also highly sensitive and precise, making it ideal for detecting rare targets and measuring small-fold changes. The Applied Biosystems™ QuantStudio™ Absolute Q™ Digital PCR System can perform all of the necessary steps for dPCR to be conducted on a single instrument. Microfluidic array plate (MAP) technology further improves dPCR by enabling exceptional consistency and accuracy, as well as reduced dead volumes. Here, explore what dPCR can achieve across a wide range of fields and applications. The two fundamental aspects of digital PCR are separating the bulk reaction mix into many isolated micro-reactions and leveraging an endpoint measurement to assess PCR product formation. Separating the targets into micro-reactions allows direct quantification of the target sequence (through Poisson statistics) without the need for reference standards and enables high precision results. It also greatly enhances assay sensitivity for selectively detecting rare target sequences within a high abundance of similar background wildtype sequences (e.g., single base pair changes). Finally, it allows researchers to assess if multiple target locations are physically linked to each other on the same molecule (e.g., plasmid, RNA, chromosome, viral vector). Evaluating the endpoint PCR signal makes digital PCR more resistant to PCR inhibitors present in the sample since variations in PCR efficiency do not impact the endpoint digital measurement. In contrast, Ct values obtained from qPCR are affected by PCR reaction efficiency, and changes in efficiency can complicate quantitative or even qualitative analyses. 6 key benefits of dPCR The advantages of dPCR described above can be summarized in the following set of key benefits: Absolute quantification High precision Rare target sensitivity Robustness against PCR inhibition Quantify low abundance targets Molecule-specific analysis Detecting rare molecular sequences and biomarkers for liquid biopsy research 5 Measuring viral titers 8 Environmental DNA/RNA applications 11 Quantifying residual DNA 13 Gene expression 16 Gene editing confirmation 18 Contents 5 Background Cell-free DNA (cfDNA), released from necrotic and apoptotic cells, is a valuable potential source of tumor-derived DNA. Because cfDNA can be obtained from liquid biopsies, it represents a noninvasive way to identify and track oncogenic mutations and other cancer-related biomarkers. Characterizing cfDNA found in liquid biopsies helps researchers detect cancer, quantify tumor burden, and measure and monitor therapeutic response. Digital PCR (dPCR), with its high sensitivity, is better suited for rare target detection compared to conventional methods. It can precisely quantify cfDNA and circulating tumor DNA (ctDNA), which are usually present in low concentrations. dPCR can not only detect rare targets, but also distinguish rare targets from more abundant wildtype counterparts with similar sequences. dPCR does this by drastically reducing target competition through compartmentalizing the PCR reaction volume into thousands of individual micro-reactions, enabling quantification of rare targets with mutant allele frequencies of 0.1% or lower. Detecting rare molecular sequences and biomarkers for liquid biopsy research Example workflow DNA extraction MagMAX™ Cell-Free Total Nucleic Acid Isolation Kit MagMAX Cell-Free DNA Isolation Kit dPCR prep Absolute Q™ Universal DNA dPCR Master Mix (5X) Absolute Q™ Liquid Biopsy dPCR Assays Plate loading QuantStudio™ Absolute Q™ MAP16 Plate Kit dPCR run QuantStudio Absolute Q Digital PCR System Data analysis QuantStudio™ Absolute Q™ Digital PCR Software Absolute quantification High precision Rare target sensitivity Robustness against PCR inhibition Quantify low abundance targets Molecule-specific analysis Benefits 6 FAM dye fluorescence VIC dye fluorescence 0.1% Mutant allele frequency 25K 20K 15K 10K 5K 0 0 1,000 2,000 3,000 4,000 5,000 6,000 7,000 The QuantStudio