Simultaneous on-line DNA sequencing on both strands with two fluorescent dyes.

We describe an automated DNA-sequencing technique which allows both the simultaneous sequencing from the two strands of double-stranded templates and the subsequent detection of the sequencing products online and in parallel. The technique is based on hardware technology also used in the ALF DNA sequencer (Pharmacia, Uppsala). A helium-neon laser was mounted into the sequencing device additionally to the standard argon laser. Two different primers, labeled with either fluorescein or Texas red, are used in a single sequencing reaction resulting in an output of two sequences. Both sequencing products are then analyzed on-line in the same lanes of a gel. This technique is especially useful for the complete sequencing of DNA fragments up to 1 kb. High accuracy sequencing of PCR products in double-stranded form can now be accomplished in only one sequencing reaction.