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 After careful evaluation of five hydrophobic ionic liquids, 1-butyl-1-methylpyrrolidinium bis(trifluoromethylsulfonyl)imide ([BMPyr+][Ntf2-]) 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. ddH2O double-distilled water, qPCR quantitative PCR
Highlights
In the life sciences, in molecular biology, one of the most important classes of molecules are nucleic acids, especially DNA
We report here a new method, based on hydrophobic ionic liquids (ILs), for fast and quantitative bacterial cell lysis with subsequent DNA extraction followed by downstream molecularbiological techniques such as quantitative PCR (qPCR)
In contrast to previously studied IL-based DNA extraction methods, the DNA intake capacity of the ILs should preferably be very small to minimize the need for multiple elution steps following cell lysis, and thereby reduce the need for complex handling and shorten the time required
Summary
In molecular biology, one of the most important classes of molecules are nucleic acids, especially DNA. Novel approaches utilizing ionic liquids (ILs) have been successfully applied to DNA extraction, further improving the previously established systems, via a second hydrophobic extraction phase, solid-phase microextraction, or magnetic IL extraction [11,12,13,14] What all these extraction methods have in common is that they can be separated into two general steps: the first step is lysis of the cell; the second step is separation and purification whereby the desired nucleic acid is extracted from the aqueous cell debris and sample residues. Number of pathogens permitted to be present in the sample or only a small number of target cells are present within a huge background [1] This problem is very well reflected by the actual time required for the two separate steps in commercial DNA purification kits for bacteria, such as the NucleoSpin® tissue kit, which was used in this study. The final method permits quantitative DNA extraction from Gram-negative bacteria with performance equivalent to that of a commercial kit in only a fraction of the time
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