Custom Solutions for Molecular Test Development

Challenge accepted Custom solutions for molecular test development Reagents, instruments, and plastics for PCR and isothermal amplification workflows For Research Use Only. Not for use in diagnostic procedures. PCR and isothermal amplification Whatever your project size, we’ll help you achieve your commercialization goals Our Commercial Supply team is your partner for successful commercial development of your future diagnostic, research, and applied products. We offer an exceptional combination of quality, globally distributed products, and professional commercial services to support and guide you at every step. “The quality and performance of Applied Biosystems™ master mixes and Invitrogen™ custom oligos are key for our kit development. The Thermo Fisher Scientific team provided us with a one-stop solution for the entire process—from assay test design, assay optimization, and kit validation, to partnering on the development of our commercialization strategy.” —Patrick Burke, PhD, Executive Vice President, Emerging Products, Myriad Genetics 2 Contents Introduction 4 Commercial supply capabilities 5 Custom services and manufacturing 5 Exceptional quality 6 Robust security of supply 7 Domain competence 8 Molecular analysis workflow 9 Nucleic acid extraction 10 Dynabeads magnetic beads 10 MagMAX kits 12 KingFisher instruments 15 Amplification 16 Reverse transcriptases 16 DNA polymerases 18 High-fidelity DNA polymerases 20 PCR-grade dNTPs 23 qPCR master mixes 24 RT-LAMP master mix 25 Lyo-ready enzymes 26 Lyo-ready Bst DNA Polymerase 31 Lyo-ready master mixes 34 Primers, probes, and oligonucleotides 35 Instrumentation 36 PCR and qPCR plastics, seals, and accessories 38 Custom manufacturing services 39 3 Introduction If you’re involved in the development of a commercial molecular assay, you’ve come to the right place. When you bring our experienced Commercial Supply team onboard, whether your project is large or small, your commercialization goals become our goals. By leveraging our exceptional combination of quality, global distribution, and professional commercial services and support, you can maximize your efficiency and scale while minimizing risk. More than just a partner for your project, you’ll have an experienced personal guide committed to your success at every stage of the product development and commercialization process. Accelerate growth and differentiate yourself in the market with: • Reagents available for incorporation into your assay • Global installed base for validating your kit • Instrument placement and reagent rentals • Private-label options 4 Commercial supply capabilities Your commercial assay is likely to require customization to fit your specificity, sensitivity, and precise workflow needs. Our products and services are not one-size-fits-all. When you partner with us, we build your assay components to your exact specifications. From assay design to workflow optimization—whatever your needs are, our custom services team is there for you. In fact, many of our custom services and products are available exclusively to our commercial partners. We can customize your products in a number of ways: • Fill volume • Formulation • Packaging • Private labeling • Bulk ordering Custom services include: • Assay design • Workflow development and optimization • Performance testing • Bridging studies • Guard banding • Verification testing • Analytical validation testing • Stability studies Manufacturing services: • Kitting • Fill and finish • Custom packaging • Private label Custom services and manufacturing Find out more at thermofisher.com/mdxcustom 5 When you rely on us for manufacturing, you’ll gain the advantage of our uniquely robust processes. You can have confidence that your product will be manufactured in accordance with regulatory compliance guidelines and our commitment to manufacturing excellence. Don’t cut corners when it comes to standards. Manufacture with us, and your product will go through rigorous testing to help ensure lot-to-lot consistency and reproducibility of your final supplies. You’ll benefit from our uncompromising quality management system, which requires our suppliers to undergo a high level of scrutiny, including thorough audits and quality control (QC) on incoming raw materials. Bring your vision into being in our state-of-the-art manufacturing centers. True to our company-wide commitment to advancing life sciences, your products will be manufactured in facilities that help ensure your: • Innovation • Flexibility • Reproducibility • Efficiency • Scalability Austin, TX Monterrey, MX Milwaukee, WI Rockford, IL Carlsbad, CA Pleasanton, CA Lillestrøm, Norway Oslo, Norway Grand Island, NY Frederick, MD Madison, WI Eugene, OR * Certain regulatory standards are available at specific sites only. Compliance* • Six Sigma and Lean Manufacturing processes • Certified quality system with ISO 9001, ISO 13485, and ISO 14001 status • US 21 CFR Part 820 Quality System (current good manufacturing practice (CGMP)) • Manufacturer of Class I, II, and III medical devices • Manufacturer of general-purpose reagents • Under USDA guidelines • Medicines and Healthcare products Regulatory Agency (MHRA) • Good laboratory practice (GLP)–accredited Exceptional quality 6 You shouldn’t have to worry about changes in raw materials that may require you to retest your product. Partnering with us helps ensure the security of your raw materials supply, which is crucial to developing and manufacturing a reliable commercial product. We have strong safeguards in place to help you reduce risk and avoid delays and setbacks in manufacturing, including: • Advanced change control notifications • Supply agreements • A global manufacturing footprint • Comprehensive controls over raw materials and processes • Multiple manufacturing sites • Financial stability • Confidentiality Vilnius, Lithuania Marsiling, Singapore Robust security of supply 7 Domain competence Any company—especially a smaller or newer company—can benefit greatly from working closely with us early in the product development process. That’s because we have extensive experience in developing and commercializing products, and we can leverage our leading-edge technologies to help benefit you. Your dedicated Business Development Manager will serve as your project champion and help connect you with the right resources in R&D, manufacturing, regulatory affairs, and more. Having a dedicated professional from Thermo Fisher on your team to guide you through the process of bringing a product to market can help you maximize efficiency and avoid costly missteps. We have partnered with companies who have developed assays in countless focus areas, including: • Oncology • Infectious disease • Reproductive health • Neurobiology • Cardiovascular disease • Hematology • Cell therapy • Agricultural and environmental • Food safety • Research tools 8 Molecular analysis workflow We offer a one-stop shop for development of molecular tests. All of our products are highly optimized to work in concert with one another, to help ensure compatibility between product lines. Choose from our trusted brands, including Thermo Scientific, Applied Biosystems, and Invitrogen. The products mentioned in this handbook represent those most commonly used by our molecular testing partners. If you can’t find exactly what you need, or would like assistance selecting the right product for your workflow, contact us and we will guide you. Nucleic acid extraction Reverse transcription Amplification • Dynabeads magnetic beads and buffers (page 10) • MagMAX kits (page 12) • KingFisher instruments (page 15) • Reverse transcriptases (page 16) • One-step master mixes (page 28) • dNTPs (page 23) • DNA polymerases (page 18) • Master mixes (page 24) • dNTPs (page 23) • Primers and probes (page 35) • Plates, tubes, and seals (page 38) Run and analysis • Thermal cyclers (page 36) • Real-time PCR instruments (page 37) “The broad range of genetic analysis products is only one of the many reasons we chose Thermo Fisher as our strategic partner for developing molecular tests. Its dedicated team—including field engineers, bioinformaticians, key account managers, CFDA registration experts, and global leaders—has been a great resource in helping us expand our business.” —Junquan Xu, President, CapitalBio Technology 9 Nucleic acid extraction We offer a wide range of extraction solutions for your unique assay or application, including Invitrogen™ Dynabeads™ magnetic beads, customizable for your workflow; or turnkey