Nucleic acid (NA) purification from clinical samples is commonly achieved using silica solid phase extraction in the presence of a chaotropic salt. Versions of these protocols have been adapted for point of care (POC) diagnostic devices in miniaturized platforms. Most such protocols require a high net amount of input NA, which is often achieved by adding exogenous carrier NA to the clinical sample. As a result, for samples containing less than 1 μg of total NA, NA recovery is low in the absence of carrier NA. Clinical samples used in POC diagnostics may contain very low NA concentrations (~1 ng/ml), which result in NA-limited interactions with the solid phase that are outside the dynamic range of POC diagnostics. This work is a study of DNA-silica interactions in the DNA-limiting regime to gain fundamental understanding of the mechanisms at play in order to increase the dynamic range and sensitivity of miniaturized NA based POC diagnostics. DNA adsorption and recovery from silica surfaces for concentrations less than 1 μg/ml are studied. A protocol was designed and developed to systematically quantify the adsorption of DNA onto a silica surface and the amount of DNA recovered by elution at very low concentrations. Various adsorption conditions were examined including a range of pH, different chaotropes, and DNA concentrations down to
2.5 pg/ml. DNA recovery was further optimized for low concentration samples by varying elution buffers. DNA-silica adsorption was enhanced by low pH and was further improved by the presence of a chaotrope. Different adsorption conditions had little effect on DNA recovery using low salt, high pH elution buffers, but DNA recovery did exceed 40% when adsorbed initially with 5 M guanidinium thiocyanate at pH 5.2. Recovery was enhanced by eluting with 95 °C formamide or 1 M NaOH, supporting the hypothesis that DNA-silica interactions are dominated by hydrophobic forces and hydrogen bonding. While heated formamide and NaOH are non-ideal elution buffers for practical POC devices, these results are important for engineering a set of optimized reagents and conditions that could maximize DNA recovery from a microfluidic POC silica system. / 2017-06-21T00:00:00Z
| Identifer | oai:union.ndltd.org:bu.edu/oai:open.bu.edu:2144/17086 |
| Date | 21 June 2016 |
| Creators | Katevatis, Constantinos Ioannis |
| Source Sets | Boston University |
| Language | en_US |
| Detected Language | English |
| Type | Thesis/Dissertation |
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