Fluorescently labeled model DNA sequences for exonucleolytic sequencing

We describe here the enzyme-catalyzed, low-density labeling of DNAs with fl uorescent dyes. Firstly, for 'natural' template DNAs, dNTPs were partially substituted in the labeling reactions by the respective fluorophore-bearing analogs. The DNAs were labeled by PCR using Tag DNA polymerase. The covale nt incorporation of dye-dNTPs decreased in the following order: rhodamine-g reen-5-duTP (Molecular Probes, the Netherlands), tetramethylrhodamine-4-dUT P (FluoroRed, Amersham Pharmacia Biotech), Cy5-dCTP (Amersham Pharmacia Bio tech). Exonucleolytic degradation by the 3' --> 5' exonuclease activity of T7 DNA polymerase (wild type) in the presence of excess reduced thioredoxin proceeded to complete breakdown of the labeled DNAs. The catalytic cleavag e constants determined by fluorescence correlation spectroscopy were betwee n 0.5 and 1.5 s(-1) at 16 degreesC, normalized for the covalently incorpora ted dye-nucleotides. Secondly, rhodamine-green-X-dUTP (Roche Diagnostics), tetramethylrhodamine-6-dUTP (Roche Diagnostics), and Cy5-dCTP were covalent ly incorporated into the antisense strand of 'synthetic' 218-b DNA template constructs (master sequences) at well defined positions, starting from the primer binding site, by total substitution for the naturally occuring dNTP s. The 218-b DNA constructs were labeled by PCR with a thermostable 3' --> 5' exonuclease deficient mutant of the Tgo DNA polymerase which we have sel ected. The advantage of the special, synthetic DNA constructs as compared t o natural DNAs lies in the possibility of obtaining tailor-made nucleic aci ds, optimized for testing the performance of exonucleolytic sequencing. The number of incorporated fluorescent nucleotides determined by complete exon ucleolytic degradation and fluorescence correlation spectroscopy were six o ut of six possible incorporations for rhodamine-green-X-duTP and tetramethy lrhodamine-6-dUTP, respectively. Their covalent and base-specific incorpora tions were confirmed by the novel analysis methodology of re-sequencing (i. e. mobility-shift gel electrophoresis, reversion-PCR and re-sequencing) fir st developed in the paper Foldes-Papp et al. (2001) and in this paper. This methodology was then used by other groups within the whole sequencing proj ect. (C) 2001 Elsevier Science B.V. All rights reserved.