Applied Biosystems™ MagMAX™ kits, optimized for easy integration with your existing workflow. Both Dynabeads magnetic beads and MagMAX kits work seamlessly with Thermo Scientific™ KingFisher™ instruments for high-throughput genomic analysis. For more unique or demanding applications, our R&D scientists will work closely with you to customize our beads and buffers to your exact specifications. Dynabeads product Surface Bead size (μm) Properties MyOne™ SILANE Hydrophilic 1.1 • Silica-like surface • Highest magnetic content MyOne™ Carboxylic Acid Hydrophilic 1.1 • Negatively charged • Chromatography functions M-270 Carboxylic Acid Hydrophilic 2.8 • Negatively charged • Chromatography functions Dynabeads product Surface Bead size (μm) Properties MyOne™ Carboxylic Acid Hydrophilic 1.1 • Low nonspecific binding of nucleic acids • Covalent coupling of oligonucleotides M-270 Carboxylic Acid Hydrophilic 2.8 • Low nonspecific binding of nucleic acids • Covalent coupling of oligonucleotides MyOne™ Streptavidin C1 Hydrophilic 1.1 • Based on carboxylic acid beads • No blocking proteins MyOne™ Streptavidin T1 Hydrophobic 1.1 • Based on tosylactivated beads • BSA as blocking protein MyOne™ Streptavidin T2 Hydrophobic 1.1 • Based on tosylactivated beads • Animal origin–free blocking protein M-270 Streptavidin Hydrophilic 2.8 • Based on carboxylic acid beads • No blocking proteins M-280 Streptavidin Hydrophobic 2.8 • Based on tosylactivated beads • BSA as blocking protein Oligo(dT)25 Hydrophilic 2.8 • mRNA (poly(A)) capture Dynabeads magnetic beads are widely used for both generic and specific capture steps of a nucleic acid workflow and are applied to a broad range of research applications, including tissue-based and liquid biopsy–based oncology, noninvasive prenatal testing, and infectious diseases. Dynabeads magnetic beads are monodisperse with tight coefficient of variation (CV) specifications and exceptional batch-to-batch reproducibility. Our beads offer easy handling, excellent dispersion abilities, and lack of magnetic remanence, making Dynabeads magnetic beads well suited for manual as well as automated protocols. Generic capture of nucleic acid • Multi-target and multi-matrix compatibility • High-efficiency capture of rare or abundant targets • Flexible and scalable workflows Sequence-specific capture of nucleic acid • Dynabeads Streptavidin products enable fast and efficient capture of biotinylated oligonucleotides • Dynabeads Carboxylic Acid products enable tunable coupling of aminated oligonucleotides In addition to a wide portfolio of off-the-shelf Dynabeads products, our proprietary technology and know-how enable the Dynabeads products team to provide customized workflow and product development. Dynabeads magnetic beads 10 “Securing access to Invitrogen Dynabeads MyOne magnetic beads and reagents for our second-generation colorectal cancer blood test enabled us to reduce the complexity of the kit significantly. The high-quality materials were able to improve reproducibility and robustness of our method.” —Uwe Staub, PhD, Chief Operating Officer, Epigenomics Biomolecule coupling Bead surface modification Workflow optimization Customizable capabilities 11 Table 1. MagMAX kits for nucleic acid purification. Nucleic acid isolated Application Sample types Features MagMAX mirVana Total RNA Isolation Kit True total RNA (total RNA enriched for small RNA) RNA-based oncology or infectious diseases Works with most samples of interest, including plasma, serum, whole blood, cell culture, tissue, and urine • No need to buy multiple kits to work with specific samples • Phenol-free (no organic extraction) • Verified for use with Applied Biosystems™ TaqMan™ Advanced miRNA Assays MagMAX DNA Multi-Sample Ultra 2.0 Kit Genomic DNA Genotyping for inherited diseases, oncology, and infectious diseases Compatible with common sample types (whole blood, tissue, saliva, buccal swabs, and oral rinse) • Streamlined protocols for numerous noninvasive biological samples MagMAX Cell-Free DNA Isolation Kit Cell-free DNA Noninvasive prenatal testing (NIPT), liquid biopsy Whole blood • Can enrich for cell-free DNA fraction MagMAX Cell-Free Total Nucleic Acid Isolation Kit Cell-free DNA and RNA NIPT, liquid biopsy Whole blood • Enrichment of all nucleic acid species present in cell-free biological samples such as plasma MagMAX FFPE DNA/RNA Ultra Kit DNA and RNA Genotyping and gene expression for solid tumors FFPE samples • No sample splitting (RNA and DNA from same FFPE section) MagMAX Viral/Pathogen Ultra Nucleic Acid Isolation Kit DNA and RNA Infectious diseases Serum and plasma; nasal, tracheal, and cloacal swabs; ear samples; whole blood; semen; oral fluid; feces • Purification from viruses and easy-to-lyse bacteria and parasites • Low and high sample input volumes MagMAX Saliva gDNA Isolation Kit Genomic DNA A variety of molecular protocols, particularly for consumer genomics Saliva • Scalable and automatable protocol for the isolation of genomic DNA (gDNA) from fresh and stabilized saliva MagMAX Wastewater Ultra Nucleic Acid Isolation kit RNA and DNA Surveillance testing for infectious agents Wastewater samples • Fast procedure allows for 24–96 samples to be processed in ~45 min • Compatible with wastewater and sewage concentrated via ultracentrifugation, precipitation, filtration, or bead-based viral enrichment, and unconcentrated samples MagMAX Microbiome Ultra Nucleic Acid Isolation kit RNA and DNA Microbial genome analysis Stool, swabs, transport media, culture media, urine, saliva, and soil • Fast procedure allows for 96 samples to be processed in ~60 min • Mechanical disruption via bead beating in 96-well plate or individual tubes MagMAX kits Learn more about MagMAX kits at thermofisher.com/magmax Our MagMAX kits are designed for the purification of high-quality nucleic acids from a variety of sample types suitable for a range of downstream applications (Table 1). Each kit is optimized for use with KingFisher instruments to help reduce overall handling and processing times while increasing consistent sample preparation. Each kit contains the necessary beads and solutions for lysis/binding, wash, and elution. 12 Figure 1. Linear recovery of gDNA across a wide range of volume inputs. gDNA was extracted from 4 donors’ samples using the MagMAX Saliva gDNA Isolation Kit with the KingFisher Flex instrument. A total of 200 μL, 400 μL, 500 μL, 1 mL, and 2 mL of stabilized saliva from each donor was purified and measured for total DNA yield. Total yield (ng) Saliva input volume (L) Saliva input volume (L) Total yield (ng) 4,000 3,000 2,000 2,000 1,000 1,000 1,000 2,000 0 4,000 3,000 2,000 1,000 0 MagMAX Saliva gDNA linear recovery MagMAX Saliva gDNA linear recovery y = 1.5906x R2 = 0.9829 y = 1.8474x R2 = 0.9931 y = 1.0596x R2 = 0.9874 y = 1.0025x R2 = 0.9876 Featured MagMAX kit: MagMAX Saliva gDNA Isolation Kit The Applied Biosystems™ MagMAX™ Saliva gDNA Isolation Kit offers a fast and user-friendly way to purify gDNA from saliva. With minimal steps, an affordable price, and comprehensive support, users can process more samples with consistent results. Saliva is much more stable at room temperature than other sample types; it also yields a greater quantity of gDNA than buccal swabs, and does so more consistently. Highlights • Fast, easy, and budget-friendly automated sample prep for saliva gDNA • 96 samples in a ~20 min instrument run with less than 45 min of hands-on time • Compatible with top 10 saliva collection devices • Consistent gDNA recovery (Figure 1) Find out more at thermofisher.com/saliva 13 The Applied Biosystems™ MagMAX™ Cell-Free DNA Isolation Kit is designed for enrichment of circulating cell-free DNA (cfDNA) and optimized for use with biological samples such as serum and plasma. The kit is based on Applied Biosystems™ MagMAX™ magnetic bead technology, enabling reproducible recovery of high-quality DNA that is suitable for a broad range of applications, including downstream analysis of circulating tumor DNA, or fetal DNA. Highlights • Flexible sample volume inputs ranging from 500 μL to 10 mL of plasma, serum, or urine • Enrichment of cfDNA over gDNA; phenol-free extraction • Purified cfDNA in less than 45 min when used with KingFisher instruments • Recovers DNA shorter than 800 bp (Figure 2) Figure 2. Enrichment of cfDNA following extraction from plasma samples. Cell-free plasma was separated from 4 normal blood samples by centrifugation for 20 min at 2,000 x g, then for 30 min at 6,000 x g. DNA was extracted from 4 mL plasma using either the MagMAX Cell-Free DNA