Formation of a resistive region at the anode end in DNA capillary electrophoresis

Abstract

We have studied the formation of a resistive region in the capillary during DNA separation. This effect is caused by an unequal change in the mobilities of cations and anions at the interface between the running buffer solution and the capillary. We studied the motion of the resistive region boundary by sequential removal of portions of the affected capillary end. We found that in the process of developing the resistive region the distribution of the electric fields in the capillary changes from uniform to extremely nonuniform, with a very high field (above 1 MV/cm) in the resistive region and a reduced field (80 V/cm) in the rest of the capillary. Though theoretically a resistive region may appear either at the anode (detection) or the cathode (injection) end of the capillary, all previous publications report the formation of the resistive region at the cathode side. In our experiments, however, the anomalous region is formed at the anode. Thus, the separated DNA peaks move towards the slowly progressing resistive region. Our results indicate that the DNA is stopped at the boundary and does not enter the region. When the resistive region is clipped off the peak motion resumes. This suggests that there exists a potential barrier at the resistive layer boundary that prevents the drift of the peaks towards the anode. The formation of the resistive region interferes with a normal separation process causing a gradual decrease of the capillary current and the deceleration and eventual quenching of the peak motion. For the ABI chemistry, we experimented with adding polymers to the electrode buffer to equate the transference numbers for anions and cations, and found the conditions at which this effect is completely eliminated.

Numerical model for DNA loading in microdevices: Stacking and autogating effects

“…These effects are particularly pronounced in the sample well and most dominantly associated with spatially and temporally varying fields near the matrix/water interface. Similar effects have been observed for a long time in capillary devices [17]. However, because of the timing-critical voltage switching aspect of crossinjectors, they have much more pronounced effects on the performance of microdevices than was previously observed in capillaries.…”

Contaminant-induced current decline in capillary array electrophoresis

“…Ion distribution was measured by the cut capillary method [12,14]. This required removing the capillary from the apparatus, which cooled it from 557C to 207C, causing the matrix to shrink about 5 mm.…”

“…The Swerdlow group [3] also studied the effect of DNA samples, and explicitly showed that rapid current decline was associated with the injection of a sufficient quantity of fragments longer than 1300 bp, and that smaller quantities did not cause the effect to occur. While sample-independent current decline has been minimized in commercial instruments through judicious selection of buffer and matrix components [13,14], the swift and severe current decline from large fragments continues to be a concern.…”

Experimental study of the formation of high‐resistivity zones at the gel/buffer interface in CE

“…In contrast to all the above-quoted papers describing formation of the resistive region at the cathode end of the capillary, we consistently observed the resistive region at the anode (detection) end. This gave us an opportunity to directly study the behavior of the DNA peaks in this region [13].…”

“…In [13], for characterization of the migration of high-resistivity zones, we measured time-dependent electric current, and then determined the length of the influenced region by a sequential removal of small (1 mm) pieces of the affected capillary end. This method suffered from two major shortcomings: first, the accuracy of the measured length of the resistive region was rather low and second, the measurement destroyed the capillary and was difficult to reproduce.…”