Journal of Analytical & Bioanalytical Techniques
Open Access

Abstract

Single DNA molecules of genomic length can be stretched by confinement in nanofluidic channels. Nanofluidic devices have been used to characterize the base pair sequence, or the methylation of DNA by imaging fluorescence barcodes of single molecules. The resolution of the fluorescence barcode imaged on DNA is maximized when the DNA is stretched to its full contour length (0.34 nm per base pair). In nanochannels, DNA stretching is provided by confinement only i.e. DNA can be fully stretched if the channel cross-section matches the persistence length of the DNA (50 nm), which can be challenging to fabricate. A nanofluidic device where 98% stretching of genomic DNA is achieved by an additional mechanism: the hydrodynamic drag of a buffer flow, was designed. At such high stretching, the number of base pairs included in the diffraction limit is minimized thus providing the best barcode resolution obtainable using conventional epifluorescence (about 1 kilobase). A device was used to image fluorescence barcodes of human DNA fragments obtained by proteolysis of metaphase chromosomes. The fluorescence barcodes are specific to the underlying base sequence of each fragment covering a minimum of 1.4 mega base pairs. It has been shown that the barcode image enables to map each fragment to its origin in the human reference genome. Moreover, it was able to detect large structural variations (from a couple of kilobase and up) present in single copies of the human genome by comparing the fluorescence pattern of a given molecule to the pattern expected from the human reference genome (hg18).

Biography

Rodolphe Marie has completed his PhD in 2004 from the Technical University of Denmark (DTU) and postdoctoral studies from Lund University in Sweden. He is an Associate Professor at DTU Nanotech, the department of micro and nanotechnology at DTU.