Absolute Q dPCR System can effectively distinguish low-abundance mutant KRAS p.G12V alleles against a background of 99.9% wild type KRAS alleles using the KRAS 520 assay for detecting the KRAS p.G12V mutation. Orange dots represent dPCR micro-reactions positive for the wildtype allele only, purple dots represent micro-reactions positive for the mutant allele only, and green dots represent micro-reactions present for both alleles. Micro-reactions negative for both alleles are indicated by black dots. dPCR can detect mutant KRAS p.G12V alleles against a background of 99.9% wild type KRAS alleles Case studies CAR-T cell monitoring using digital PCR technologies Raquel Muñoz García, PhD Biomedicine, Instituto de Biomedicina de Sevilla, Hospital Virgen del Rocio, Spain Using digital PCR to quantify plasma genotyping Yanan Kuang, PhD Dana Farber Cancer Institute Watch webinar Watch webinar 7 Ordering information Description Contents Cat. No QuantStudio Absolute Q Digital PCR System (see website) A52864 Absolute Q Universal DNA dPCR Master Mix (5X) Single tube containing ready-to-use, 5X-concentrated dPCR master mix; sufficient for 200 dPCR reactions A72710 QuantStudio Absolute Q MAP16 Plate Kit 12x 16-well microinjection-molded plates for use with the QuantStudio Absolute Q Digital PCR System A52865 MagMAX Cell-Free Total Nucleic Acid Isolation Kit MagMAX Cell-Free Total Nucleic Acid lysis/binding solution, wash solution concentrate, elution solution, magnetic beads, proteinase K A36716 MagMAX Cell-Free DNA Isolation Kit MagMAX Cell-Free DNA magnetic beads, lysis/binding solution, wash solution, elution solution A29319 Absolute Q Liquid Biopsy dPCR Assays (see website) 8 Measuring viral titers Background Cell and gene therapies use viral vectors for gene transfer, including adeno-associated viruses (AAV), lentiviruses, retroviruses, herpesviruses, and adenoviruses. These viral vectors need to be accurately quantified to facilitate correct formulation when creating new therapeutic approaches. Although quantitative PCR (qPCR) is commonly used for nucleic acid qualification, digital PCR (dPCR) can offer a comprehensive alternative. dPCR provides absolute quantification across a range of nucleic acid concentrations without the need for a standard curve, thus removing a source of variability and simplifying workflows. dPCR can further leverage the use of micro-reactions to provide addition information beyond just target concentration. It can also interrogate specific locations along a length of DNA to determine intactness, letting scientists assess if nucleic acid targets are physically linked, or intact, on the same template molecule. This enables researchers to evaluate if packaged viral genomes are intact or fragmented. DNA/RNA extraction Sample collection dPCR prep Absolute Q Universal DNA dPCR Master Mix (5X) Absolute Q 1-Step RT-dPCR Master Mix (4X) Absolute Q Viral Titer dPCR Assays Plate loading QuantStudio Absolute Q MAP16 Plate Kit dPCR run QuantStudio Absolute Q Digital PCR System Data analysis QuantStudio Absolute Q Digital PCR Software Benefits Absolute quantification High precision Rare target sensitivity Robustness against PCR inhibition Quantify low abundance targets Molecule-specific analysis Example workflow 9 NTC 0 250 500 750 1000 1250 1500 1 244 703 1,376 2 192 516 1,136 0 202 542 1,138 R esult cp/μL NTC 123 427 876 0 182 532 1,245 0 120 404 860 1e-001 131 435 913 QuantStudio Absolute Q Digital PCR System Competitor A QuantStudio 3D Competitor B AAV CMV Target 0 1,000 2,000 3,000 4,000 0 1,000 2,000 3,000 4,000 Expected concentration (copies/μL) AAV particles Control DNA O bserv e d con c entration (c o pies/μL) A B Sample 1 Sample 2 Sample 3 Sample 1 Sample 2 Sample 3 Absolute Q Viral Titer dPCR Assay for AAV demonstrates the precision of dPCR, with observed results mirroring expected results (A). The Absolute Q AAV and Absolute Q CMV dPCR assays provide AAV and CMV