Optimization of DNA electrophoretic behavior in poly(vinyl pyrrolidone) sieving matrix for DNA sequencing.
Poly(vinyl pyrrolidone) solution was used as a separation matrix in capillary electrophoresis for DNA sequencing. Four-label four-color detection was performed for base calling. Dye-labeled DNA showed large mobility shifts at normal conditions for DNA separation. Temporal correction of mobility shifts was achieved by normalizing with respect to pure peaks that are without spectral interference or temporal overlap at each color channel. To achieve even better performance, a DNA separation condition that does not require corrections for mobility shifts was found. Dichlororhodamine-labeled DNA fragments showed ideal electrophoretic behaviors according to DNA size in the presence of 10 M urea. The base-calling accuracy of dichlororhodamine-labeled M13mp18 and PGEM/U DNA were 99.3% for 333 bases and 99% for 315 bases, respectively. Base calling of unknown DNA samples obtained in the presence of 10 M urea showed 99.1% accuracy.
Song
J M
JM
Ames Laboratory-USDOE and Department of Chemistry, Iowa State University, Ames 50011, USA.
Yeung
E S
ES
D004396:Coloring Agents; D004247:DNA / Q000032:analysis*; D019075:Electrophoresis, Capillary / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry; D005470:Fluorometry; D006860:Hydrogen Bonding; D009691:Nucleic Acid Denaturation; D011205:Povidone / Q000737:chemistry*; D012235:Rhodamines / Q000737:chemistry; D017422:Sequence Analysis, DNA / Q000379:methods*; D014508:Urea / Q000494:pharmacology
D004396:Coloring Agents; D005456:Fluorescent Dyes; D012235:Rhodamines; D014508:Urea; D004247:DNA; D011205:Povidone