Fluorescent labelling of sequencing primers for automated oligonucleotide synthesis

Abstract

A fluorescein derivative is described which can be used as a normal phosphoramidite in oligonucleotide synthesis, giving high yields of fluorescein labelled sequencing primers. The labelled primers were used in automated DNA-sequence analysis without modification of existing protocols, the computer-processed sequences being reproducibly readable up to 400 bases. The procedure described makes the fluorescent labelling of oligonucleotides much easier, and the time of labeling can be significantly reduced. It speeds up the “primer walking” approach of automated DNA sequencing.

Key Words:

• DNA sequencing
• fluorescence
• primer labelling
• oligonucleotide synthesis
• labelled phosphoramidites