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 magnetiz...

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Veröffentlicht in:Analytical chemistry (Washington) 2006-06, Vol.78 (11), p.3632-3637
Hauptverfasser: Ueberfeld, Jörn, El-Difrawy, Sameh A, Ramdhanie, Korisha, Ehrlich, Daniel J
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Sprache:eng
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Zusammenfassung: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 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 ∼1-s duration. The method should be broadly extendable to DNA and protein separations in both microdevice and capillary electrophoresis.
ISSN:0003-2700
1520-6882
DOI:10.1021/ac052201x