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• W50714092 endingPage "9085" @default.
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• W50714092 abstract "Fluorescent DNA of high molecular weight is an important tool for studying the physical properties of DNA and DNA−protein interactions, and it plays a key role in modern biotechnology for DNA sequencing and detection. While several DNA polymerases can incorporate large numbers of dye-linked nucleotides into primed DNA templates, the amplification of the resulting densely labeled DNA strands by polymerase chain reaction (PCR) is problematic. Here, we report a method for high density labeling of DNA in PCR reactions employing the 5′-triphosphate of 1,3-diaza-2-oxo-phenoxazine (tCo) and Deep Vent DNA polymerase. tCo is a fluorescent cytosine analogue that absorbs and emits light at 365 and 460 nm, respectively. We obtained PCR products that were fluorescent enough to directly visualize them in a gel by excitation with long UV light, thus eliminating the need for staining with ethidium bromide. Reactions with Taq polymerase failed to produce PCR products in the presence of only small amounts of dtCoTP. A comparative kinetic study of Taq and Deep Vent polymerase revealed that Taq polymerase, although it inserts dtCoTP with high efficiency opposite G, is prone to forming mutagenic tCo-A base pairs and does not efficiently extend base pairs containing tCo. These kinetics features explain the poor outcome of the PCR reactions with Taq polymerase. Since tCo substitutes structurally for cytosine, the presented labeling method is believed to be less invasive than labeling with dye-linked nucleotides and, therefore, produces DNA that is ideally suited for biophysical studies." @default.
• W50714092 created "2016-06-24" @default.
• W50714092 creator A5024903730 @default.
• W50714092 creator A5031137637 @default.
• W50714092 creator A5056457744 @default.
• W50714092 creator A5082952838 @default.
• W50714092 date "2009-10-07" @default.
• W50714092 modified "2023-10-16" @default.
• W50714092 title "High Density Labeling of Polymerase Chain Reaction Products with the Fluorescent Base Analogue tCo" @default.
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• W50714092 doi "https://doi.org/10.1021/ac9017555" @default.
• W50714092 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/2784673" @default.
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• W50714092 hasPublicationYear "2009" @default.
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