Harnessing Magnetic Beads in Molecular Diagnostics

ebook Harnessing magnetic beads in molecular diagnostics How magnetic beads streamline diagnostic workflows and ensure accurate results There’s no doubt about it, molecular diagnostics is revolutionizing healthcare. We now have fast molecular tests for a whole range of conditions from infectious diseases to cancer and complex genetic disorders (1). When looking to develop cutting-edge assays, a powerful, yet sometimes overlooked stage is nucleic acid purification (NAP). NAP can be both streamlined and enhanced using magnetic beads. During nucleic acid purification, magnetic beads can isolate high-integrity DNA and RNA to boost the accuracy of downstream applications like PCR and next-generation sequencing (NGS). Thus, magbeads can help you get the most out of your molecular assays with precision diagnostics and reliable disease monitoring. Traditional methods for extracting nucleic acids—like solid phase absorption involving silica spin columns and organic phase extraction followed by precipitation— can be effective but come with certain drawbacks. For instance, these methods often involve complex handling steps, provide variable level of reproducibility, and have limited compatibility with automation (2). Using magnetic beads can avoid each of these issues. They simplify the purification process while supporting both manual and automated workflows. Automated systems using magnetic bead protocols reduce human error, enhance reproducibility, and speed up the extraction process. Another key benefit of magnetic beads is their adaptability. They can be coated with various chemistries tailored to your specific diagnostic needs. In this e-book, we will explore the role of magnetic beads in molecular diagnostics, focusing on two main applications: nucleic acid extraction and purification and NGS. We will also discuss the benefits and challenges of using magnetic beads in these settings and provide practical magnetic bead recommendations to help you maximize assay performance and diagnostic accuracy. 2 Magnetic beads in nucleic acid purification Magnetic beads, especially those coated with silica, are a popular tool for DNA and RNA extraction and purification. Extraction involves isolating nucleic acids from cells or tissues, while purification involves removing contaminants to obtain highly pure nucleic acids. Silica-coated magnetic beads provide a highly selective nucleic acid binding surface in the presence of chaotropic salts such as guanidinium thiocyanate. The presence of salt can reduce the electrostatic repulsive force between the charged silica surface, and at the same time, the chaotropic salt can promote the hydrogen bonding and also increase hydrophobic interactions between nucleic acids and silica surface (3). As a result, negatively charged DNA or RNA molecules are able to effectively interact with the silica surface on the beads. Following DNA or RNA binding, magbeads are washed multiple times to remove any contaminants. Finally, the DNA or RNA is eluted in a low-salt buffer, resulting in a highly purified sample ideal for a whole range of diagnostic applications. Unlike spin columns, these simple wash and elution steps avoid the need for centrifugation, simplifying the process (2). Fig 1. How chaotropic agents enhance nucleic acid extraction with magnetic beads. Other bead surface chemistries can be used in NAP. For example, carboxylatemodified beads are used in solid-phase reversible immobilization (SPRI). These beads function similarly to silica beads but offer unique characteristics such as sizedependent nucleic acid binding. Size selection of DNA, which requires specific buffer conditions, is routinely used in NGS workflows Oligo(dT)-coated beads are another type of magnetic bead used in NAP. These beads are specifically designed to capture polyadenylated mRNA molecules by hybridizing to their poly(A) tails. Their specificity is essential in RNA-seq and gene expression studies and when isolating mRNA from complex samples. Learn more about how you can boost nucleic acid purification from clinical samples with magnetic beads OH SiO2 Fe2O4 OH OH • Guanidine isothiocyanate • Guanidine hydrochloride • Nal • Urea Chaotropic salt OH OH OH OH OH ONa+ SiO2 Fe2O4 ONa+ ONa O- + Na+ ONa+ ONa+ ONa+ ONa+ ONa+ SiO2 Fe2O4 ONa+ ONa O- + Na+ ONa+ ONa+ ONa+ ONa+ 3 Challenges with magnetic beads in DNA and RNA extraction Despite their advantages in DNA and RNA extraction, magnetic beads can suffer from nonspecific binding. Without protocol optimization, unintended molecules like proteins or cellular debris can adhere to the bead surface. This binding often occurs when the bead-to-sample ratio is imbalanced or when suboptimal wash conditions are used. Nonspecific binding can harm molecular diagnostic assays with contaminants reducing nucleic acid purity. Additional challenges in nucleic acid extraction include the need for precise bead handling. Poor pipetting technique or insufficient magnetic separation may result in bead carryover. Carryover can introduce contaminants or skew results in sensitive molecular diagnostic tests like PCR. Optimizing chaotropic salt concentration can also be challenging yet is essential for effective DNA binding and purity. While high concentrations of chaotropic salts improve nucleic acid binding to the beads, they can complicate elution. Excessive salts require high-pH, low-ionic-strength buffers to effectively recover nucleic acids. However, if not controlled, excessive