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.
De Bellis
G
G
Consiglio Nazionale delle Ricerche, Istituto di Tecnologie Biomediche, Avanzate, LITA, Segrate, Italy.
Pergolizzi
R
R
Debernardi
S
S
Invernizzi
L
L
Luzzana
M
M
D017931:DNA Primers / Q000378:metabolism*; D005453:Fluorescence; D017422:Sequence Analysis, DNA / Q000379:methods*
D017931:DNA Primers