A Parallelized Nanofluidic Device for High-Throughput Optical DNA Mapping of Bacterial Plasmids.

Sriram Kk,Yii-Lih Lin,Tsegaye Sewunet,Marie Wrande,Christian G Giske,Fredrik Westerlund,Linus Sandegren

doi:10.3390/mi12101234

Abstract

Optical DNA mapping (ODM) has developed into an important technique for DNA analysis, where single DNA molecules are sequence-specifically labeled and stretched, for example, in nanofluidic channels. We have developed an ODM assay to analyze bacterial plasmids—circular extrachromosomal DNA that often carry genes that make bacteria resistant to antibiotics. As for most techniques, the next important step is to increase throughput and automation. In this work, we designed and fabricated a nanofluidic device that, together with a simple automation routine, allows parallel analysis of up to 10 samples at the same time. Using plasmids encoding extended-spectrum beta-lactamases (ESBL), isolated from Escherichia coli and Klebsiella pneumoniae, we demonstrate the multiplexing capabilities of the device when it comes to both many samples in parallel and different resistance genes. As a final example, we combined the device with a novel protocol for rapid cultivation and extraction of plasmids from fecal samples collected from patients. This combined protocol will make it possible to analyze many patient samples in one device already on the day the sample is collected, which is an important step forward for the ODM analysis of plasmids in clinical diagnostics.

Highlights

Optical DNA mapping (ODM) using fluorescence imaging of single long DNA molecules, pioneered by David Schwartz and others in the early 1990s [1,2], has developed into a versatile tool to study genomic DNA
We developed a nanofluidic device with the capability to perform ODM
[24,26]; we have shown, that detecting identical important, for example, for epidemiological studies of bacterial transmission and plasmids in several samples can be a proxy for ongoing clonal outbreaks [25,27]

Summary

Introduction

Optical DNA mapping (ODM) using fluorescence imaging of single long DNA molecules, pioneered by David Schwartz and others in the early 1990s [1,2], has developed into a versatile tool to study genomic DNA. Work involving DNA mapping was performed by stretching fluorescently labelled single DNA molecules on a positively charged or hydrophobic glass surface [3,4,5]. The advent of micro- and nanofluidic devices opened a new possibility of stretching DNA molecules in highly confined environments, providing the opportunity to manipulate single DNA molecules that are not deposited on a surface [6,7,8,9]. Studies of DNA molecules in nanochannels focused primarily on understanding the physics of confined DNA [9,10,11]. This, in turn, opened the possibility to use nanofluidic channels for ODM studies of DNA [13,14]. Various enzymatic labeling schemes, such as nick labeling [15], nick-flap labeling [16], and methyltransferase labeling [17], have

Methods

Results

Conclusion