Fluorescent high-density labeling of DNA: error-free substitution for a normal nucleotide Export

@article{citeulike:2195665, 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.}, author = {Foldes-Papp, Zeno and Angerer, Bernhard and Ankenbauer, Waltraud and Rigler, Rudolf}, booktitle = {DNA-Sequencing at the Single Molecule Level}, citeulike-article-id = {2195665}, citeulike-linkout-0 = {http://dx.doi.org/10.1016/S0168-1656(00)00416-8}, citeulike-linkout-1 = {http://www.sciencedirect.com/science/article/B6T3C-42JHD6H-6/2/7fcc76aaf42cbb3156eeb5c319674c0a}, day = {13}, doi = {10.1016/S0168-1656(00)00416-8}, journal = {Journal of Biotechnology}, keywords = {dutp, labeling, microarray}, month = {April}, number = {3}, pages = {237--253}, posted-at = {2008-01-04 21:28:10}, priority = {2}, title = {Fluorescent high-density labeling of DNA: error-free substitution for a normal nucleotide}, url = {http://dx.doi.org/10.1016/S0168-1656(00)00416-8}, volume = {86}, year = {2001} }

TY - JOUR ID - citeulike:2195665 L3 - citeulike-article-id:2195665 N2 - 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. IS - 3 JF - Journal of Biotechnology EP - 253 TI - Fluorescent high-density labeling of DNA: error-free substitution for a normal nucleotide VL - 86 T2 - DNA-Sequencing at the Single Molecule Level SP - 237 KW - dutp KW - labeling KW - microarray AU - Foldes-Papp, Zeno AU - Angerer, Bernhard AU - Ankenbauer, Waltraud AU - Rigler, Rudolf PY - 2001/04/13/ UR - http://dx.doi.org/10.1016/S0168-1656(00)00416-8 ER -