This process is experimental and the keywords may be updated as the learning algorithm improves. This is a preview of subscription content, log in to check access. Angela Karp 1 Peter G. Isaac 2 David S. Ingram 3 1. Personalised recommendations. Cite chapter How to cite? Taub , Tom S. Drug Metabolism and Disposition , 48 8 , Resistive amplitude fingerprints during translocation of linear molecules through charged solid-state nanopores. The Journal of Chemical Physics , 3 , Conrad , Katerina Kourentzi , Richard C.
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At pH 8, the loss was maximized. It has been theorized that chaotropes increase DNA-silica affinity by dehydrating the surface and promoting hydrogen bonding between the DNA and silica surface [ 19 ]. Focusing on events downstream of the DNA adsorption process, our elution results suggest that an increase in DNA adsorption capacity at low pH does not necessarily leads to a higher DNA recovery rate. Instead, the best DNA recovery results were obtained by taking advantage of a weaker DNA-silica-chaotrope adsorption complex facilitated at the intermediate pH of 5.
Thus, a DNA-silica-chaotrope adsorption event at pH 5. In arriving to this conclusion, we first employed buffer EB and 0. Finally, eluting with 1 M NaOH resulted in a Since NaOH completely destroys the DNA-silica-chaotrope complex via silica surface dissolution, the high level of NaOH-dependent DNA liberation suggests that hydrophobic interactions plays a larger role than hydrogen bonding or ionic interactions in the processes of adsorption complex formation at pH 3.
We expected the lack of ionic interactions was due to the fact that phosphate and silanol surface groups are protonated and charge-neutralized at pH 3. Thus, if the destruction of all possible hydrogen bonds with hot formamide only amounted to liberating a small portion of the entire pool of adsorbed DNA molecules, then hydrophobic interactions are the likely dominant force that holds the DNA-silica-chaotrope complex together.
The experiments were conducted such that DNA, rather than the available surface, was the limiting factor for adsorption. Our results support the model in which the adsorption capacity increases with decreasing pH. This is in contrast to other experimental designs in literature that have demonstrated recovery of significant amounts of DNA after adsorbing at pH 4 or 8, with the caveat that the amount of unrecoverable DNA within the system was not addressed.
To elaborate, these studies were conducted using large concentrations of input DNA, such that the net unrecoverable amount of DNA was negligible compared to the net recovered [ 6 , 25 , 26 ]. These differences become vital when one is trying to use silica columns to bind and release minute amounts of total DNA from NA-dilute clinical samples. Of course, most commercial kits address this problem by adding exogenous DNA to artificially increase the overall DNA load.
However, this approach is not ideal in POC applications, since adding reagents increases cost, complexity, and volume to devices that need to be as inexpensive, simple, and small as possible. Our results suggest that the standard method for eluting DNA using buffer EB from silica following chaotrope-mediated adsorption may not be ideal for low concentration of DNA.
Based on these results, DNA recovery depends on 1 the concentration of DNA in the adsorption solution, 2 the adsorption solution pH, 3 the presence of a chaotrope, and 4 the elution buffer. It also depends on the ratio of input DNA to the available surface area of silica when comparing to previous results. This however was controlled for in these experiments. Thus, designing a device to use this technology requires not only an understanding of the DNA-silica-chaotrope interaction, but the dynamic range of characteristics associated with clinical samples and how sample preparation steps affect these characteristics.
Our data suggests that we can increase the range of initial sample conditions for which this technology can be implemented. Controlling for presence of a chaotrope, DNA adsorption increased with increasing acidity of the aqueous adsorption solution.
Adsorption at pH 3 resulted in DNA-silica-chaotrope complex that was too strong for buffer EB to disrupt effectively, while adsorption at pH 8 was not strong enough for DNA to adsorb sufficiently.
Hot formamide and 1 M NaOH resulted in increased recovery of However, neither of these buffers is practical for POC use. As our knowledge of these interactions improve, so will the range of clinical applications for this technology. Formal analysis: CK. Funding acquisition: CMK. Investigation: CK. Project administration: CMK. Resources: CMK.
Software: CK. Validation: CK. Visualization: CK AF. Writing — original draft: CK. Browse Subject Areas? Click through the PLOS taxonomy to find articles in your field. Abstract DNA extraction from clinical samples is commonly achieved with a silica solid phase extraction column in the presence of a chaotrope.
Introduction Nucleic acid NA molecular diagnostics are highly specific and sensitive assays that detect the presence or absence of target-specific DNA or RNA sequences from crude biological samples [ 1 , 2 ]. Background DNA extraction kits from silica-based solid phase columns often utilizes a chaotropic buffer that serves both as a protein denaturant and cofactor that promotes NA adsorption.
Materials and methods Materials All experiments were conducted using Davisil amorphous silica particles Sigma-Aldrich, St. Download: PPT. DNA-silica system Using NA extraction and elution protocols from standard Qiagen extraction kits and previously published POC diagnostic devices as templates, the overall experimental procedures are shown in Fig 1.
Elution curves To understand the DNA-silica-chaotrope interaction, elution curves were generated for different conditions. DNA purification Since the polymerase chain reaction PCR was used to quantify the DNA amounts lost and recovered, an additional ethanol precipitation step was applied to all filtrates to remove PCR-inhibiting salts [ 36 ].
The DNA-silica-chaotrope interaction at low pH is dominated by hydrophobic forces In our series of experiments, the largest percentage of input DNA recovered was Fig 5. Elution of DNA from silica using varying elution buffers. References 1. Point of care diagnostics: status and future. Anal Chem. Advances in microfluidic PCR for point-of-care infectious disease diagnostics.
Biotechnol Adv. Bhattacharyya A, Klapperich CM. Thermoplastic microfluidic device for on-chip purification of nucleic acids for disposable diagnostics.
Qiagen, Qiagen. QIAprep Miniprep Handbook. J Biomed Biotechnol. Evaluation of silica resins for direct and efficient extraction of DNA from complex biological matrices in a miniaturized format. Anal Biochem. Nucleic acid purification using microfabricated silicon structures. Biosens Bioelectron. Improved DNA extraction efficiency from low level cell numbers using a silica monolith based micro fluidic device. Anal Chim Acta. Factors determining flow rate in chromatographic columns.
View Article Google Scholar Toner M, Irimia D. Annu Rev Biomed Eng. Continuous flow microfluidic device for cell separation, cell lysis and DNA purification. Forensic DNA analysis on microfluidic devices: a review. J Forensic Sci. Clin Chem. Zhang Y, Cremer PS.
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