Isolation Kit (red trace) or kit Q (blue trace). The eluted DNA was loaded onto a High Sensitivity DNA Chip and run on the Agilent™ Bioanalyzer™ 2100 system. Sample 1 Sample 2 Sample 3 Sample 4 Size (bp) Size (bp) Size (bp) Size (bp) 35 100 150 200 300 400 500 600 1,000 2,000 10,380 35 100 150 200 300 400 500 600 1,000 2,000 10,380 35 100 150 200 300 400 500 600 1,000 2,000 10,380 35 100 150 200 300 400 500 600 1,000 2,000 10,380 60 50 40 30 20 10 0 140 120 100 80 60 40 20 0 140 120 100 80 60 40 20 0 140 120 100 80 60 40 20 0 RFU RFU RFU RFU Find out more at thermofisher.com/cfdnaisolation 14 Table 2. KingFisher instruments for automated sample preparation. Duo Prime system Apex system Presto system Instrument format Compact benchtop Benchtop Benchtop + robotic liquid handler Throughput range Low to medium Medium to high Ultrahigh Samples per run, plate format 6 or 12 per run 96 or 24 per run 96 or 24 per run Heating or cooling options Heating and cooling Heating and cooling Heating only KingFisher instruments KingFisher purification systems automate and increase the throughput of bead-based purification protocols. Designed to deliver high-quality results with minimal hands-on time, KingFisher systems use permanent magnetic rods and disposable tip combs to collect, transfer, and mix magnetic particles. KingFisher instruments support more applications than any other single sample preparation instrument. They are elegantly designed to use multiple magnetic bead reagents in 96- or 24-well plate format (Table 2). • Throughput: 6–96 samples per run • Plate format: 96- or 24-well plates • Protocols: optimized protocols from trusted reagent brands, or custom protocols • Reagents: support multiple magnetic bead reagents Find a model that supports you best at thermofisher.com/kingfisher 15 Amplification We offer a comprehensive portfolio of reverse transcriptases (RTs), from the wild-type Moloney murine leukemia virus (M-MuLV) RT to the Invitrogen™ SuperScript™ line of RTs with exceptional characteristics such as enhanced sensitivity and reduced reaction time (Table 3). Our proprietary in vitro protein evolution technology has enabled the introduction of multiple favorable mutations into the traditional M-MuLV RT. This has dramatically improved the enzyme’s thermostability, tolerance for inhibitors, and processivity. Benefits • Robust performance with challenging samples • Enhanced thermostability and processivity • High efficiency and sensitivity, even in the presence of inhibitors • Minimal false-positive results with low residual host-cell DNA • Lyophilization compatibility with lyo-ready formulation Find out more at thermofisher.com/reverse-transcription Reverse transcriptases Table 3. RT selection chart. Characteristic SuperScript RT, 1-Step RT-qPCR SuperScript IV RT SuperScript III RT Maxima RT RevertAid RT (M-MuLV) Optimal reaction temperature 50°C 50°C 50°C 50°C 37–42°C RNase H activity Yes No Reduced Yes Yes RNase H– version available No No No Yes Yes Reaction time 5 min 10 min 15 min 30 min 60 min Inhibitor tolerance High High Good Good Low Sensitivity High High Medium Good Medium Lyo-ready* Yes Yes Yes Yes Yes 1-step RT-qPCR Yes No Yes No Yes 2-step RT-qPCR No Yes Yes Yes Yes 1- or 2-step RT-PCR, LAMP, other No Yes No Yes No * Lyo-ready is a lyophilization-compatible enzyme formulation without glycerol. 16 Figure 3. Linearity across 10 orders of magnitude of RNA input. RT-qPCR targeting human 18S rRNA was performed using Invitrogen™ SuperScript™ IV VILO™ Master Mix and an Applied Biosystems™ TaqMan™ Assay with inputs of 1 fg–1 μg HeLa total RNA. E (efficiency of the reaction) = 94.2%, R2 = 0.999. 0.01 0.1 1.0 10.0 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 Rn 0 5 10 15 20 25 30 35 40 Ct 0.15 1 μg 100 ng 10 ng 1 ng 100 pg 10 pg 1 pg 100 fg 10 fg 1 fg RNA quantity (pg) Cycle 10−2 100 102 104 106 0.01 0.1 1.0 10.0 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 Rn 0 5 10 15 20 25 30 35 40 Ct 0.15 1 μg 100 ng 10 ng 1 ng 100 pg 10 pg 1 pg 100 fg 10 fg 1 fg RNA quantity (pg) Cycle 10−2 100 102 104 106 Table 4. Quality testing to help ensure high performance of the RT in your RNA-based assays. Parameter* Method used Unit concentration One unit of the enzyme incorporates 1 nmol of dTMP into a polynucleotide fraction in 10 min at 37°C Endodeoxyribonucleases and exodeoxyribonucleases Incubation of labeled single- and double-stranded oligonucleotides with enzyme Endodeoxyribonucleases (nicking activity) Incubation of supercoiled plasmid DNA with enzyme Ribonucleases Incubation of radiolabeled RNA transcript with enzyme Functional testing Functionally tested in first-strand cDNA synthesis Human gDNA Quantitative PCR test, which uses amplification of selected human gDNA fragments E. coli gDNA Quantitative PCR test, which uses amplification of E. coli 23S rRNA gene fragment * Scope of standard quality-control program may vary for a specific enzyme. Featured RT: SuperScript IV RT Invitrogen™ SuperScript™ IV RT is an M-MuLV RT mutant with exceptional robustness and reliability in cDNA synthesis. This RT enzyme exhibits strong inhibitor tolerance, high processivity, thermostability, highly efficient full-length cDNA synthesis, and reduced RNase H activity. SuperScript IV RT demonstrates its ability to carry out reliable cDNA synthesis in the presence of common PCR reaction inhibitors, which are typical in situations where biological samples of optimal quality are not available. Highlights • Robust cDNA synthesis with a variety of gene targets, including degraded RNA and RNA samples of suboptimal purity • High sensitivity and linearity in a 10-minute reaction • Linear dynamic range across a broad range of RNA input (Figure 3), crucial for detection of low-abundance targets • Extensive quality testing (Table 4) Find out more at thermofisher.com/ssiv 17 DNA polymerases We are dedicated to providing PCR enzymes that meet the most demanding customer requirements. Our knowledge and experience in enzymology and in vitro evolution allow us to develop enzymes with new or improved properties. Our customers can select enzymes from a spectrum of DNA polymerases based on a combination of hot-start technology, speed, level of tolerance for PCR inhibitors, or lyophilization compatibility (Table 5). High performance, lot-to-lot consistency, and extensive quality-control testing enable high sensitivity, accuracy, and reproducibility of PCR and real-time PCR (qPCR) assays developed with our enzymes. Benefits • High sensitivity and specificity for detection of low-copy DNA targets • Robust amplification of difficult-to-amplify targets, including those of suboptimal purity • Lyophilization- and microfluidics-compatible formulations available • Tailor-made solutions available for your specific applications, including product custom formulation • Available as a stand-alone enzyme or in a master mix format Table 5. Taq DNA polymerase selection chart. Characteristic Platinum II Taq DNA Polymerase** Platinum Taq DNA Polymerase** AmpliTaq Gold DNA Polymerase LibertyTaq DNA Polymerase Wild-type Taq DNA Polymerase Phire Hot Start II DNA Polymerase Hot-start PCR Antibody-based Antibody-based Chemically modified Proprietary No Affibody-based TaqMan probe–compatible Yes Yes Yes Yes Yes No Reactivation time 2 min 2 min 10 min 0 min 0 min 0 min Extension rate 15 sec/kb 30–60 sec/kb 30–60 sec/kb 30–60 sec/kb 30–60 sec/kb 10–15 sec/kb Sensitivity High High High Good Good Good Specificity High High High Good Good Good Inhibitor tolerance High Standard Standard Standard Standard Standard Lyo-ready* Yes Yes Yes Yes Yes Yes * Lyo-ready is a lyophilization- and microfluidics-compatible enzyme formulation without glycerol. ** DNA-free version available. Find out more at thermofisher.com/pcrenzymes Taq DNA polymerases 18 Table 6. Quality testing to help ensure high performance of DNA polymerases in your assays. Parameter* Method used Unit concentration Incorporation of labeled dNTP into polynucleotide fraction by enzyme during the selected time interval Endodeoxyribonucleases and exodeoxyribonucleases Incubation of labeled single- and double-stranded oligonucleotides with enzyme Ribonucleases Incubation of labeled RNA transcript with enzyme Residual activity assay Extension of labeled, double-stranded oligonucleotide with 5ʹ overhangs by enzymes without heat activation Functional testing Quantitative PCR containing 10-fold dilutions over 5 orders of magnitude of human and E. coli gDNA Human gDNA Quantitative PCR test, which uses amplification of selected human gDNA fragments E. coli gDNA