FLUORESCENCE-BASED AUTOMATED DNA-SEQUENCING BY LIMITED PRIMER LABELING

The applicability of automated DNA sequencing systems to sequencing st rategies that require a large number of primers is limited by the nece ssity for expensive fluorescence-labeled oligonucleotides. Here we pre sent a simple procedure that allows the use of unlabeled oligonucleoti des to perform fluorescence-based DNA sequencing. This method is based on a limited primer extension that incorporates three deoxynucleotide s, one of which carries a fluorescent moiety. The elongated fluorescen t primer is then used in a standard T7 sequencing reaction. This label ing ruocedure is both economical and straightforward and offers a vali d alternative to current fluorescence-labeling protocols. Results of t his method with different DNA templates demonstrate the reliability of the protocol.