salts and low-ionic-strength buffers can inhibit PCR amplification steps. Workflow and bead selection considerations 1 Optimize bead-to-sample ratio to reduce nonspecific binding To reduce nonspecific binding, carefully adjust the bead-to-sample ratio. If you have too many beads, excess surface area can result in nonspecific binding. But having too few beads can reduce nucleic acid yield. Use trial runs with different ratios to help you determine the ideal balance for each sample type. 2 Tweak washing protocols to optimize purity and yields Effective washing is crucial for high purity nucleic acids. Use ethanolbased wash buffers and multiple wash cycles to remove proteins and other contaminants. Optimize buffer stringency and wash times to retain target nucleic acids while eliminating impurities. For RNA isolation, using RNase-free reagents and containers is also essential. 3 Ensure precise handling and separation for optimal purity Precise sample handling and pipetting minimizes bead carryover, which helps optimize purity. Use consistent magnetic separation times and gentle pipetting techniques to ensure clear separation between the beads and supernatant. 4 Adjust the chaotropic salt concentration for effective recovery Adjust chaotropic salt concentration to enhance nucleic acid binding, while also keeping an eye on PCR inhibition. For elution, use a low-ionic-strength, high-pH buffer to efficiently release DNA or RNA without degrading target molecules. Testing elution conditions across different salt concentrations and temperature can improve recovery. 4 Magnetic beads in next-generation sequencing steps Next-generation sequencing is a powerful technology that allows for rapid, highthroughput sequencing of DNA and RNA. One disease area NGS has transformed is cancer. Advanced NGS approaches provide detailed insights into genetic variations, transcriptomic changes, and structural aberrations. From identifying biomarkers to understanding tumor evolution, NGS workflows are driving personalized oncology (4). Magnetic beads streamline NGS library preparation, supporting key steps like size selection, library normalization, and target enrichment—all steps necessary to achieve high quality libraries. By reducing manual steps and consumable use, magbeads make NGS workflows more efficient and reproducible while minimizing costs across various diagnostic applications. Find more information below on how key NGS workflow steps are transformed with magnetic beads. 5 Library normalization Magnetic beads are also widely used for library normalization, a step that makes sure each sequencing run sample has a consistent concentration of library fragments. Library normalization is especially important in multiplexing experiments where libraries originating from different samples are pooled and sequenced in the same run. These samples need balanced library inputs for equal representation during sequencing. In magbead-based normalization, a fixed bead volume is added to each sample. This fixed volume controls the amount of DNA bound across samples for consistent sequencing depth and coverage. With magnetic bead-based normalization, you can achieve more consistent read depths compared to traditional methods. Normalization improves data uniformity and reduces the need for post-sequencing adjustments (6). However, bead-based normalization requires the sample to exceed the beads’ binding capacity, which isn’t ideal for precious or low-yield samples. Target enrichment and hybrid capture In targeted sequencing applications, such as exome sequencing or gene panel analysis, streptavidin-coated magnetic beads are a convenient option for hybrid capture and target enrichment. In this process, biotinylated probes bind with target genomic regions, defined by panel design. Magnetic beads efficiently capture these hybridized targets and enable elimination of off-target DNA fragments through stringent washing steps. This approach enriches regions of interest, providing deeper coverage that is useful for identifying mutations, structural variants, or RNA alterations such as fusion transcripts in leukemia. These deeper molecular insights are invaluable for guiding therapeutic decisions (7). Fig 2. A standard volume of magnetic beads can bind a consistent quantity of nucleic acid molecules, aiding NGS library normalization. Size selection One of the main uses of magbeads in NGS is size selection. In this library preparation step, precision is key to ensure appropriate-length RNA fragments are selected for profiling. Size selection is crucial across several disease areas, including cancer, with the improved identification of gene fusions and structural variations that are commonly observed in tumor cells (5). As an alternative to gel electrophoresis, magbead-based size selection is fast and automation-friendly, taking only minutes per sample. This is a big improvement compared to the lengthy setup, run, and extraction time with gel electrophoresis. A popular choice for magnetic bead size selection is SPRI technology. This approach uses carboxyl-coated beads to bind nucleic acids reversibly in the presence of polyethylene glycol (PEG) and salts. With SPRI technology, you can achieve precise size selection by adjusting the buffer solution and bead-to-sample ratio. These abilities makes it easier to selectively bind and elute DNA fragments within an accurate size window (2). SPRI’s reversible binding can also simplify NGS library cleanup, reducing sample loss and improving DNA