Quantitative PCR test, which uses amplification of E. coli 23S rRNA gene fragment * Scope of standard quality-control program may vary for a specific enzyme. Figure 4. Multiplex PCR with Platinum II Taq Hot-Start DNA Polymerase. Fifteen targets (99, 131, 160, 199, 251, 300, 345, 400, 516, 613, 735, 908, 1,005, 1,190, and 1,606 bp) were simultaneously amplified from 0, 1.6, 8, 40, 200, and 1,000 ng of human genomic DNA, in 50 μL reactions. The Thermo Scientific™ GeneRuler™ 100 bp Plus DNA Ladder was used as a size marker (M). Featured Taq DNA polymerase: Platinum II Taq Hot-Start DNA Polymerase Invitrogen™ Platinum™ II Taq Hot-Start DNA Polymerase combines an enzyme engineered for speed and inhibitor tolerance with a proprietary buffer that allows for a universal primer annealing temperature. These attributes, combined with a robust hot-start feature, open new possibilities in DNA- and RNA-based assay designs (Figure 4). Highlights • Platinum hot-start technology offers exceptional specificity, sensitivity, and yields and retains these features in the glycerol-free format • Engineered Taq DNA polymerase allows fast cycling, even in inhibitor-rich environments (various transport media, hemin, EDTA, citrate, isopropanol) • Lyo-ready formulation is available; for the lyo-ready version, an optimized protocol is available for one-step RT-qPCR with Lyo-Ready SuperScript RT, 1-Step RT-qPCR • Extensive quality testing (Table 6) Find out more at thermofisher.com/platinumiitaq M M 19 Table 7. High-fidelity DNA polymerase selection chart. Characteristic Platinum SuperFi II DNA Polymerase Phusion Plus DNA Polymerase Phusion Hot Start II DNA Polymerase Phusion U Hot Start DNA Polymerase Fidelity compared to Taq polymerase >300x >100x 52x 25x Hot-start PCR Antibody-based Affibody-based Affibody-based Affibody-based Extension rate 15–30 sec/kb 15–30 sec/kb 15–30 sec/kb 15–30 sec/kb Inhibitor tolerance High Good Medium Medium dUTP tolerance No No No Yes Multiplexing Yes Yes Yes Yes Lyo-ready* No No Yes On request * Lyo-ready is a lyophilization-compatible enzyme formulation without glycerol. Thermo Scientific™ and Invitrogen™ high-fidelity DNA polymerases are designed to amplify DNA fragments with exceptional robustness and fidelity, and to generate PCR products with high accuracy and speed, even with the most difficult templates. Choose from a collection of our high-fidelity enzymes: their formats, buffers, and dNTP solutions vary depending on the sophistication of your DNA-based assay and your needs for flexibility (Table 7). Find out more at thermofisher.com/pcrenzymes Benefits • Highest fidelity on the market (>100x Taq polymerase) • Robust amplification of versatile targets, up to 20 kb • Exceptional tolerance of PCR inhibitors • Shorter cycling times for faster time-to-results • High yields without optimization • Minimal nonspecific amplification • Available as a stand-alone enzyme or in a master mix format High-fidelity DNA polymerases 20 Table 8. Quality testing to help ensure optimal performance of high-fidelity DNA polymerases. Parameter* Method used Unit concentration One unit of enzyme incorporates 10 nmol of dNTPs into a polynucleotide fraction at 74°C in 30 min Endodeoxyribonucleases Incubation of supercoiled plasmid DNA with 10 U of enzyme at 37°C for 4 hr and analysis on agarose gel Residual activity assay Extension of labeled, double-stranded oligonucleotide with 5ʹ overhangs for 4 hr at 37°C in the presence of dNTPs Functional testing in PCR PCR amplification of 7.5 kb fragments from human gDNA and 20 kb fragments from lambda DNA, and analysis on agarose gel Human gDNA Quantitative PCR test, which uses amplification of selected human gDNA fragments * Scope of standard quality-control program varies for different enzymes. Figure 5. Fidelity comparison across commercially available enzymes relative to Taq enzyme. A 3.9 kb sequence was amplified by PCR using different DNA polymerases, and the resulting PCR amplicons were then fragmented with a MuA transposase. Unique molecular identifiers (UMI), which consist of 12 random nucleotides, were introduced during fragmentation to individually tag each product. After next-generation sequencing, reads were aligned to the correct sequence, grouped by UMI families, and errors were called. Errors were identified only if they were present in all reads in the UMI family; otherwise they were discarded as sequencing errors. The polymerase fidelities were normalized to that of Taq DNA polymerase. Figure 6. Platinum SuperFi II DNA Polymerase shows high tolerance to common PCR inhibitors. A 2 kb human genomic DNA fragment was amplified from 50 ng of human genomic DNA using Platinum SuperFi II DNA Polymerase or high-fidelity DNA polymerases from other suppliers: A, Q5™ Hot Start High-Fidelity; B, PrimeSTAR™ GXL; C, Merck KOD Hot Start; D, KAPA™ HiFi HotStart PCR Kit, in reaction mixtures containing 1, no inhibitor; 2, humic acid (4 μg/mL); 3, hemin (20 μM); or 4, bile salt (1 mg/mL). The size marker is the Invitrogen™ TrackIt™ 1 Kb Plus DNA Ladder. Featured high-fidelity DNA polymerase: Platinum SuperFi II DNA Polymerase Invitrogen™ Platinum™ SuperFi™ II DNA Polymerase is designed for success in PCR, combining the highest fidelity with trusted Invitrogen™ Platinum™ hot-start technology. Featuring >300x the fidelity of Taq polymerase, Platinum SuperFi II DNA Polymerase is well suited for applications benefiting from supreme sequence accuracy. Highlights • Exceptionally high fidelity (>300x higher fidelity than Taq polymerase) (Figure 5) • High specificity and increased yields with Platinum hot-start technology • Robust amplification of a wide range of targets with tolerance of common PCR inhibitors (Figure 6) • Convenient workflow with room-temperature reaction setup and 24-hour benchtop stability of preassembled reactions • Extensive quality testing (Table 8) Platinum SuperFi II enzyme NEB Q5 Hot Start High-Fidelity enzyme Takara PrimeSTAR GXL enzyme Merck KOD Hot Start enzyme KAPA HiFi HotStart enzyme Qiagen HotStar HiFidelity enzyme Taq enzyme 0 100 200 300 Fidelity compared to Taq enzyme Find out more at thermofisher.com/platinumsuperfi 21 Figure 7. EquiPhi29 DNA Polymerase demonstrates low bias when amplifying 3 bacterial genomes. A mixture of bacterial genomes with low-GC (S. aureus, 33% GC), moderate-GC (E. coli, 51% GC), and high-GC (P. aeruginosa, 68% GC) content was amplified using EquiPhi29 and Phi29 DNA Polymerases as well as a DNA polymerase from another supplier. For each genome, the GC content of the reference genome, in 100 bp windows indicated in gray, was plotted versus the coverage normalized to the unamplified genome mix, indicated in green. In the absence of sequencing bias, all windows should be equally distributed close to the normalized coverage of 1, indicated in light blue. The normalized coverage obtained after amplification using different polymerases is shown. EquiPhi29 DNA Polymerase amplifies DNA with the lowest bias across all GC content levels, compared to other DNA polymerases. Fraction of normalized coverage Supplier B Phi29 DNA polymerase Phi29 DNA Polymerase EquiPhi29 DNA Polymerase, 45°C EquiPhi29 DNA Polymerase, 42°C Unamplified genome mix control 3 2 1 0 GC content (%) 20 40 60 80 Find out more at thermofisher.com/equiphi29 EquiPhi29 DNA Polymerase Thermo Scientific™ EquiPhi29™ DNA Polymerase is a proprietary phi29 isothermal DNA polymerase mutant developed through in vitro protein evolution. This enzyme is significantly improved over phi29 DNA polymerase in protein thermostability, reaction speed, product yield, and amplification bias while retaining all the benefits of the wild-type enzyme, including high processivity (>70 kb), strong strand displacement activity, and 3ʹ to 5ʹ exonuclease (proofreading) activity acting preferentially on single-stranded DNA or RNA. Highlights • Lowest amplification bias offered in the market (Figure 7) • Extremely high yields of amplified DNA, even from minute amounts of template • Highly accurate DNA synthesis in a short amount of time • Available in lyo-ready format 22 Table 9. dNTP specifications. Parameter Method Concentration 100 mM pH 7.3–7.5 Appearance Clear colorless solution Purity (HPLC) ≥99% triphosphate Base purity (HPLC) ≥99.5% deoxynucleoside Pyrophosphate ≤0.003 pmol PPi/pmol dNTP Endodeoxyribonucleases, exodeoxyribonucleases, and ribonucleases Undetectable after incubation of labeled oligonucleotide with dNTP Endodeoxyribonuclease and nicking activities Undetectable after incubation of supercoiled plasmid DNA with dNTP Human DNA