Dideoxy linear PCR on a commercial fluorescent automated DNA sequencer.
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.
Manoni
M
M
Consiglio Nazionale delle Ricerche, Istituto di Tecnologie, Biomediche Avanzate, Milano, Italy.
Pergolizzi
R
R
Luzzana
M
M
De Bellis
G
G
D001322:Autoanalysis; D001483:Base Sequence; D004247:DNA / Q000737:chemistry*; D003838:Deoxyadenine Nucleotides; D003845:Deoxycytosine Nucleotides; D003848:Deoxyguanine Nucleotides; D054306:Dideoxynucleotides; D005456:Fluorescent Dyes; D009711:Nucleotides; D010957:Plasmids; D016133:Polymerase Chain Reaction / Q000379:methods*; D013942:Thymine Nucleotides
D003838:Deoxyadenine Nucleotides; D003845:Deoxycytosine Nucleotides; D003848:Deoxyguanine Nucleotides; D054306:Dideoxynucleotides; D005456:Fluorescent Dyes; D009711:Nucleotides; D013942:Thymine Nucleotides; C037773:2',3'-dideoxyadenosine triphosphate; C037406:2',3'-dideoxycytidine 5'-triphosphate; C031709:2',3'-dideoxyguanosine 5'-triphosphate; D004247:DNA; C017216:2',3'-dideoxythymidine triphosphate