Plant & Animal Genome IX Conference Plant & Animal Genome IX Conference
Poster: Sequencing & EST
P4
Automated fluorescent sequencing using the ABI Prism BigDye Terminator chemistry is the method of choice for high throughput DNA sequence determination. If not removed prior to analysis, certain reaction components will interfere with data collection. Buffer salts will give rise to aberrant electrophoretic migration and unincorporated dye-labeled terminators and related species interfere with the signal from sequencing extension products in the 0-150 base range (fig. 1). Common purification methods are based on gel filtration and precipitation by ethanol. These procedures are problematic for high throughput robotic applications in that they require (multiple) centrifugation and/or vacuum drying steps. Promega has developed a series "silicaparamagnetic" particles for the purification of nucleic acids such as plasmid DNA and PCR fragments. This technology can also be applied to the high throughput purification of Big Dye[tm] Terminator DNA Sequencing reactions prior to automated sequence analysis. Procedures have been developed on the Beckman Biomek 2000 and Tecan Genesis RSP 150 robotic workstations using the standard 96-well amplification plate format. Sequence quality is similar to or better than the manual methods and offers the distinct advantage of a truly hands off protocol. No user interventions are required from the time the 96-well amplification plate is put on the robot deck until the samples are loaded on the DNA sequencer.