Undetectable via qPCR test, which uses amplification of Alu repeats in human genomic DNA E. coli DNA Undetectable via qPCR test, which uses amplification of E. coli 23S rRNA gene fragment Functional testing Functionally tested in two-step RT-qPCR using different starting amounts of RNA transcript in reverse transcription reactions followed by amplification with hot-start Taq DNA polymerase Find out more at thermofisher.com/dntps PCR-grade dNTPs We are one of the few primary manufacturers of nucleotides. Our deoxyribonucleoside triphosphates (dNTPs) have been extensively tested and verified for use in a wide variety of molecular biology applications, including highly sensitive techniques such as RT-qPCR and next-generation sequencing. Highlights • >99% purity confirmed by HPLC (Table 9) • Manufactured using dedicated equipment for each dNTP • Free of contaminating RNases, DNases, and qPCR, PCR, and RT inhibitors • High stability with a 48-month shelf life at –20°C; stable after >100 freeze/thaw cycles • No limitations to manufacturing scale • Available as individual dNTPs, sets, or mixes 23 qPCR master mixes Real-time PCR master mixes Applied Biosystems™ TaqPath™ and TaqMan™ master mixes provide turnkey solutions for real-time PCR. They contain buffer, dNTPs, passive reference dye, thermostable hot-start DNA polymerase, and other components formulated for reliable 5ʹ nuclease–based real-time PCR. Just add your sample and TaqMan Assay components, and start your reactions. We offer a range of real-time PCR master mixes optimized for specific applications, and provide TaqMan master mixes for Research Use Only (RUO), as well as TaqPath master mixes, which are general purpose reagents (GPRs), for laboratory use. Find out more at thermofisher.com/taqman Genotyping master mixes Applied Biosystems™ TaqPath™ ProAmp™ Master Mixes are versatile master mixes developed for high-throughput genotyping and copy number variation (CNV) analysis applications that require accurate results from samples containing PCR inhibitors. TaqPath ProAmp Master Mixes are designed to deliver accurate and reproducible results from genomic DNA targets. The easy workflow and reproducible performance even in the presence of inhibitors offer confidence in results. Find out more at thermofisher.com/proamp Gene expression analysis master mixes Depending on your workflow, you may choose to perform reverse transcription and amplification in one or two reactions, and would need a 1-step or 2-step gene expression master mix, respectively. Applied Biosystems™ TaqPath™ qPCR Master Mix, CG, is an excellent choice for 2-step gene expression analysis. For superb performance, use SuperScript IV VILO Master Mix to prepare cDNA prior to amplification with the TaqPath qPCR Master Mix. Find out more at thermofisher.com/taqpath PowerUp SYBR Green Master Mix The Applied Biosystems™ PowerUp™ SYBR™ Green Master Mix is a preformulated, optimized universal 2X master mix that is well suited for more economical assays in less demanding applications. Coupled with user-supplied primer sets and templates, PowerUp SYBR Green Master Mix is designed to amplify targets for accurate gene expression analysis. It uses Invitrogen™ SYBR™ Green I dye (a dye that binds to doublestranded DNA) to detect PCR products as they accumulate. Find out more at thermofisher.com/sybr 24 Highlights • Doesn’t require thermal cycler (only heating block) • Fast turnaround time (15–30 min) (Figures 8 and 9) • Multiple options for detection (real-time, endpoint) • Simple workflow (from sample to result in a few steps) • Suitable for low-resource or in-field settings Invitrogen™ SuperScript™ IV RT-LAMP Master Mix uses a reverse transcriptase to create a cDNA template from RNA and a DNA polymerase with strong strand displacement activity. This results in fast, simple, and specific amplification under isothermal conditions. • Includes a proprietary evolved Bst DNA polymerase for high reaction speed RT-LAMP master mix 0 10 20 30 40 50 60 70 100 70 50 30 NTC Time to result (min) Copy number of synthetic SARS-CoV-2 RNA Detecting synthetic SARS-CoV-2 RNA using real-time readout SuperScript IV RT-LAMP Master Mix NEB WarmStart LAMP Master Mix OptiGene GspSSD2.0 Isothermal Mastermix Lucigen LavaLAMP RNA Master Mix • Includes SuperScript IV reverse transcriptase: – High tolerance of a variety of inhibitors – Robust and specific cDNA synthesis – Fast reverse transcription reaction • Optimized reaction buffer to ensure high specificity and sensitivity • Invitrogen™ RNaseOUT™ inhibitor to prevent RNA degradation • Can be used to amplify viral RNA (e.g., from measles, influenza, SARS-CoV-2, and other pathogens) • Compatible with real-time and endpoint detection; includes Invitrogen™ SYTO™ 9 dye for real-time detection • Convenient single-tube format available, with 100, 400, and 1,000 reactions Figure 8. SuperScript IV RT-LAMP Master Mix amplifies RNA faster; viral pathogen detection in as little as 5 minutes with an evolved Bst DNA polymerase. SuperScript IV RT-LAMP Master Mix provides the fastest RNA target amplification at constant speed, even with low target copy numbers. Amplification speed was determined using synthetic SARS-CoV-2 RNA, the same primer set, and SYTO 9 dye for real-time detection. The NEB WarmStart™ LAMP Kit (DNA & RNA), OptiGene GspSSD2.0 Isothermal Mastermix, and Lucigen LavaLAMP™ RNA Master Mix were used following the manufacturers’ recommended protocols. Figure 9. SuperScript IV RT-LAMP Master Mix demonstrates outstanding reaction speed, amplifying target SARS-CoV-2 RNA purified from samples in less than 10 minutes with 100% specificity. SuperScript IV RT-LAMP Master Mix could amplify SARS-CoV-2 RNA from positive samples in under 10 minutes. Amplification speed was determined using purified SARS-CoV-2 RNA from samples as confirmed by gold-standard RT-qPCR, the same primer, and SYTO 9 dye for real-time detection. The NEB WarmStart LAMP Kit (DNA & RNA), OptiGene GspSSD2.0 Isothermal Mastermix, and Lucigen LavaLAMP RNA Master Mix were used following the manufacturers’ recommended protocols. 100 100 62.5 12.5 100 62.5 37.5 0 0 10 20 30 40 50 60 70 80 90 100 SuperScript IV RT-LAMP Master Mix WarmStart LAMP 2X Master Mix (NEB) Detecting SARS-CoV-2 RNA in samples using real-time readout Isothermal Mastermix (OptiGene) LavaLAMP RNA Master Mix (Lucigen) Percentage of purified clinical SARS-CoV-2 RNA detected 15 min 10 min 25 Benefits • High concentration—allows flexibility in final formulations with primers, probes, and excipients • Cost savings—room-temperature shipping and longer shelf life of the assay • Performance—same excellent results you expect from wet formulations (Figure 10) From Invitrogen™ SuperScript™ reverse transcriptases (RTs) (Figure 11) to Platinum II Taq DNA Polymerase, we offer a large portfolio of lyo-ready enzyme preparations to meet the performance requirements of RT-qPCR and qPCR assays in dry formats (Table 10). These enzymes are formulated without glycerol and perform consistently across different formats— standard glycerol, lyo-ready, lyophilized, and reconstituted. Lyo-ready enzymes Figure 10. Amplification plots before (control sample) and after (samples I and II) lyophilization of lyo-ready SuperScript III RT. ΔRn 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 Cycle 41 39 37 35 33 31 29 27 25 PGK1 Two-step RT-qPCRreactions were performed to detec1t the PGK1 gene in blood stored with heparin o rEDTA. Lyo-ready SuperScriptRTs deliver superior tolerance to reactio ninhibitors compared to other suppliers’ products. Lyo-Ready RT Performance: E‡ciency with Di‡cult Samples EDTA blood Heparin blood Lyo-ready SuperScript IV RT Lyo-ready SuperScript III RT Supplier 1 Supplier 2 Supplier 3 Average Ct Figure 11. Lyo-ready RT performance: efficiency with difficult samples. Two-step RT-qPCR reactions were performed to detect PGK1 transcripts in blood stored with heparin or EDTA. Lyo-ready SuperScript RTs deliver superior tolerance to reaction inhibitors compared to other suppliers’ products. 