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Hydrophobic ionic liquids for quantitative bacterial cell lysis with subsequent DNA quantification.

ABSTRACT: DNA is one of the most frequently analyzed molecules in the life sciences. In this article we describe a simple and fast protocol for quantitative DNA isolation from bacteria based on hydrophobic ionic liquid supported cell lysis at elevated temperatures (120-150 °C) for subsequent PCR-based analysis. From a set of five hydrophobic ionic liquids, 1-butyl-1-methylpyrrolidinium bis(trifluoromethylsulfonyl)imide was identified as the most suitable for quantitative cell lysis and DNA extraction because of limited quantitative PCR inhibition by the aqueous eluate as well as no detectable DNA uptake. The newly developed method was able to efficiently lyse Gram-negative bacterial cells, whereas Gram-positive cells were protected by their thick cell wall. The performance of the final protocol resulted in quantitative DNA extraction efficiencies for Gram-negative bacteria similar to those obtained with a commercial kit, whereas the number of handling steps, and especially the time required, was dramatically reduced. Graphical Abstract After careful evaluation of five hydrophobic ionic liquids, 1-butyl-1-methylpyrrolidinium bis(trifluoromethylsulfonyl)imide ([BMPyr ⁺ ][Ntf ₂^- ]) was identified as the most suitable ionic liquid for quantitative cell lysis and DNA extraction. When used for Gram-negative bacteria, the protocol presented is simple and very fast and achieves DNA extraction efficiencies similar to those obtained with a commercial kit. ddH ₂ O double-distilled water, qPCR quantitative PCR.

SUBMITTER: Fuchs-Telka S

PROVIDER: S-EPMC5333376 | biostudies-literature | 2017 Feb

REPOSITORIES: biostudies-literature

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Hydrophobic ionic liquids for quantitative bacterial cell lysis with subsequent DNA quantification.

Fuchs-Telka Sabine S Fister Susanne S Mester Patrick-Julian PJ Wagner Martin M Rossmanith Peter P

Analytical and bioanalytical chemistry 20161221 6

DNA is one of the most frequently analyzed molecules in the life sciences. In this article we describe a simple and fast protocol for quantitative DNA isolation from bacteria based on hydrophobic ionic liquid supported cell lysis at elevated temperatures (120-150 °C) for subsequent PCR-based analysis. From a set of five hydrophobic ionic liquids, 1-butyl-1-methylpyrrolidinium bis(trifluoromethylsulfonyl)imide was identified as the most suitable for quantitative cell lysis and DNA extraction beca ...[more]

PMID: 28004172

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Project description:Deoxyribonucleic acid (DNA) carries the genetic information essential for the growth and functioning of living organisms, playing a significant role in life sciences research. However, the long-term storage and preservation of DNA, while ensuring its bioactivity, are still current challenges to overcome. In this work, aqueous solutions of ionic liquids (ILs) were investigated as potential preservation media for double stranded (dsDNA). A screening of several ILs, by combining the cholinium, tetrabutylammonium, tetrabutylphosphonium, and 1-ethyl-3-methylimidazolium, cations with the anions bromide, chloride, dihydrogen phosphate, acetate, and glycolate, was carried out in order to gather fundamental knowledge on the molecular features of ILs that improve the dsDNA stability. Different IL concentrations and the pH effect were also addressed. Circular dichroism (CD) spectroscopy was used to evaluate the conformational structure and stability of dsDNA. IL-DNA interactions were appraised by UV-Vis absorption spectrophotometry and 31P nuclear magnetic resonance (NMR) spectroscopy. The results obtained demonstrate that pH has a significant effect towards the dsDNA stability. Amongst the ILs investigated, cholinium-based ILs are the most promising class of ILs to preserve the dsDNA structure, in which electrostatic interactions between the cholinium cation and the DNA phosphate groups play a significant role as demonstrated by the 31P NMR data, being more relevant at higher IL concentrations. On the other hand, the denaturation of dsDNA mainly occurs with ILs composed of more hydrophobic cations and able to establish dispersive interactions with the nucleobases environment. Furthermore, the IL anion has a weaker impact when compared to the IL cation effect to interact with DNA molecules. The experimental data of this work provide relevant fundamental knowledge for the application of ILs in the preservation of nucleic acids, being of high relevance in the biotechnology field.

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Hydrophobic ionic liquids for quantitative bacterial cell lysis with subsequent DNA quantification.

Publications

Hydrophobic ionic liquids for quantitative bacterial cell lysis with subsequent DNA quantification.

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