Fluorescent high-density labeling of DNA: error-free substitution for a normal nucleotide.
The enzymatic incorporation of deoxyribonucleoside triphosphates by a thermostable, 3'-->5' exonuclease deficient mutant of the Tgo DNA polymerase was studied for PCR-based high-density labeling of 217-bp "natural" DNA in which fluorescent-dUTP was substituted completely for the normal dTTP. The amplified DNA carried two different sorts of tethered dye molecules. The rhodamine-green was used for internal tagging of the DNA. Since high-density incorporation of rhodamine-green-X-dUTP led to a substantial reduction (quenching) of the rhodamine-green fluorescence, a second "high" quantum yield label, Cy5, was inserted via a 5'-tagged primer in order to identify the two-color product. A theoretical concept of fluorescence auto- and cross-correlation spectroscopy developed here was applied to quantify the DNA sequence formed in terms of both the number of two-color fluorescent molecules and the number of covalently incorporated rhodamine-green-X-dUMP residues. The novel approach allowed to separate optically the specific DNA product. After complete, exonucleolytic degradation of the two-color DNA we determined 82-88 fluorescent U* labels incorporated covalently out of 92 maximum possible U* incorporations. The heavily green-labeled DNA was then isolated by preparative mobility-shift electrophoresis, re-amplified in a subsequent PCR with normal deoxyribonucleoside triphosphates, and re-sequenced. By means of this novel methodology for analyzing base-specific incorporations that was first developed here, we found that all fluorescent nucleotides and the normal nucleotides were incorporated at the correct positions. The determined labeling efficiency of 0.89-0.96 indicated that a fraction of the substrate analog was not bearing the fluorophore. The results were used to guide developments in single-molecule DNA sequencing. The labeling strategy (principal approach) for PCR-based high-density tagging of DNA, which included an appropriate thermostable DNA polymerase and a suitable fluorescent dye-dNTP, was developed here.
Department of Medical Biophysics, MBB, Karolinska Institute, S-17177 Stockholm, Sweden. Zeno.Foldes-Papp@kfunigraz.ac.at
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Földes-Papp
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Angerer
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Ankenbauer
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Rigler
D001483:Base Sequence; D004247:DNA / Q000032:analysis / Q000737:chemistry*; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry / Q000235:genetics; D004586:Electrophoresis / Q000379:methods; D005456:Fluorescent Dyes / Q000032:analysis / Q000737:chemistry*; D008969:Molecular Sequence Data; D009154:Mutation; D016133:Polymerase Chain Reaction / Q000379:methods; D012235:Rhodamines / Q000737:chemistry; D013050:Spectrometry, Fluorescence / Q000379:methods*
D005456:Fluorescent Dyes; D012235:Rhodamines; D004247:DNA; D004259:DNA-Directed DNA Polymerase