Nucleic Acids Research, 1994, Vol. 22, No. 19 3997-4001
© 1994
METHODS |
Internal fluorescence labeling with fluorescent deoxynucleotides in two-label peak-height encoded DNA sequencing by capillary electrophoresis
Department of Chemistry, University of Alberta Edmonton, Alberta T6G 2G2, Canada 1Boehringer Mannheim GmbH, R&D Biochemicals D-82377 Penzberg, Germany
*To whom correspondence should be addressed
Received March 31, 1994. Revised July 14, 1994. Accepted July 14, 1994.
Fluorescently labeled deoxynucleotides were used for internal labeling of DNA sequencing fragments generated in a two-color peak-height encoded protocol. Sequenase and a manganese-containing buffer were used to generate uniform peak heights. Tetramethyl rhodamine — dATP was used in a labeling step, followed by termination with ddATP and ddCTP in a 3:1 ratio. Fluorescein - dATP was used in a second reaction, followed by termination with ddGTP and ddTTP in a 3:1 ratio. The fragments were pooled and separated by capillary gel electrophoresis. The results were compared with peak-height encoded sequencing based on fluorescently labeled primers. The dye-labeled primers produced higher resolution separations for shorter fragments. However, dye-labeled primer fragments suffered from an earlier onset of biased reptation and produced shorter sequencing reads. Fragments from 50 to at least 500 bases in length could be sequenced with the internal labels.