recovery. These features are especially beneficial when sequencing rare or limited samples. Lysis Solution contains nucleic acids as well as other cell debris Sample Prepare sample Binding Positively charged magnetic beads selectively bind to the DNA in optimized conditions Washing Removal of non-nucleic acid contaminants Elution Elution of purified DNA directly to the solution Learn more about magnetic beads for NGS 6 Workflow and bead selection considerations 1 Bead-to-sample ratio for effective size selection and effective washing Adjusting volume ratio between the bead and buffer solution and the sample solution is essential for effective size selection. A higher bead-to-sample ratio binds smaller fragments, while a lower ratio selects for larger ones. Experiment with different bead-to-sample ratios to achieve your desired fragment sizes and refer to the manufacturer’s protocol for each sequencing platform. 2 Optimize washing steps to minimize contaminants Effective washing with ethanol-based buffers is essential to remove unbound DNA and contaminants like salts. Washing should be thorough but gentle to avoid bead aggregation or loss of target fragments. The stringency of washes including temperature adjustments reduce nonspecific binding and thus reduce the amount of off-target reads. Air drying beads after washing minimizes residual ethanol that can interfere with sequencing reactions. 3 Optimize buffers in SPRI workflows for binding power The reversible binding power of SPRI hinges on the concentrations of PEG and salts used in the binding buffer. Even tiny variations in these concentrations can affect DNA fragment size distribution. Sample components (such as MgCl2 , ethanol, glycerol) also affect size selection because pH will change size selection profile. Therefore, it’s essential to use consistent buffer formulations across samples. 4 Adopt automation for high-throughput workflows For labs handling large sample volumes, automating bead-based workflows can be a savior. Automation can not only reduce hands-on time, but can boost consistency, reduce error, and increase throughput, all of which are valuable in clinical or diagnostic NGS applications. Challenges with magnetic beads in NGS workflows While magnetic beads offer many advantages in NGS workflows, you may face certain challenges that negatively affect your sequencing results. For example, during size selection, improper bead handling can lead to aggregation. Aggregation can disrupt the DNA fragment binding process, resulting in inconsistent size selection Stringent washing steps are also key for successful magbead-based NGS preparation. Inadequate washing may leave behind contaminants that interfere with downstream sequencing reactions, potentially reducing read quality and introducing noise into the data. Explore magnetic bead technology, engineered for high yield and consistent size distributions in NGS 7 Magnetic beads in molecular diagnostics There are many benefits to incorporating magnetic beads in molecular diagnostics. These small but mighty separation tools can maximize precision and efficiency in a range of workflows from nucleic acid purification to NGS library preparation. With high-purity DNA/RNA extraction and consistent size selection, magnetic beads are a sure-fire way of boosting the reliability and scalability of your diagnostic assays. With these benefits in mind, integrating magbeads with molecular technologies will ultimately pave the way for improved diagnostic accuracy and patient outcomes. And with continued advancements predicted for bead chemistry and molecular diagnostics, who knows what the future holds for magnetic beads? Ready to transform your molecular diagnostics workflows? Browse Cytiva’s full range of magnetic beads for nucleic acids and proteins 8 References 1. Dwivedi S, Purohit P, Misra R, et al. Diseases and Molecular Diagnostics: A Step Closer to Precision Medicine. Indian Journal of Clinical Biochemistry. 2017;32(4):374. doi:10.1007/S12291-017-0688-8 2. Mirna Lorena S, Cynthia PC, Maria Isabela AR, et al. Nucleic Acids Isolation for Molecular Diagnostics: Present and Future of the Silica-based DNA/RNA Purification Technologies. Separation & Purification Reviews. 2023;52(3):193-204. doi:10.1080/15422119.2022.2053159 3. Chen W-Y, Matulis D, Hu W-P, Lai Y-F, Wang W-H. Studies of the interactions mechanism between DNA and silica surfaces by isothermal titration calorimetry. J Taiwan Inst Chem Eng. 2020;116:62-66. doi:10.1016/j.jtice.2020.11.019 4. Sabour L, Sabour M, Ghorbian S. Clinical Applications of Next-Generation Sequencing in Cancer Diagnosis. Pathol Oncol Res. 2017;23(2):225-234. doi:10.1007/S12253-016-0124-Z 5. Jaksik R, Drobna-Śledzińska M, Dawidowska M. RNA-seq library preparation for comprehensive transcriptome analysis in cancer cells: The impact of insert size. Genomics. 2021;113(6):4149-4162. doi:https://doi.org/10.1016/j.ygeno.2021.10.018 6. Li Y, Liu S, Wang Y, et al. Research on a Magnetic Separation-Based Rapid Nucleic Acid Extraction System and Its Detection Applications. Biosensors (Basel). 2023;13(10). doi:10.3390/bios13100903 7. Oberacker P, Stepper P, Bond DM, et al. Bio-On-Magnetic-Beads (BOMB): Open platform for high-throughput nucleic acid extraction and manipulation. PLoS Biol. 2019;17(1):e3000107. doi:10.1371/JOURNAL.PBIO.3000107 9 cytiva.com Cytiva and the Drop logo are trademarks of Life Sciences IP Holdings Corp. or an affiliate doing business as Cytiva. 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