quantification precision across multiple dPCR platforms and multiple concentrations (B). dPCR provides high quantification precision across a range of concentrations 10 Ordering information Description Contents Cat. No QuantStudio Absolute Q Digital PCR System (see website) A52864 Absolute Q Universal DNA dPCR Master Mix (5X) Single tube containing ready-to-use, 5X-concentrated dPCR master mix; sufficient for 200 dPCR reactions A72710 QuantStudio Absolute Q MAP16 Plate Kit 12x 16-well microinjection-molded plates for use with the QuantStudio Absolute Q Digital PCR System A52865 Absolute Q 1-Step RT-dPCR Master Mix (4X) Single tube containing ready-to-use, 4X concentrated dPCR master mix; sufficient for 200 dPCR reactions A55146 Absolute Q AAV dPCR Assay Single tube containing ready-to-use, 20X-concentrated assay reagent; sufficient for 700 dPCR reactions A52740 Absolute Q Viral Titer dPCR Assay - CMV Single tube containing ready-to-use, 20x-concentrated assay reagent; sufficient for 700 dPCR reactions A52741 Absolute Q Viral Titer dPCR Assays (see website) Case studies Achieving consistent adeno-associated virus quantification with the QuantStudio Absolute Q AutoRun dPCR Suite Kimberly Gomez, Thermo Fisher Scientific Himani Patel, Thermo Fisher Scientific Watch webinar 11 DNA/RNA extraction MagMAX Wastewater Ultra Nucleic Acid Isolation Kits Sample collection dPCR prep Absolute Q Universal DNA dPCR Master Mix (5X) Absolute Q dPCR SARS-CoV-2 Wastewater Surveillance Kit Plate loading QuantStudio Absolute Q MAP16 Plate Kit dPCR run QuantStudio Absolute Q Digital PCR System Data analysis QuantStudio Absolute Q Digital PCR Software Example Workflow Background Wastewater surveillance is an effective method for evaluating pathogen spread within a community. This came to the forefront during the COVID-19 pandemic, with researchers tracking SARS-CoV-2 RNA in community wastewater to help inform public health policies. Wastewater surveillance often involves measuring low concentration targets in samples containing PCR inhibitors. dPCR is well-suited to accurately detecting and quantifying small relative changes in lowconcentration targets.1 dPCR is also less sensitive to contaminants that may affect amplification efficiency. Researchers are now leveraging dPCR to help monitor potential health issues such as pathogens/parasites and antimicrobial resistance. It is also useful for identifying rare native species and invasive species. The QuantStudio Absolute Q dPCR system enables background subtraction and machine learning-backed false positive rejection, enabling high confidence in quantification results—valuable in situations where few positive micro-reactions are expected per sample. Environmental DNA/RNA applications Benefits Absolute quantification High precision Rare target sensitivity Robustness against PCR inhibition Quantify low abundance targets Molecule-specific analysis 12 Water samples from fish hatcheries were analyzed for invasive mud snail DNA. qPCR Ct values were >39, but dPCR returned clear positive results. dPCR avoids the variability found in high qPCR Ct values that can make it difficult to detect trends between samples. dPCR can overcome PCR inhibitors to detect environmental DNA Ordering information Description Contents Cat. No QuantStudio Absolute Q Digital PCR System (see website) A52864 Absolute Q Universal DNA dPCR Master Mix (5X) Single tube containing ready-to-use, 5X-concentrated dPCR master mix; sufficient for 200 dPCR reactions A72710 QuantStudio Absolute Q MAP16 Plate Kit 12x 16-well microinjection-molded plates for use with the QuantStudio Absolute Q Digital PCR System A52865 Absolute Q dPCR SARS-CoV-2 Wastewater Surveillance Kit Absolute Q 1-step RT-dPCR Master Mix (4X-concentrated), Absolute Q Wastewater Surveillance Assay (20X-concentrated); sufficient for 200 dPCR reactions A55241 MagMAX Wastewater Ultra Nucleic Acid Isolation Kit Lysis buffer, binding solution, wash buffer, elution solution, DNA/RNA binding beads, proteinase K A52606 MagMAX Wastewater Ultra Nucleic Acid Isolation Kit with Virus Enrichment Dynabeads wastewater virus enrichment, lysis buffer, binding solution, wash buffer, elution solution, DNA/ RNA binding beads, proteinase K A52610 Reference 1. Li H, et al., Application of droplet digital PCR to detect the pathogens of infectious diseases. Biosci Rep. 2018;38(6):BSR20181170. 