Medizinische Universität Graz Austria/Österreich - Forschungsportal - Medical University of Graz
Gewählte Publikation:
Földes-Papp, Z; Angerer, B; Ankenbauer, W; Rigler, R.
Fluorescent high-density labeling of DNA: error-free substitution for a normal nucleotide.
J Biotechnol. 2001; 86(3):237-253
Doi: 10.1016%2FS0168-1656%2800%2900416-8
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- Führende Autor*innen der Med Uni Graz
- Földes-Papp Zeno
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- Abstract:
- 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.
- Find related publications in this database (using NLM MeSH Indexing)
- Base Sequence -
- DNA - analysis
- DNA-Directed DNA Polymerase - chemistry
- Electrophoresis - methods
- Fluorescent Dyes - analysis
- Molecular Sequence Data - analysis
- Mutation - analysis
- Polymerase Chain Reaction - methods
- Rhodamines - chemistry
- Spectrometry, Fluorescence - methods
- Find related publications in this database (Keywords)
- single-molecule DNA sequencing
- high-density labeling
- base-specific incorporation
- thermostable 3 '-> 5 ' exonuclease deficient mutant of Tgo DNA polymerase
- two-color fluorescence cross-correlation spectroscopy
- mobility-shift gel electrophoresis
- rever