One label, one tube, Sanger DNA sequencing in one and two lanes on a gel.

Sequencing in less than four lanes on a gel, using only one fluorescent label for the four bases (without variations in electrophoretic mobilities or problems with spectral overlap) in one tube was introduced in (1). In that process T7 DNA polymerase and buffer containing magnesium ions were used. By adjusting the ddNTPs ratios the sequence is deduced from the peak magnitudes corresponding to the four bases. The resolution was limited by nonuniformity of the peaks and by decrease of overall labelling due to the two-step process used in that sequencing protocol. In the method described in this note, applicable both to fluorescent and radioactive endlabelling, we have used the one-step protocol with manganese ions in one tube as described (2). This protocol results in uniform overall labelling (within 15% up to base 300). Biochemicals, annealing, termination and stop mixes, as well as diluted enzyme were as described in (4). Briefly, 1.25 ng of M13 ssDNA in 5 /tl solution were combined with 2 /tl fluorescently labelled primer (2 /*M) and 6 jtl annealing mix. The mixture was denatured at 65°C for 3 minutes and cooled down to 25 °C. Then 4U of T7 DNA polymerase in 2 /d were added and mixed. Nucleotide mixes, as described below, were added and strand synthesis proceeded for 10 minutes at 37°C. The reaction was stopped with 20 fd stop mix containing 10 mM NaOH for complete denaturation. Before loading, samples were heated at 65°C for 3 minutes and cooled down on ice. The amount of sample is sufficient for 4 gel runs on the EMBL automated fluorescent DNA Sequencer or the A.L.F. Sequencer (Pharmacia LKB). One lane, one tube reaction: Nucleotide mixes were prepared from the termination mixes (4) in a ratio of T:C:G:A = 2:2:1:0.5 (6 /tl of T termination mix, 6 /il of C-mix, 3 yl of G-mix, 1.5 /i\ of A-mix). Two lane sequencing reactions: Nucleotide mixes were prepared as above, in a ratio of 1:1 for the T>C and 2:1 for the G> A reaction. Fig. 1 (6) shows raw data obtained in the standard (2) four lanes one-step protocol (Fig. 1A), in two lanes (Fig. IB) and