Novel fluorescent nonnucleoside triphosphates as terminators of enzyme-directed DNA synthesis

Near infrared (NIR) fluorescent acycloterminators were tested as substrates in the Sanger enzymatic method of DNA sequencing. The acyclic triphosphates of adenosine, uridine, guanosine, and cytidine (AcyNTP) were labeled with a heptamethine carbocyanine dye via a propargylamino linker to the purine or pyrimidine base. Dye-labeled AcyNTPs which are lacking in the sugar moiety positions equivalent to the C-2 and C-3 of the ribose functioned similarly to chain-terminating dideoxynucleotides (ddNTPs). These fluorescent nonnucleotide analogs were incorporated by a mutant, thermostable Tag DNA polymerase with the same efficacy and fidelity as traditional ddNTPs. Sequence read length and base-calling accuracy were comparable for both dye-acycloterminator and dye-primer sequencing methods. fn two primer walking projects, cycle sequencing with fluorescent AcyNTPs achieved a mean sequence read length of 1,090 bases with 99.1% accuracy at one kilobase read length. The cyanine dye-labeled acycloterminators produced electropherograms in which weak T peaks follow G peaks. In cases of polymorphism, such peak height variability may make it difficult to distinguish the presence or absence of a heterozygote at a specific site.