26 Find out more at thermofisher.com/lyoreadyenzymes Reverse transcriptases (RTs) • Invitrogen™ Lyo-ready SuperScript™ Reverse Transcriptase, 1-step RT-qPCR • Invitrogen™ Lyo-ready SuperScript™ III RT • Invitrogen™ Lyo-ready SuperScript™ IV RT • Thermo Scientific™ Lyo-ready Maxima™ H Minus RT • Thermo Scientific™ Lyo-ready Maxima™ RT • Thermo Scientific™ Lyo-ready M-MLV RT • Thermo Scientific™ Lyo-ready RevertAid™ H Minus RT • Thermo Scientific™ Lyo-ready RevertAid™ M-MLV RT Taq DNA polymerases • Invitrogen™ Lyo-ready Platinum™ II Taq DNA polymerase • Invitrogen™ Lyo-ready Platinum™ Taq DNA Polymerase • Invitrogen™ Lyo-ready LibertyTaq™ DNA Polymerase • Applied Biosystems™ Lyo-ready AmpliTaq Gold™ DNA Polymerase • Thermo Scientific™ Lyo-ready Phire™ Hot Start II DNA Polymerase Isothermal enzymes • Invitrogen™ Lyo-ready Bst DNA Polymerase • Thermo Scientific™ Lyo-ready EquiPhi29™ DNA Polymerase RNase inhibitors • Invitrogen™ Lyo-ready RNaseOUT™ RNase Inhibitor • Thermo Scientific™ Lyo-ready RiboLock™ RNase Inhibitor Proofreading DNA polymerases • Invitrogen™ Lyo-ready Platinum™ SuperFi™ DNA Polymerase • Thermo Scientific™ Lyo-ready Phusion™ Hot Start II DNA Polymerase Table 10. Selection of lyo-ready enzymes for molecular assays. 27 Rn Amplification plot S gene, target 2 (Cy5 dye) 2.50 2.25 2.00 1.75 1.50 1.25 1.00 0.75 0.50 0.25 0.00 Cycle Rn Amplification plot N gene (JOE dye) 2.0 1.9 1.8 1.7 1.6 1.5 1.4 1.3 1.2 1.1 1.0 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0.0 –0.1 Cycle Rn Amplification plot Eff. 96% R²: 0.99 Cq 22.1 Cq 20.0 Cq 21.8 Eff. 97% R²: 0.99 Eff. 102% R²: 0.99 S gene, target 1 (FAM dye) 2.0 1.9 1.8 1.7 1.6 1.5 1.4 1.3 1.2 1.1 1.0 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0.0 –0.1 Cycle 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 2 4 6 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 Figure 13. Lyo-ready 1-step RT-qPCR on SARS-CoV-2 positive samples: triplex detection. Viral RNA was extracted from swab samples positive for SARS-CoV-2 using the Applied Biosystems™ MagMAX™ Viral/Pathogen Nucleic Acid Isolation Kit (Cat. No. A48310). RNA copies (tested 200,000, 20,000, 2,000, 200, and 20 copies) per reaction were calculated according to the standard curve created using Twist Synthetic SARS-CoV-2 Control RNA (Twist Bioscience). Each dilution point of all three targets (2 targets for the S gene, 1 target for the N gene) was checked in three technical replicates. Tests were performed using a protocol for lyo-ready 1-step RT-qPCR with the following cycling conditions: 15 min at 50°C followed by 2 min at 95°C, then 5 sec at 95°C and 20 sec at 60°C for 45 cycles. Assays were run on the Applied Biosystems™ QuantStudio™ 7 Flex Real-Time PCR System. Quantification cycle (Cq) values are shown for the 200,000 copies/reaction dilution point. Specially designed lyo-ready one-step RT-qPCR system for quantitative analysis of RNA Specially designed Invitrogen™ 5X Lyo-ready Platinum™ II PCR Buffer, combined with Invitrogen™ Lyo-ready Platinum™ II Taq Hot-Start DNA Polymerase and Invitrogen™ Lyo-ready SuperScript™ Reverse Transcriptase, 1-Step RT-qPCR, is an excellent choice to obtain results with reduced time and cost, and minimal optimization (Figure 12). Lyo-ready Platinum II Taq Hot-Start DNA Polymerase • Engineered Taq DNA polymerase with increased tolerance to reaction inhibitors originating from sample materials or nucleic acid purification steps. The lyo-ready enzyme formulation offers the feasibility to lyophilize while retaining all favorable properties of the standard enzyme preparation (with glycerol). • The polymerase activity is blocked at ambient temperatures and restored after the initial denaturation step at 95°C. This automatic “hot start” provides increased sensitivity, specificity, and yield while allowing reaction assembly at room temperature. • Lyo-ready Platinum II Taq Hot-Start DNA Polymerase extends 1 kb in 15 seconds. The extension step can be prolonged without a negative effect on specificity. • Like the standard Taq DNA polymerase, it has both 5΄ to 3΄ polymerase and 5΄ to 3΄ exonuclease activities. 5X Lyo-ready Platinum II PCR Bu er Purified RNA Fluorescence signal Lyo-ready Platinum II Taq Hot-Start DNA Polymerase 1-step RT-qPCR Lyo-ready SuperScript Reverse Transcriptase, 1-Step RT-qPCR Figure 12. Workflow for lyo-ready 1-step RT-qPCR. Lyo-ready SuperScript Reverse Transcriptase, 1-Step RT-qPCR • Engineered version of Moloney murine leukemia virus (M-MuLV) reverse transcriptase that features increased thermal stability and maintained RNase H activity. • The enzyme can be used to synthesize first-strand cDNA at temperatures up to 55°C (optimally at 50°C), providing increased specificity and higher yields of cDNA. Lyo-ready Platinum II PCR Buffer • Optimized to achieve excellent performance for the reverse transcriptase and polymerase in 1-step RT-qPCR. • The buffer is also optimized for increased tolerance to reaction inhibitors originating from sample material or nucleic acid purification steps. The optimized reaction buffer and stand-alone enzymes allow reduction in time and resources spent on RT-qPCR assay development. In addition, the multiplexing capability of the system allows testing on a broader range of targets in a single reaction tube (Figures 13–15). Tolerance to inhibitors helps ensure reaction sensitivity and accuracy (Figure 16). 28 Eciency (%) Lyo-ready 1-step RT-qPCR QuantiTect Virus Kit LightCycler Multiplex RNA Virus Master 120 115 110 105 100 95 90 85 80 75 70 65 60 5 0 S gene, target 1 (FAM dye) N gene (Cy5 dye) S gene, target 2 (VIC dye) Figure 14. Comparison of efficiency of 1-step RT-qPCR in triplex detection (2 targets for the S gene, 1 target for the N gene) of Twist Synthetic SARS-CoV-2 Control RNA (Twist Bioscience). Four dilutions (containing 20,000, 2,000, 200, and 20 copies per reaction) were tested in triplicate, and the qPCR efficiencies of the reactions were compared using lyo-ready 1-step RT-qPCR products (Thermo Fisher Scientific), the QuantiTect™ Virus Kit (Qiagen), and LightCycler™ Multiplex RNA Virus Master (Roche Life Science). The tests were performed according to the recommended standard protocols of each supplier. 4 3 2 1 35 30 25 20 15 Human SARS-CoV-2 Parainfluenza virus type 1 Adenovirus type 4 Chikungunya virus Cq Log dilution Figure 15. Detection of targets in pentaplex. One-step RT-qPCR was performed in a pentaplex reaction containing HeLa S3 total RNA, synthetic SARS-CoV-2 RNA, parainfluenza virus type 1 RNA, adenovirus type 4 DNA, and chikungunya virus RNA. Nucleic acids from parainfluenza virus, adenovirus, and chikungunya virus were purified from Vircell inactivated virus suspension using the MagMAX Viral/Pathogen Nucleic Acid Isolation Kit (Cat. No. A48310). All targets were tested at four 10-fold dilutions. Each sample was tested in triplicate for all five targets at each dilution point. Tests were performed on the QuantStudio 7 Flex Real-Time PCR System. Target Fluorophore (channel) Template Efficiency R2 Slope Human TAMRA HeLa S3 RNA 97.7 1.000 –3.4 SARS-CoV-2 ROX Synthetic SARS-CoV-2 RNA 100.1 0.999 –3.3 Parainfluenza virus type 1 JOE Parainfluenza virus type 1 RNA 102.7 0.998 –3.3 Adenovirus type 4 FAM Adenovirus type 4 DNA 95.3 0.997 –3.4 Chikungunya virus Cy5 Chikungunya virus RNA 96.3 0.999 –3.4 29 SARS-CoV-2 RNA, N gene Plasma 5% Control Plasma 10% Cycle Rn Cq Eciency 0.996 0.996 0.997 R2 99% 101% 101% Saliva quantity Target gene 20,000 copies 2,000 copies 200 copies 20 copies S 0% Lyo-ready RT-qPCR system with SalivaReady Solution N S 5% N S 10% N S 0% Vendor 1 kit N 3/3 detected 1/3 or 2/3 detected Not detected S 5% N S 10% N S 0% N S 5% N S 10% N Vendor 2 kit Figure 18. Direct one-step RT-qPCR with the lyo-ready one-step RT-qPCR system and SalivaReady Solution in the presence of blood plasma. Each reaction mixture was premixed with the SalivaReady Solution and contained from 20,000 to 20 copies of synthetic SARS-CoV-2 RNA. The lyo-ready one-step RT-qPCR enzyme pair was able to successfully detect down to 20 copies of SARS-CoV-2 RNA in the presence of up to 10% of blood plasma in the reaction mix. Figure 17. Direct one-step RT-qPCR with the lyo-ready one-step RT-qPCR system and SalivaReady Solution in the presence of saliva. Each reaction mixture was premixed with SalivaReady Solution and contained from 20,000 to 20 copies of synthetic SARS-CoV-2 RNA. In total, three technical repeats were done for each sample. The color of the rectangle indicates the number of detected targets in each triplicate. Compared with kits from other vendors on the market, our lyo-ready RT-qPCR system is able to successfully detect SARS-CoV-2 RNA in the presence of up to 10% of saliva in the reaction mix. Lyo-ready system in direct one-step RT-qPCR The lyo-ready system (Lyo-ready Platinum II Taq Hot-Start DNA Polymerase and Lyo-ready SuperScript Reverse Transcriptase, 1-Step RT-qPCR) is optimized to work in an inhibitor-rich environment. Using this lyo-ready system with the Applied Biosystems™ SalivaReady™ Solution enables detection of down to 20 copies of RNA from the