Mechanical DNA sequencing

Vincent Croquette lab, from the Physics department and IBENS in collaboration with S. Benkovic lab at Penn State University, present the proof of concept for a single-molecule platform that
allows DNA identification and sequencing. In contrast to most present methods, their scheme is not based on the detection of the fluorescent nucleotides but on DNA hairpin length.
By pulling on magnetic beads tethered by a DNA hairpin to the surface, the molecule can be unzipped, because the force can break the hydrogen bonds between complementary bases. In this open state the hairpin can hybridize with complementary oligonucleotides that can be added in solution, which transiently block the hairpin re-zipping when the pulling force is reduced. By measuring the distance from the surface to the bead of a blocked hairpin, one can determine the position of the hybrid along the molecule with nearly single-base precision. Our approach can be used to identify a DNA fragment of known sequence in a mix of various fragments and to sequence an unknown DNA fragment by hybridization or ligation.

Principle of the assay : a) a hairpin DNA molecule is assembled from a dsDNA fragment (in blue) by ligation of a loop at one end and a fork at the other end. The arms of this fork can be bound to a magnetic bead by a biotin bond and to the sample surface by digoxigenin bond. b) Approaching magnets from the beads exerts a force which will pull open the DNA when it exceeds 15pN, fully opening the molecule. In this open state, the molecule extension is maximal and oligonucleotides (brow, purple and violet) will hybridize with the open DNA. By removing the magnets, the force decreases and the hairpin refolds like a zipper leading to the reduction of the molecule extension. When the zipper bumps in the oligonucleotides it gets blocked transiently leading to a pause (iii, iv, v) before the molecule fully refolds (vi). The blockage positions allow to localize the sequences complementary to that of the oligonucleotides.

This work can be found at : Nature Methods,

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