Primer Design for Automated DNA Sequencing Utilizing T7 DNA Polymerase and Internal Labeling with FIuorescein45-dATP

摘要

We have identified additional criteria for the walking primer design thai improve the success rate of automated fluorescent DNA sequencing using the internal labeling technique and 77 DNA polymerase. These criteria resulted from the evaluation of aver220 sequences generated with walking primers and fluorescein-15-dATP as internal label in the course of the European Community (EC) yeast genome sequencing project. In this project primers were designed using standard commercial software. Intensities ofsequencing signals varied aver a broad range from very strong to very weak, depending on the primers used. This led us to evaluate primer performance relative lo (i) the template sequence immediately downstream of the primer binding sire and (ii) theprimer sequence itself. Our experiments show that the position of the first labeled dATP to be incorporated downstream of the primer into the growing strand is substantial for the signal intensity of the sequence. The closer to the primer that the first'A' is incorporated, the stronger the peak intensities are. An additional feature of sequencing with native 77 DNA polymerase is its ability to remove a 3'-terminaI 'A' of the primer by the 3'→5' cxoimclease activity and to exchange the nucleotide with a labeled dATP by the polymerase activity.