samples with up to 10% of saliva or blood plasma content (Figures 17 and 18). Figure 16. Detection of viral targets with inhibited samples. One-step RT-qPCR was performed according to each supplier’s protocol and recommendations for cycling conditions. Performance was measured using the standard curve created from a dilution series of synthetic SARS-CoV-2 RNA (20,000, 2,000, 200, and 20 copies/reaction), with each dilution quantitated in triplicate. Mean Cq values are indicated for the first measurable dilution of each target of the triplex (two targets for the S gene, one target for the N gene). Each inhibitor was added to the prepared master mix at the levels indicated. Tests were performed on the QuantStudio 7 Flex Real-Time PCR System. * The Twist Synthetic SARS-CoV-2 RNA control was used in the experiment. It is synthesized by transcribing six non-overlapping 5 kb fragments (generated from Twist Gene Fragments) into ssRNA. These provide coverage of greater than 99.9% of the viral genome. Beta strain South African. All standard Twist Synthetic SARS-CoV-2 RNA controls are Biosafety Level 1. ** Universal transport medium for viruses, Chlamydia, Mycoplasma, and Ureaplasma (COPAN Diagnostics). S2 25.1 N 25.4 24.8 S1 S2 N S1 S2 N S1 25.3 25.2 25.0 25.8 26.6 Lyo-ready 1-step RT-qPCR system Triplex: S gene (S1 and S2), N gene Cq of control (synthetic SARS-CoV-2 RNA, 20,000/reaction)* Control + inhibitors ƒCq „ 1 1 < ƒCq „ 2 ƒCq > 2 Vendor 1 kit Vendor 2 kit 10 μM hemin 2% isopropanol 2.5 μM UTM** ‘Cq = (Cq of control) – (Cq of inhibitor-treated sample): 30 Figure 19. Reaction speed tests with RNA targets. Figure 20. Reaction speed tests with DNA targets. The Lyo-ready Bst DNA Polymerase is an engineered DNA polymerase with strong strand-displacement activity for fast loop-mediated isothermal amplification (LAMP) reaction speed, increased sensitivity, and high tolerance to inhibitors from challenging samples. It supports maximum flexibility to optimize LAMP reactions and amplifies various pathogen targets, including human adenovirus, measles virus, and SARS-CoV-2. Also, it is a glycerol-free product compatible with assay lyophilization and microfluidics-based technologies. Highlights • Fast—amplifies targets in as little as 10 minutes • Sensitive—down to 50 copies • Robust—amplifies even inhibitor-containing samples • Flexible—formulation is glycerol-free and compatible with lyophilization Lyo-ready Bst DNA Polymerase 0 5 10 15 20 25 30 5,000 copies 1,000 copies 500 copies 1,000 copies 500 copies SARS-CoV-2 Measles virus Time to detection (min) Lyo-ready Bst DNA Polymerase NEB Bst 3.0 DNA Polymerase NEB Bst 2.0 WarmStart DNA Polymerase OptiGene GspSSD2.0 DNA Polymerase ArcticZymes IsoPol BST+ DNA Polymerase 20 25 30 35 40 45 50 5,000 copies 1,000 copies 500 copies 1,000 copies 500 copies Adenovirus type 41 M. pneumoniae Time to detection (min) Lyo-ready Bst DNA Polymerase NEB Bst 3.0 DNA Polymerase NEB Bst 2.0 WarmStart DNA Polymerase OptiGene GspSSD2.0 DNA Polymerase ArcticZymes IsoPol BST+ DNA Polymerase Lucigen Bst DNA Polymerase 0 5 10 15 High reaction speed The fast speed of Lyo-ready Bst DNA Polymerase provides target detection in as little as 10 minutes in a LAMP reaction. Lyo-ready Bst DNA Polymerase showed higher reaction speed than other commercially available DNA polymerases (Figures 19 and 20). All of the DNA polymerases were used following their respective manufacturers’ recommended protocols, and the reverse transcriptase was added to reactions for RNA targets. Error bars represent the standard deviation of reaction speed calculated from three separate experiments for each tested sample (n = 3). 31 High sensitivity The high sensitivity of Lyo-ready Bst DNA Polymerase enables amplification from a limited amount of DNA or RNA. Lyo-ready Bst DNA Polymerase showed higher sensitivity than other commercially available DNA polymerases (Figures 21 and 22). All of the DNA polymerases were used following their respective manufacturers’ recommended protocols, and the reverse transcriptase was added to reactions for RNA targets. Error bars represent the standard deviation of reaction speed calculated from three separate experiments for each tested sample (n = 3). Figure 21. Sensitivity tests with RNA targets. Figure 22. Sensitivity tests with DNA targets. 0 5 10 15 20 25 30 35 40 100 copies 50 copies 100 copies 50 copies Time to detection (min) Lyo-ready Bst DNA Polymerase NEB Bst 3.0 DNA Polymerase NEB Bst 2.0 WarmStart DNA Polymerase OptiGene GspSSD2.0 DNA Polymerase ArcticZymes IsoPol BST+ DNA Polymerase SARS-CoV-2 Measles virus 0 10 20 30 40 50 60 100 copies 50 copies 100 copies 50 copies Adenovirus type 41 M. pneumoniae Time to detection (min) Lyo-ready Bst DNA Polymerase NEB Bst 3.0 DNA Polymerase NEB Bst 2.0 WarmStart DNA Polymerase OptiGene GspSSD2.0 DNA Polymerase ArcticZymes IsoPol BST+ DNA Polymerase Lucigen Bst DNA Polymerase 32 Resistance to inhibitors Lyo-ready Bst DNA Polymerase maintains high performance in the presence of inhibitors (Figure 23), such as copurified compounds from biological samples (e.g., xylan, hemin, humic acid, urea, heparin) and universal transport medium (UTM), or reagents used for sample preparation (e.g., ethanol, salts in high concentrations). Figure 23. Performance in the presence of inhibitors. Inhibitors were added to LAMP reactions together with 1,000 copies of synthetic adenovirus type 41 DNA. The relative increase in time to detection shows the change in detection time between the reactions with inhibitors and the control reaction where no inhibitor was added. 0 50 100 150 200 250 300 350 Xylan 2,000 μg/mL Hemin 5 μM Humic acid 0.8 ng/μL Urea 5% Heparin 0.2 U UTM 5 μL Ethanol 5% Total 250 mM KCI Total 300 mM KCI Relative increase in time to detection (%) Biological inhibitors Inhibitors from sample preparation 33 Table 11. Lyo-ready master mixes. Characteristic TaqMan Lyo-ready 1-Step RT-qPCR Master Mix TaqMan Lyo-ready qPCR Master Mix TaqMan Lyo-ready Multiplex qPCR Master Mix Concentration 5X 5X 5X Inhibitor tolerance Medium Medium Medium Multiplexing Yes Yes Yes dNTP mix Standard Standard Optimized for genotyping and contamination control Passive reference ROX None ROX None None Sample Cat. No. (volume) C1402SMP (1.5 mL) C1403SMP (1.5 mL) C1407SMP (1.5 mL) C1408SMP (1.5 mL) C1400SMP (1.5 mL) Figure 24. Comparison of qPCR amplification curves using Applied Biosystems™ TaqMan™ Lyo-ready 1-Step Master Mix and Applied Biosystems™ TaqMan™ Fast Virus 1-Step Master Mix. The Applied Biosystems™ VetMAX™ Xeno™ Internal Positive Control was used as the target, in a dilution series spanning 7 orders of magnitude. The TaqMan Lyo-ready 1-Step Master Mix maintains or exceeds the performance of TaqMan Fast Virus 1-Step Master Mix in Cq and fluorescence (ΔRn) values. The TaqMan Lyo-ready 1-Step Master Mix can be formulated with Applied Biosystems™ ROX™ passive reference dye (as shown here) or without the passive reference dye. 10 1 0.1 0.01 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 Cycle ΔRn Lyo-ready master mixes Our low-glycerol, lyo-ready qPCR master mixes (Table 11) are one-tube solutions that are ready to be mixed with primers, probes, and excipients, for the subsequent lyophilization step. There is no need for time-consuming development, optimization, and sourcing of multiple separate qPCR reaction components. These master mixes retain the reproducibility, sensitivity, and specificity required for commercial assays (Figure 24). Once lyophilized, the assays demonstrate little to no loss of sensitivity or specificity, and have been shown to reproducibly detect a single copy of target per reaction and provide a linear dynamic range of over 7 orders of magnitude. 