Journal of Biotechnology 86 (2001) 203 – 224 Fluorescently labeled model DNA sequences for exonucleolytic sequencing Zeno Fo ¨ ldes-Papp a, *, Bernhard Angerer b , Per Thyberg a , Michael Hinz c , Stefan Wennmalm a , Waltraud Ankenbauer b , Hartmut Seliger c , Arne Holmgren d , Rudolf Rigler a a Department of Medical Biophysics, MBB, Karolinska Institute, S -17177 Stockholm, Sweden b Roche Diagnostics GmbH, Nonnenwald 2, D-82372 Penzberg, Germany c Section for Polymeric Chemistry, Uniersity Ulm, D-89069 Ulm, Germany d Medical Nobel Institute for Biochemistry, MBB, Karolinska Institute, S -17177 Stockholm, Sweden Received 27 August 1999; received in revised form 25 April 2000; accepted 22 May 2000 Abstract We describe here the enzyme-catalyzed, low-density labeling of DNAs with fluorescent 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 Taq DNA polymerase. The covalent incorporation of dye-dNTPs decreased in the following order: rhodamine-green-5-dUTP (Molecular Probes, the Netherlands), tetramethylrho- damine-4-dUTP (FluoroRed, Amersham Pharmacia Biotech), Cy5-dCTP (Amersham Pharmacia Biotech). Exonucle- olytic 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 cleavage constants determined by fluorescence correlation spectroscopy were between 0.5 and 1.5 s -1 at 16°C, normalized for the covalently incorporated dye-nucleotides. Secondly, rhodamine-green-X-dUTP (Roche Diagnostics), tetramethylrho- damine-6-dUTP (Roche Diagnostics), and Cy5-dCTP were covalently 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 dNTPs. 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 selected. The advantage of the special, synthetic DNA constructs as compared to natural DNAs lies in the possibility of obtaining tailor-made nucleic acids, optimized for testing the performance of exonucleolytic sequencing. The number of incorporated fluorescent nucleotides determined by complete exonucleolytic degradation and fluorescence correlation spectroscopy were six out of six possible incorporations for rhodamine-green-X-dUTP and tetramethylrhodamine-6- dUTP, respectively. Their covalent and base-specific incorporations were confirmed by the novel analysis methodol- ogy of re-sequencing (i.e. mobility-shift gel electrophoresis, reversion-PCR and re-sequencing) first developed in the www.elsevier.com/locate/jbiotec * Corresponding author. Present address: Clinical Immunology and Jean Dausset Laboratory, Graz University M.S. and Hospital, Auenbrugger Platz 8, A-8036 Graz, Austria. Tel.: +43-316-3852547; fax: +43-316-3854790. E-mail address: Zeno.Foldes-Papp@kfunigraz.ac.at (Z. Fo ¨ ldes-Papp). 0168-1656/01/$ - see front matter © 2001 Elsevier Science B.V. All rights reserved. PII:S0168-1656(00)00414-4