Automated bead-trapping apparatus and control system for single-molecule DNA sequencing.
We have been investigating a microfluidics platform for high-speed, low-cost sequencing of single DNA molecules using novel "charge-switch" nucleotides. A significant challenge is the design of a flowcell suitable for manipulating bead-DNA complexes and sorting labeled polyphosphate molecules by charge. The flowcell is part of a single-molecule detection instrument, creating fluorescence images from labeled polyphosphates. These images would ultimately be analyzed by signal processing algorithms to identify specific nucleotides in a DNA sequence. Here we describe requirements of the fluidics system for loading, identifying, tracking, and positioning beads. By dynamically modulating pressure gradients in the plenum chambers of a multi-channel network, we could guide individual beads with high precision to any desired coordinate and reversibly trap them in stepped channels. We show that DNA immobilized on pressure-trapped beads can be physically extended into a downstream channel under electric force for analysis. Custom dynamic algorithms for automated bead control are described.
Bashford
Greg
G
Department of Biological Systems Engineering, University of Nebraska-Lincoln, Lincoln, NE 68583, USA. gbashford2@unl.edu
Lamb
Don
D
Grone
Dan
D
Eckles
Bob
B
Kornelsen
Kevin
K
Middendorf
Lyle
L
Williams
John
J
D004867:Equipment Design; D019544:Equipment Failure Analysis; D046210:Microfluidic Analytical Techniques / Q000295:instrumentation*; D008856:Microscopy, Fluorescence / Q000295:instrumentation*; D020411:Oligonucleotide Array Sequence Analysis / Q000295:instrumentation*; D012371:Robotics / Q000295:instrumentation* / Q000379:methods; D017422:Sequence Analysis, DNA / Q000295:instrumentation* / Q000379:methods