High-Throughput Purification of Viral RNA Based on Novel Aqueous Chemistry for Nucleic Acid Isolation

Hourfar, M. K.; Michelsen, Uwe; Schmidt, Michael; Berger, Annemarie; Seifried, Erhard; Roth, W. Kurt

doi:10.1373/clinchem.2005.048603

Cited by 35 publications

(38 citation statements)

References 21 publications

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“…The MagPrep silica particles are silica-coated magnetic nanoparticles commercialized specifically for use in nucleic acid purification. 1,4 However, the surface chemistry of these particles is proprietary, and might not represent a pure silica surface. Because of this, we also used finely ground pure silica as a second solid matrix for bulk depletion experiments.…”

Section: Resultsmentioning

confidence: 99%

Multiphasic DNA Adsorption to Silica Surfaces under Varying Buffer, pH, and Ionic Strength Conditions

et al. 2012

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Reversible interactions between DNA and silica are utilized in the solid phase extraction and purification of DNA from complex samples. Chaotropic salts commonly drive DNA binding to silica, but inhibit DNA polymerase amplification. We studied DNA adsorption to silica using conditions with or without chaotropic salts through bulk depletion and Quartz Crystal Microbalance (QCM) experiments. While more DNA adsorbed to silica using chaotropic salts, certain buffer conditions without chaotropic salts yielded a similar amount of eluted DNA. QCM results indicate that under stronger adsorbing conditions, the adsorbed DNA layer is initially rigid, but becomes viscoelastic within minutes. These results qualitatively agreed with a mathematical model for a multiphasic adsorption process. Buffer conditions that do not require chaotropic salts can simplify protocols for nucleic acid sample preparation. Understanding how DNA adsorbs to silica can help optimize nucleic acid sample preparation for clinical diagnostic and research applications.

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Section: Resultsmentioning

confidence: 99%

Multiphasic DNA Adsorption to Silica Surfaces under Varying Buffer, pH, and Ionic Strength Conditions

et al. 2012

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“…To overcome these challenges, several alternative solid phase extraction methods not based on chaotropic salts and organic solvents have been reported. 2–5 …”

Section: Introductionmentioning

confidence: 99%

DNA Adsorption to and Elution from Silica Surfaces: Influence of Amino Acid Buffers

et al. 2013

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Solid phase extraction and purification of DNA from complex samples typically requires chaotropic salts that can inhibit downstream polymerase amplification if carried into the elution buffer. Amino acid buffers may serve as a more compatible alternative for modulating the interaction between DNA and silica surfaces. We characterized DNA binding to silica surfaces, facilitated by representative amino acid buffers, and the subsequent elution of DNA from the silica surfaces. Through bulk depletion experiments, we found that more DNA adsorbs to silica particles out of positively compared to negatively charged amino acid buffers. Additionally, the type of the silica surface greatly influences the amount of DNA adsorbed, and the final elution yield. Quartz crystal microbalance experiments with dissipation monitoring (QCM-D) revealed multiphasic DNA adsorption out of stronger adsorbing conditions such as arginine, glycine, and glutamine, with DNA more rigidly bound during the early stages of the adsorption process. The DNA film adsorbed out of glutamate was more flexible and uniform throughout the adsorption process. QCM-D characterization of DNA elution from the silica surface indicates an uptake in water mass during the initial stage of DNA elution for the stronger adsorbing conditions, which suggests that for these conditions the DNA film is partly dehydrated during the prior adsorption process. Overall, several positively charged and polar neutral amino acid buffers show promise as an alternative to methods based on chaotropic salts for solid phase DNA extraction.

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“…Multiplexing at the NA level [37] can be implemented in LF format by adding different haptens to the amplification reagent mix [26]. Integration of magnetic beads as an alternative to the silica membrane isolation of nucleic acids has been developed and tested (results not shown) and may improve sensitivity since it allows omitting EtOH-based wash steps [38] and NA elution from the protocol with direct input of all bound NA targets in a microliter size amplification reaction. Moreover, magnetic beads may also be applied to capture and concentrate targets other than NA for multiplex analysis.…”

Section: Discussionmentioning

confidence: 99%

Development of a Generic Microfluidic Device for Simultaneous Detection of Antibodies and Nucleic Acids in Oral Fluids

Chen

Abrams

Geva

et al. 2013

BioMed Research International

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A prototype dual-path microfluidic device (Rheonix CARD) capable of performing simultaneously screening (antigen or antibody) and confirmatory (nucleic acid) detection of pathogens is described. The device fully integrates sample processing, antigen or antibody detection, and nucleic acid amplification and detection, demonstrating rapid and inexpensive “sample-to-result” diagnosis with performance comparable to benchtop analysis. For the chip design, a modular approach was followed allowing the optimization of individual steps in the sample processing process. This modular design provides great versatility accommodating different disease targets independently of the production method. In the detection module, a lateral flow (LF) protocol utilizing upconverting phosphor (UCP) reporters was employed. The nucleic acid (NA) module incorporates a generic microtube containing dry reagents. Lateral flow strips and PCR primers determine the target or disease that is diagnosed. Diagnosis of HIV infection was used as a model to investigate the simultaneous detection of both human antibodies against the virus and viral RNA. The serological result is available in less than 30 min, and the confirmation by RNA amplification takes another 60 min. This approach combines a core serological portable diagnostic with a nucleic acid-based confirmatory test.

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High-Throughput Purification of Viral RNA Based on Novel Aqueous Chemistry for Nucleic Acid Isolation

Cited by 35 publications

References 21 publications

Multiphasic DNA Adsorption to Silica Surfaces under Varying Buffer, pH, and Ionic Strength Conditions

Multiphasic DNA Adsorption to Silica Surfaces under Varying Buffer, pH, and Ionic Strength Conditions

DNA Adsorption to and Elution from Silica Surfaces: Influence of Amino Acid Buffers

Development of a Generic Microfluidic Device for Simultaneous Detection of Antibodies and Nucleic Acids in Oral Fluids

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