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 manipulate samples much smaller than a microliter. As a result, only a small fraction of the analyte contained with the sample volume is transferred to the capillary. This problem is particularly acute in DNA sequencing applications, where on-column stacking is difficult and where the sequencing sample is relatively expensive to prepare. We report a method that transfers 75% of the DNA contained within a 3 microliters 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 precipitation. The use of low ionic strength formamide achieves two tasks. First, it produces a very high resistance sample, which increases the voltage drop across the sample and decreases the field across the capillary. This electric field manipulation ensures that DNA fragments do 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 the capillary is carried by DNA fragments. In the limit of complete elimination of impurity ions from the loading solvent, current passing through the sample is carried only by DNA fragments and loading becomes a coulometric process.
Figeys
D
D
Department of Chemistry, University of Alberta, Edmonton, Canada.
Ahmadzedeh
H
H
Arriaga
E
E
Dovichi
N J
NJ
D004277:DNA, Single-Stranded / Q000737:chemistry; D019075:Electrophoresis, Capillary / Q000379:methods*; D005559:Formamides / Q000737:chemistry; D009994:Osmolar Concentration; D017422:Sequence Analysis, DNA / Q000379:methods*
D004277:DNA, Single-Stranded; D005559:Formamides; C031066:formamide