PSEUDO-COULOMETRIC LOADING IN CAPILLARY ELECTROPHORESIS DNA-SEQUENCING

While injection volumes in capillary electrophoresis are typically in the nanoliter range, it is difficult to physically prepare and manipul ate samples much smaller than a microliter. As a result, only a small fraction of the analyte contained with the sample volume is transferre d to the capillary. This problem is particularly acute in DNA sequenci ng applications, where on-column stacking is difficult and where the s equencing sample is relatively expensive to prepare. We report a metho d that transfers 75% of the DNA contained within a 3 mu l sample onto a capillary for DNA sequencing. This method relies on the use of very low ionic strength formamide to resuspend the DNA after an ethanol pre cipitation. The use of low ionic strength formamide achieves two tasks . First, it produces a very high resistance sample, which increases th e voltage drop across the sample and decreases the field across the ca pillary. This electric field manipulation ensures that DNA fragments d o not migrate down the capillary during the loading process, allowing long injection periods without excessive band-broadening. Second, the low ionic strength of the formamide increases the transference number of the DNA; more of the current passing through the injection tip of t he capillary is carried by DNA fragments. In the limit of complete eli mination of impurity ions from the loading solvent, current passing th rough the sample is carried only by DNA fragments and loading becomes a coulometric process.