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

The enzymatic incorporation of deoxyribonucleoside triphosphates by a therm ostable, 3' --> 5' exonuclease deficient mutant of the Tgo DNA polymerase w as studied for PCR-based high-density labeling of 217-bp 'natural' DNA in w hich fluorescent-dUTP was substituted completely for the normal dTTP. The a mplified DNA carried two different sorts of tethered dye molecules. The rho damine-green was used for internal tagging of the DNA. Since high-density i ncorporation of rhodamine-green-X-dUTP led to a substantial reduction (quen ching) of the rhodamine-green fluorescence, a second 'high' quantum yield l abel, Cy5, was inserted via a 5'-tagged primer in order to identify the two -color product. A theoretical concept of fluorescence auto- and cross-corre lation 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. Afte r complete, exonucleolytic degradation of the two-color DNA we determined 8 2-88 fluorescent U* labels incorporated covalently out of 92 maximum possib le U* incorporations. The heavily green-labeled DNA was then isolated by pr eparative mobility-shift electrophoresis, re-amplified in a subsequent PCR with normal deoxyribonucleoside triphosphates, and re-sequenced. By means o f this novel methodology for analyzing base-specific incorporations that wa s first developed hire, we found that all fluorescent nucleotides and the n ormal nucleotides were incorporated at the correct positions. The determine d labeling efficiency of 0.89-0.96 indicated that a ii-action of the substr ate analog was not bearing the fluorophore. The results were used to guide developments in single-molecule DNA sequencing. The labeling strategy (prin cipal approach) for PCR-based high-density tagging of DNA, which included a n appropriate thermostable DNA polymerase and a suitable fluorescent dye-dN TP, was developed here. (C) 2001 Elsevier Science B.V. All rights reserved.