34 Primers, probes, and oligonucleotides Find out more at thermofisher.com/taqman Applied Biosystems™ TaqMan™ probes include a minor groove binder (MGB) moiety at the 3ʹ end that increases the primer melting temperature (Tm) of the probe and stabilizes probe–target hybrids, enabling shorter probe length. The nonfluorescent quencher (NFQ) absorbs (quenches) signal from the fluorescent dye label at the other end of the probe. The properties of the NFQ, combined with the short length of the MGB probe, result in lower background signal than with non-MGB NFQ probes. Lower background signal means increased sensitivity and precision in your data. MGB probes and unlabeled primers are also available as analyte-specific reagents (ASRs) to help meet requirements for test development. Applied Biosystems™ probe and primer ASRs are manufactured in a registered facility in accordance with current good manufacturing practices (CGMPs) with a quality management system certified to ISO 13485:2003. Applied Biosystems™ TaqMan™ QSY™ probes incorporate a proprietary 3ʹ QSY quencher to provide maximal PCR performance in a multiplex format. TaqMan QSY probes are available with Applied Biosystems™ FAM™, VIC™, and our proprietary ABY™ and JUN™ dyes, allowing amplification of up to four targets in a single reaction. All four dyes are optimized for the filter sets on Applied Biosystems™ real-time PCR instruments and work together with minimal spectral overlap for optimal performance. TaqMan Assays are optimized primer and probe sets in one convenient tube. We provide a complete range of custom-synthesized oligonucleotide primers and probes, all built to your specifications. Simply identify your target of interest or exact sequence, and our team can design your assay. 35 Table 12. Applied Biosystems thermal cycler selection chart. ProFlex PCR System VeritiPro Thermal Cycler SimpliAmp Thermal Cycler MiniAmp Plus and MiniAmp Thermal Cyclers Automated Thermal Cycler Thermal cyclers Key benefits Ultimate flexibility and throughput Robust and accurate PCR optimization Elegantly simple and precise Routine PCR, elevated Designed for easy robotic integration User interface 8.4-inch touchscreen 8.0-inch touchscreen 8.4-inch touchscreen 5.0-inch touchscreen Automated via robotics Maximum sample throughput 480,000 reactions 384 reactions 96 reactions 96 reactions 384 reactions Maximum block ramp rate 6.0°C/sec 6.0°C/sec 4.0°C/sec MiniAmp Plus Thermal Cycler: 3.5°C/sec MiniAmp Thermal Cycler: 3.0°C/sec 3.5°C/sec Temperature optimization 6-zone VeriFlex Block on 96-well system 2-zone VeriFlex Block on 3 x 32-well system 6-zone VeriFlex Block on 96-well system 3-zone VeriFlex Block on 96-well system MiniAmp Plus Thermal Cycler: 3-zone VeriFlex Block on 96-well system MiniAmp Thermal Cycler: none None Find out more at thermofisher.com/thermalcyclers With Applied Biosystems™ thermal cyclers and real-time PCR platforms, you get exceptional value with excellent performance, reliability, and world-class support. When you validate your assay on Applied Biosystems instruments, you can leverage the expansive, global installed base in end-user laboratories, or place our instruments in a reagent rental agreement. As an option, you can even have an Applied Biosystems instrument private-labeled and integrated into your workflow and deliver it to your customers with your own label attached. Your customers will also gain access to the same validation and IQ/OQ services that our direct instrument customers enjoy. Instrumentation Thermal cyclers We offer a broad portfolio of high-quality thermal cyclers for commercial applications (Table 12). 36 Real-time PCR instruments The Applied Biosystems family of instruments is designed for compatibility with TaqMan Assays and master mixes. Our genetic analysis instruments, along with a workflow to match your requirements, support your lab’s assay development needs. Table 13 lists our most popular instruments for assay development and commercialization. Find out more at thermofisher.com/qpcrsystems Table 13. Applied Biosystems real-time PCR instruments for molecular assays. QuantStudio 5 system QuantStudio 7 Flex system 7500 Fast system Description Modern, interactive, affordable, dual mode A high-throughput platform that can grow with you A widely used workhorse instrument Footprint (W x D x H) 27 x 50 x 40 cm 53 x 70 x 75 cm 34 x 45 x 49 cm Optical system Bright white LED White halogen lamp White halogen lamp Blocks Fixed block: 96-well 0.2 mL Interchangeable: 96-well 0.2 mL, 96-well 0.1 mL, 384-well, TaqMan Array Card Fixed block: 96-well 0.1 mL 37 PCR and qPCR plastics, seals, and accessories For more than 25 years, we have been at the forefront of innovation in the manufacturing of high-grade plastic consumables, with a focus on providing plastic solutions for molecular biology applications, including PCR and qPCR. We have the essentials to help ensure that your development and manufacturing projects are a success: • State-of-the-art injection molding manufacturing facilities that meet Class 10,000 or 100,000 cleanroom standards • Experience in thin-walled polypropylene plastics and sealing films for PCR and qPCR, and sample storage • Custom product design, rapid prototyping, and manufacturing • In-house toolmaking and tool maintenance • Options for barcodes that are unique to your product, in any barcode format • Customized product packaging and labeling • Application-specific quality control • ISO 9001 compliance, with complete traceability and process controls High efficiency, reduced variability Uniform, ultrathin walls enable consistent maximum heat transfer for equally high performance from every sample. Secure, easy sealing Specially designed caps create a tight seal that is still easy to open and close. Tube strips with attached caps that open and close independently are also available. Innovative product design White plastics for enhanced qPCR detection Thermo Scientific™ white qPCR plastics are designed to provide sensitive and accurate fluorescence detection by preventing refraction out of the tube and increasing the signal-to-noise ratio. Evaporation protection Raised rim around each well enables secure sealing and safeguards against evaporation. Find out more at thermofisher.com/oemplastics 38 Streamline your supply chain and avoid investing in costly manufacturing capabilities, with our custom manufacturing services. We provide raw materials, manufacturing of components, and packaging of molecular assay kits according to your design and requirements. • Choice of tube and bottle sizes • Variety of plate formats • Fill and finish • Custom kit configurations • Private labeling Custom manufacturing services We have experience in OEM product development, formulation, aseptic processing, kit assembly, and packaging. We can bring individual components such as master mixes, assays, and controls together in a skillfully designed package with your company logo. Multiple levels of regulated manufacturing are available, e.g., CGMP, general purpose reagents, research use only, cleanroom, controlled environment, and advanced quality management systems ISO 9001 and ISO 13485. Armadillo PCR Strip Plates Thermo Scientific™ Armadillo™ PCR Strip Plates are automation-friendly plates that can be segmented into smaller pieces easily and safely without special tools. Each 96-well plate can be divided into individual 8-tube strips or blocks of 8-tube strips (Figure 25). Armadillo PCR Strip Plates are a flexible solution for hybrid workflows that include both automated and manual processes for producing and distributing reagents and assays. Easy visual organization 3 color choices Easy-to-read well identification text Black text for excellent contrast No warping, even after thermal cycling Hard shell for mechanical stability and flatness Snug fit to thermal blocks Thin-wall polypropylene for excellent mechanical fit and heat transfer Easily and safely divided into 8-tube strips Available with clear or white wells Figure 25. Features of Armadillo PCR Strip Plates. The 96-well format is suitable for high-throughput production with automated filling, sealing, and lyophilization. Individual 8-tube strips are convenient for low-throughput downstream applications. In addition to being compatible with the leading PCR and qPCR instruments, the Armadillo PCR Strip Plates are suitable for robotic handling. Find out more at thermofisher.com/armadillostripplates Features include: • Available in 96-well standard and low-profile formats, 5-plate sample packs, and 25- or 150-plate packs • Constructed to ANSI/SBS standards • Certified DNA-, DNase-, and RNase-free • Validated with Applied Biosystems™ instruments and compatible with industry-leading instruments 39 For Research Use Only. Not for use in diagnostic procedures. © 2019, 2023 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. Agilent and Bioanalyzer are trademarks of Agilent Technologies, Inc. Q5 and WarmStart are trademarks of New England Biolabs, Inc. Cy is a registered trademark of Cytiva. LavaLAMP is a trademark of Lucigen. IsoPol is a trademark of ArcticZymes AS. PrimeSTAR is a trademark of Takara Bio. QuantiTect is a trademark of Qiagen. KAPA and LightCycler are trademarks of Roche. TaqMan is a trademark of Roche Molecular Systems, Inc., used under permission and license. Vircell is a trademark of Vircell SL. COL025625 1123 Find out more at thermofisher.com/oem