Rapid purification of fluorescent dye-labeled products in a 96-well format for high-throughput automated DNA sequencing

• Published in: Nucleic acids research 1995-12, Vol.23 (23), p.4930-4931
• Main Authors: Krakowski, K, Bunville, J, Seto, J, Baskin, D, Seto, D
• Format: Article
• Language: English
• Subjects: automation; dna templates; DNA-directed DNA polymerase; electrophoresis; Fluorescent Dyes - isolation & purification; gel filtration chromatography; labeling techniques; resins; Resins, Plant; sequence analysis; Sequence Analysis, DNA - instrumentation; Sequence Analysis, DNA - methods
• ISSN: 0305-1048; 1362-4962
• DOI: 10.1093/nar/23.23.4930

Large-scale DNA sequencing projects can be limited by various technical bottlenecks. As more DNA templates are generated and successfully processed, the mechanisms and technologies continue to be improved and optimized. Fluorescence-based automated sequencing has replaced radioisotope-based manual DNA sequencing as the primary approach in large-scale genome projects. For this approach, the preferred DNA sequencing chemistry had been fluorescence dye-primer based reactions. Reiterations of this automated protocol generated large amounts of DNA sequence data with its corresponding biological information. This, in turn, spurred the continued improvements and modifications that allow the rapid production of data that is generated routinely today using thermostable DNA polymerases and single-stranded or double-stranded DNA templates. Dye terminator chemistries are more flexible. This approach, coupled with a thermostable DNA polymerase and the need for less template ( similar to 0.1-1 mu g) as well as the availability of single-tube reactions per sample, allows for greater sample throughput. Its utility in primer walking for difficult templates and for gap closures has led to the widespread use of Taq polymerase-mediated dye-terminator chemistries in genome sequencing laboratories. This is in spite of the additional manipulations required. For example, due to the inefficiency of Taq DNA polymerase to incorporate the dye-labeled terminators, there is a need to remove the unincorporated molecules from the DNA sequencing ladder, as these unincorporated dyes interfere with the resolution of bands near the priming site and with base calling. This step is tedious and not readily amenable to automation. We describe a method to rapidly, efficiently and inexpensively remove these contaminating unincorporated dye-terminators from the DNA sequencing ladder prior to electrophoresis on a 373A or 377 ABI Sequencer.