13 Quantifying residual DNA Background Biomanufacturing often employs biological elements from many different species. For example, a Chinese hamster ovary cell can be transfected using an Escherichia coli plasmid to produce a human protein. It is imperative that residual DNA from the manufacturing process—whether from the host cell or the plasmid—not be present in the final product. Removing host cell and process-based impurities during biopharmaceutical purification is important to support regulatory compliance. Digital PCR (dPCR) provides scientists with a rapid, streamlined, sensitive, and precise method for detecting residual DNA. The QuantStudio Absolute Q dPCR system is compatible with resDNASEQ™ assays, which are designed and analytically evaluated to help biopharmaceutical manufactures meet regulatory requirements. The Applied Biosystems resDNASEQ dPCR E1A DNA Fragment Length Kit enables accurate absolute quantitation of residual DNA fragment sizes in HEK293 production systems and other cell lines transformed with the E1A gene. The Applied Biosystems resDNASEQ dPCR E. coli DNA Kit enables sensitive and specific quantitation of E. coli DNA. Benefits Absolute quantification High precision Rare target sensitivity Robustness against PCR inhibition Quantify low abundance targets Molecule-specific analysis Example workflow Upstream Downstream Testing for residual DNA occurs during process development and final QC Plasmid development and production Cell expansion Plasmid transfection Viral vector production Purification Fill and finish dPCR quantification of residual DNA 14 dPCR E.coli assay performance 90 copies/rxn 27 copies/rxn 90,000 copies/rxn 9,000 copies/rxn 900 copies/rxn 9 copies/rxn The dPCR E. coli assay shows high detection sensitivity and broad dynamic range, detecting E. coli host cell DNA at concentrations as high as 90,000 and as low as 9 copies per reaction. dPCR E. coli and E1A assay linearity The dPCR E. coli (A) and HEK293 cells EIA (B) assays show high linearity to enable quantitative results across a broad range of DNA concentrations. A B 15 Ordering information Description Contents Cat. No QuantStudio Absolute Q Digital PCR System (see website) A52864 Absolute Q Universal DNA dPCR Master Mix (5X) Single tube containing ready-to-use, 5X-concentrated dPCR master mix; sufficient for 200 dPCR reactions A72710 QuantStudio Absolute Q MAP16 Plate Kit 12x 16-well microinjection-molded plates for use with the QuantStudio Absolute Q Digital PCR System A52865 resDNASEQ dPCR E1A DNA Fragment Length Kit resDNASEQ E1A DNA Control and dPCR Reagents A55852 resDNASEQ dPCR E. coli DNA Kit resDNASEQ E. coli DNA Control and dPCR Reagents A57017 resDNASEQ dPCR CHO DNA Kit resDNASEQ CHO DNA dPCR Reagents A59366 resDNASEQ dPCR Sf9 and Baculovirus DNA Kit dPCR Sf9 + Baculovirus DNA dPCR Reagents A59319 resDNASEQ Residual Host Cell DNA Kits (see website) 16 Gene expression Background Real-time PCR (qPCR) is commonly used to evaluate differential gene expression. A key strength of qPCR is its wide dynamic range, facilitating easy comparisons between genes with large differences in their relative abundances. For many analyses of gene expression, qPCR provides sufficient precision for meaningful comparisons of expression fold-change values. However, qPCR data can at times lack the precision needed to confidently interrogate smaller fold-changes in a gene’s expression level. Digital PCR (dPCR) can provide high precision measurements compared