Solid-support sample loading for DNA sequencing.
We present a new method for simplified low-quantity DNA loading onto microelectrophoresis devices. The method is based on combined solid-phase extraction, purification, and transport of DNA reversibly bound on paramagnetic microspheres. DNA is adsorbed onto the microspheres, captured with a magnetized permalloy wire, and then directly injected as a highly focused sample plug into the separation channel. This method circumvents both the minimum volume requirement of pipettors (since only solid beads are transported) and the timing complications of double-T microfluidic injection. Injections from Sanger samples of <100 pg total suspended weight match the signal strength of our previous conventional injections at >10-times the starting DNA sample. Sequencing traces show a resolution that matches or exceeds double-T injections. A kinetic model reproduces the time-dependence of the injection signals and proves that total nonidealities in the method produce injection-broadened plugs of approximately 1-s duration. The method should be broadly extendable to DNA and protein separations in both microdevice and capillary electrophoresis.
Ueberfeld
Jörn
J
Whitehead Institute for Biomedical Research, Nine Cambridge Center, Cambridge, Massachusetts 02142, USA.
El-Difrawy
Sameh A
SA
Ramdhanie
Korisha
K
Ehrlich
Daniel J
DJ
D001483:Base Sequence; D004247:DNA / Q000032:analysis* / Q000378:metabolism; D004566:Electrodes; D004583:Electrons; D007700:Kinetics; D008855:Microscopy, Electron, Scanning; D008957:Models, Genetic
D004247:DNA