Dideoxy linear PCR on a commercial fluorescent automated DNA sequencer
Manoni, M.; Pergolizzi, R.; Luzzana, M.; De Bellis, G.
Biotechniques 12(1): 48-50; 52-53
1992
ISSN/ISBN: 0736-6205 PMID: 1734922 Document Number: 396432
The use of automated fluorescent DNA sequencer systems and PCR-based DNA sequencing methods play an important role in the actual effort to improve the efficiency of large-scale DNA analysis. Here we show the application of the linear PCR using a single fluorescent primer and dideoxynucleotide terminators in four separate sequencing reactions on the EMBL/Pharmacia's fluorescent automated DNA sequencer. We have used dideoxy/deoxynucleoside triphosphate ratios and linear amplification cycle conditions to obtain an accurate sequencing response of up to, and over, 500 bases from just 400 ng of double-stranded DNA template without chemical denaturation. The sequencing protocol described in this paper is effectively suited for enhancement of sensitivity and performance of the automated DNA sequencing system.