Effects of metal ions on concentration of DNA in high-conductivity media by capillary electrophoresis.
On-line concentration and separation of DNA prepared in low- or high-conductivity media has been demonstrated using poly(ethylene oxide) (PEO) solution in the presence of electroosmotic flow. DNA fragments migrating against EOF stacked at the boundary between the sample zone and PEO solutions, mainly because of sieving and increases in the viscosity. Unlike conventional methods, the large DNA fragments were detected earlier toward the cathode end in this study. The limit of detection (LOD) at a signal-to-noise ratio=3 for phiX174 RF DNA-Hae III digest prepared in 50 mM Tris-borate, pH 10.0, was down to 0.171 ng/ml, with an 860-fold improvement (compared to that obtained by 10-s injection at 25 V/cm) in the sensitivity, when injecting about 2.58 microl. By applying a short plug (2.3 cm) of 0.5 mM AgNO3 prepared in 1.5% PEO solution after sample injection, the analysis of up to 0.75 microl DNA prepared in phosphate-buffered saline (PBS) has been carried out without any tedious desalting processes. This results in an LOD of 6.86 ng/ml for the DNA sample and a 155-fold improvement in the sensitivity. Moreover, this method has allowed the analysis of 0.75 micro] of polymerase chain reaction products amplified after 18 cycles with good reproducibility.
Huang
Chih-Ching
CC
Department of Chemistry, National Taiwan University, Taipei.
Chiu
Tai-Chia
TC
Chang
Huan-Tsung
HT
D004247:DNA / Q000737:chemistry*; D004553:Electric Conductivity; D019075:Electrophoresis, Capillary / Q000379:methods*; D006801:Humans; D008670:Metals / Q000737:chemistry*; D016133:Polymerase Chain Reaction; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D008670:Metals; D004247:DNA