PRIMER DESIGN FOR AUTOMATED DNA-SEQUENCING UTILIZING T7 DNA-POLYMERASE AND INTERNAL LABELING WITH FLUORESCEIN-15-DATP

We have identified additional criteria for the walking primer design t hat improve the success rate of automated fluorescent DNA sequencing u sing the internal labeling technique and T7 DNA polymerase. These crit eria resulted from the evaluation of over 220 sequences generated with walking primers and fluorescein-15-dATP as internal label in the cour se of the European Community (EC) yeast genome sequencing project. In this project primers were designed using standard commercial software. Intensities of sequencing signals varied over a broad range from very strong to very weak, depending on the primers used. This led us to ev aluate primer performance relative to (i) the template sequence immedi ately downstream of the primer binding site and (ii) the primer sequen ce itself. Our experiments show that the position of the first labeled dATP to be incorporated downstream of the primer into the growing str and is substantial for the signal intensity of the sequence. The close r to the primer that the first 'A' is incorporated, the stronger the p eak intensities are. An additional feature e of sequencing with native T7 DNA polymerase is its ability to remove a 3'-terminal 'A' of the p rimer by the 3'-->5' exonuclease activity and to exchange the nucleoti de with a labeled dATP by the polymerase activity.