Fluorescence-based automated DNA sequencing by limited primer labeling.

The applicability of automated DNA sequencing systems to sequencing strategies that require a large number of primers is limited by the necessity for expensive fluorescence-labeled oligonucleotides. Here we present a simple procedure that allows the use of unlabeled oligonucleotides to perform fluorescence-based DNA sequencing. This method is based on a limited primer extension that incorporates three deoxynucleotides, one of which carries a fluorescent moiety. The elongated fluorescent primer is then used in a standard T7 sequencing reaction. This labeling procedure is both economical and straightforward and offers a valid alternative to current fluorescence-labeling protocols. Results of this method with different DNA templates demonstrate the reliability of the protocol.