to qPCR, allowing confident discernment of smaller relative changes, even well below 2-fold. This ability can benefit investigations where slight changes in a gene’s expression have meaningful biological impacts, as well as experiments monitoring trends in expression levels over time. In addition, lower expressing genes assessed by qPCR yield higher Ct values. The increased variability associated with high Ct values can make gene expression analysis difficult even when interrogating moderate fold-change values. The ability of dPCR to deliver precise quantification at low target-concentrations complements gene expression studies where one or more targets are of low abundance. Benefits Absolute quantification High precision Rare target sensitivity Robustness against PCR inhibition Quantify low abundance targets Molecule-specific analysis DNA/RNA extraction Sample collection dPCR prep Absolute Q Universal DNA dPCR Master Mix (5X) Plate loading QuantStudio Absolute Q MAP16 Plate Kit dPCR run QuantStudio Absolute Q Digital PCR System Data analysis QuantStudio Absolute Q Digital PCR Software Example workflow 17 Ordering information Description Contents Cat. No QuantStudio Absolute Q Digital PCR System (see website) A52864 Absolute Q Universal DNA dPCR Master Mix (5X) Single tube containing ready-to-use, 5X-concentrated dPCR master mix; sufficient for 200 dPCR reactions A72710 QuantStudio Absolute Q MAP16 Plate Kit 12x 16-well microinjection-molded plates for use with the QuantStudio Absolute Q Digital PCR System A52865 Case studies Applications of multiplex digital PCR: Monitoring wastewater for SARS-CoV-2 and measuring bacterial gene expression Dr. Joan L. Slonczewski, Kenyon College Watch webinar 18 Gene editing confirmation Background The continued development of gene editing tools allows scientists to create more biologically relevant disease models and explore a greater range of potential therapeutic options. However, gene editing is rarely completely efficient or accurate. Low gene editing efficiency and off-target gene edits pose risks in terms of both product safety and workflow efficiency. Scientists therefore need to verify constructs for cell and gene therapy, assess the number of integrated vector genomes, and quantify the number of successful gene editing events or confirm the presence of phased variants. Digital PCR (dPCR) can be a powerful tool in gene editing assessment and validation. dPCR’s endpoint detection facilitates easy multiplexing for quantitative assays. Additionally, isolation of target molecules into micro-reactions enables multi-target evaluation of gene editing results for conducting analyses such as drop-off assays, as well as assessment of off-target insertion events. Benefits Absolute quantification High precision Rare target sensitivity Robustness against PCR inhibition Quantify low abundance targets Molecule-specific analysis Design assay Absolute Q Custom Digital PCR Assays Sample collection dPCR prep Absolute Q Universal DNA dPCR PCR Master Mix (5X Plate loading QuantStudio Absolute Q MAP16 Plate Kit dPCR run QuantStudio Absolute Q Digital PCR System Data analysis QuantStudio Absolute Q Digital PCR Software Example workflow Ordering information Description Contents Cat. No QuantStudio Absolute Q Digital PCR System (see website) A52864 Absolute Q Universal DNA dPCR Master Mix (5X) Single tube containing ready-to-use, 5X-concentrated dPCR master mix; sufficient for 200 dPCR reactions A72710 QuantStudio Absolute Q MAP16 Plate Kit 12x 16-well microinjection-molded plates for use with the QuantStudio Absolute Q Digital PCR System A52865 A digital PCR-based protocol to detect and quantify RNA editing events at hotspots Dr. Remi Buisson, University of California Irvine Watch webinar For Research Use Only. Not for use in diagnostic procedures. © 2024 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. EXT6981 0724 Learn more at thermofisher.com/dPCR