Improved fluorescent cycle sequencing protocol allows reading nearly 1000 bases.
Recently available thermostable DNA polymerases result in enhanced resolution and accuracy compared to thermal enzymes used previously in fluorescent cycle sequencing. These new enzymes produce less variations in peak intensities, enhance gel resolution and are less sensitive to unspecific termination caused by either DNA structure or impurities in the DNA preparation. Optimization of nucleotide ratios and the usage of high concentrations of detergents in the sequencing reaction result in sequence readings up to 1000 bases and improve overall reliability of the sequencing protocol; this works successfully in about 90% of cases.
Zimmermann
J
J
European Molecular Biology Laboratory, Heidelberg, FRG.
Wiemann
S
S
Voss
H
H
Schwager
C
C
Ansorge
W
W
D004259:DNA-Directed DNA Polymerase; D005453:Fluorescence; D006358:Hot Temperature; D017422:Sequence Analysis, DNA / Q000379:methods*
D004259:DNA-Directed DNA Polymerase