Issue 14, 2015

Topological events in single molecules of E. coli DNA confined in nanochannels

Abstract

We present experimental data concerning potential topological events such as folds, internal backfolds, and/or knots within long molecules of double-stranded DNA when they are stretched by confinement in a nanochannel. Genomic DNA from E. coli was labeled near the ‘GCTCTTC’ sequence with a fluorescently labeled dUTP analog and stained with the DNA intercalator YOYO. Individual long molecules of DNA were then linearized and imaged using methods based on the NanoChannel Array technology (Irys® System) available from BioNano Genomics. Data were collected on 189 153 molecules of length greater than 50 kilobases. A custom code was developed to search for abnormal intensity spikes in the YOYO backbone profile along the length of individual molecules. By correlating the YOYO intensity spikes with the aligned barcode pattern to the reference, we were able to correlate the bright intensity regions of YOYO with abnormal stretching in the molecule, which suggests these events were either a knot or a region of internal backfolding within the DNA. We interpret the results of our experiments involving molecules exceeding 50 kilobases in the context of existing simulation data for relatively short DNA, typically several kilobases. The frequency of these events is lower than the predictions from simulations, while the size of the events is larger than simulation predictions and often exceeds the molecular weight of the simulated molecules. We also identified DNA molecules that exhibit large, single folds as they enter the nanochannels. Overall, topological events occur at a low frequency (∼7% of all molecules) and pose an easily surmountable obstacle for the practice of genome mapping in nanochannels.

Graphical abstract: Topological events in single molecules of E. coli DNA confined in nanochannels

Supplementary files

Article information

Article type
Paper
Submitted
19 Feb 2015
Accepted
13 May 2015
First published
15 May 2015
This article is Open Access
Creative Commons BY license

Analyst, 2015,140, 4887-4894

Author version available

Topological events in single molecules of E. coli DNA confined in nanochannels

J. G. Reifenberger, K. D. Dorfman and H. Cao, Analyst, 2015, 140, 4887 DOI: 10.1039/C5AN00343A

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