High-density polymerase-mediated incorporation of fluorochrome-labeled nucleotides.
DNA-polymerase-mediated incorporation of different fluorochrome-labeled nucleotides (FdNTPs) was investigated with the goals of optimizing the high-density labeling of probes and exploring DNA sequencing strategies that rely on the controlled, sequential addition of such compounds. By systematically evaluating variables--including polymerase type, buffer conditions, and fluorochrome chemistries--a rational strategy for the sequential addition of labeled nucleotides to a DNA template was demonstrated. A simple structural model of the polymerase-DNA template complex that considered the fluorochrome moiety of the FdNTPs and the linker length also guided this strategy. Complementary results that portend the use of simple photobleaching to enable the reliable quantitation of consecutive additions are presented.
Ramanathan
Arvind
A
Laboratory for Molecular and Computational Genomics, Department of Chemistry, Laboratory of Genetics, University of Wisconsin-Madison, UW-Biotechnology Center, 425 Henry Mall, Madison, WI 53706, USA.
Pape
Louise
L
Schwartz
David C
DC
D002021:Buffers; D003432:Cross-Linking Reagents; D004259:DNA-Directed DNA Polymerase; D005456:Fluorescent Dyes / Q000737:chemistry*; D015341:Nucleic Acid Probes / Q000138:chemical synthesis*; D009711:Nucleotides; D012235:Rhodamines; D017422:Sequence Analysis, DNA
D002021:Buffers; D003432:Cross-Linking Reagents; D005456:Fluorescent Dyes; D015341:Nucleic Acid Probes; D009711:Nucleotides; D012235:Rhodamines; C034970:bacteriophage T7 induced DNA polymerase; D004259:DNA-Directed DNA Polymerase