Direct colony sequencing of plasmid DNA by dye terminator methods.

Direct colony sequencing of plasmid DNA is a powerful technique. Radioactive or fluorescent dye primer methods are the reported direct colony sequencing methods (2–4,6). Recently, dye terminator sequencing chemistry (7) has been improved by the introduction of a modified enzyme, AmpliTaq DNA polymerase FS (8), as well as a new d-Rhodamine and BigDye terminator (PE Biosystems, Foster City, CA, USA) (5) that enhances sensitivity while reducing the background. The direct colony sequencing strategy is presented here and suggests that it is possible to use a BigDye terminator as well as dye primer sequencing. Plasmid pBluescript KS+ (Stratagene, La Jolla, CA, USA) was transformed to DH5α-competent cells using the method of Hanahan (1). The appropriate volume of transformed competent cells was transferred to an LB agar plate containing 100 μg/mL ampicillin and incubated overnight at 37°C. A single bacterial colony with a diameter of 2–2.5 mm was picked up and then suspended in a reagent mixture containing 6 μL BigDye Terminator Ready reaction mixture (PE Biosystems); 2 μL (3.2 pmol) -20 M13 primer and 7 μL double-distilled water. The reactions were carried out in a model 480 DNA thermal cycler (PE Biosystems) at 94°C for 3 min and then for 25–40 cycles of 96°C for 30 s; 50°C for 15 s; and 60°C for 2 min. Each Sephadex G50 spin column (Amersham Pharmacia Biotech, Piscataway, NJ, USA) loaded with the reaction product was microcentrifuged at 730× g for 2 min to Benchmarks