SIMULTANEOUS ONLINE DNA-SEQUENCING ON BOTH STRANDS WITH 2 FLUORESCENTDYES

We describe an automated DNA-sequencing technique which allows both th e simultaneous sequencing from the two strands of double-stranded temp lates and the subsequent detection of the sequencing products online a nd in parallel. The technique is based on hardware technology also use d in the ALF DNA sequencer (Pharmacia, Uppsala). A helium-neon laser w as mounted into the sequencing device additionally to the standard arg on laser. Two different primers, labeled with either fluorescein or Te xas red, are used in a single sequencing reaction resulting in an outp ut of two sequences. Both sequencing products are then analyzed on-lin e 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 seq uencing of PCR products in double-stranded form can now be accomplishe d in only one sequencing reaction. (C) 1995 Academic Press, Inc.