Visualizing the entire DNA from a chromosome in a single frame.

C Freitag,A N Nilsson,C Noble,T Ambjörnsson,A Granéli,J Fritzsche,J O Tegenfeldt,M Reiter-Schad,K U Mir,F Persson

doi:10.1063/1.4923262

Abstract

The contiguity and phase of sequence information are intrinsic to obtain complete understanding of the genome and its relationship to phenotype. We report the fabrication and application of a novel nanochannel design that folds megabase lengths of genomic DNA into a systematic back-and-forth meandering path. Such meandering nanochannels enabled us to visualize the complete 5.7 Mbp (1 mm) stained DNA length of a Schizosaccharomyces pombe chromosome in a single frame of a CCD. We were able to hold the DNA in situ while implementing partial denaturation to obtain a barcode pattern that we could match to a reference map using the Poland-Scheraga model for DNA melting. The facility to compose such long linear lengths of genomic DNA in one field of view enabled us to directly visualize a repeat motif, count the repeat unit number, and chart its location in the genome by reference to unique barcode motifs found at measurable distances from the repeat. Meandering nanochannel dimensions can easily be tailored to human chromosome scales, which would enable the whole genome to be visualized in seconds.

Highlights

Conventional genomics methods, such as those that were used in the sequencing of the human reference genome handle and analyze molecules in bulk
Analyzing genomic information by imaging long linear single DNA molecules was first introduced by the groups of Bensimon1 and Schwartz2 by stretching the DNA on surfaces
The bends were in turn designed with a 1 lm long straight part connected to two 90 turns to minimize stress on the DNA molecules in the meander-channels

Summary

Introduction

Conventional genomics methods, such as those that were used in the sequencing of the human reference genome handle and analyze molecules in bulk. Methods such as cloning or PCR are needed to isolate and produce sufficient quantities of DNA to be detected. Analyzing genomic information by imaging long linear single DNA molecules was first introduced by the groups of Bensimon (dynamic molecular combing) and Schwartz (optical mapping) by stretching the DNA on surfaces. Optical mapping has been employed at the finishing stage of genome sequencing projects. While these methods stretch DNA on a surface, subsequent fluidic approaches have analyzed DNA in extensional flows.. DNA molecules can be tethered in both or a single end by chemical bonds to create DNA curtains as pioneered by Greene. The

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Conclusion