Effect of sample preparation and pH-mediated sample stacking on the analysis of multiplexed short tandem repeats by capillary electrophoresis.

Abstract

An amplification system was used in this study to evaluate the effect of sample preparation and its injection on the electrophoretic separation by capillary electrophoresis using a commercially available capillary electrophoresis instrument. The effect of dilution of amplified samples in water and in different sources of formamide (with conductivity values ranging from 47 to 1000 microS) was evaluated, as was contamination of the sample with high DNA concentrations or buffer salts. Although resolution remained constant in the different solvents tested, the sensitivity increased in samples diluted in water and high-purity formamide. An on-column sample preconcentration method for capillary-based DNA analysis was evaluated to increase the sensitivity of low-quality samples. This technique, pH-mediated sample stacking, is based on the injection of NaOH immediately before sample injection: A neutralization reaction occurs between OH- and tris+ ions so that a low-conductivity zone is created at the head of the capillary. DNA fragments are concentrated at the front of this zone. Using coated capillaries with hydroxycellulose 2% (MW 250,000) as a separation matrix, an improvement in sensitivity was detected in all the solvents studied. The gain in sensitivity was higher for more conductive solvents, and was not correlated with the size of the DNA fragments.