UBC Theses and Dissertations
Contaminant-induced current decline in capillary array electrophoresis Coope, Robin
This research clarifies, for the first time, the mechanism and impact of current decline in capillary array electrophoresis (CAE). High throughput capillary array electrophoresis instruments for DNA sequencing suffer to varying degrees from failure associated with electrophoretic current decline and inhibition or delay in the arrival of fragments at the detector. This effect is known to be associated with residual quantities of large, slow moving fragments of template or genomic DNA carried through from sample preparation and sequencing reactions. Here, we document and investigate the existence of an expanding ionic depletion region induced by overloading the capillary with low-mobility DNA fragments, and the effect of growth of this region on electrophoresis run failure. This depletion region forms upstream of the smaller sequencing fragments, and its expansion was found not to affect the quality of the sequencing peaks at the detector. Rather the current decline associated with depletion region growth reduces the velocity of the downstream sequencing fragments, so fewer fragments arrive at the detector during the run. It is shown, through analytical and numerical models, how increasing quantities of slow moving DNA cause the concentration of background electrolyte downstream to decline. With the concentration of such fragments beyond a threshold quantity, the anode-side boundary of the nascent depletion region is shown to propagate toward the anode at a rate faster than the contaminant DNA migration. Under such conditions the depletion region expands, the current declines, and the electrophoresis run suffers from a reduced yield of sequence data or fails completely. While the upstream boundary of the depletion region propagates with the DNA, the propagation rate of the downstream boundary is found to be inversely proportional to the amount of ionic depletion, and independent of the motion of the DNA. Observations suggest that these downstream boundaries may propagate as a result of an imbalance in current carriers brought about by the exposure of bound charges in the matrix or capillary wall, which may be coupled to a rise in pH.
Item Citations and Data