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Proceedings Paper

Novel fluorescent nonnucleoside triphosphates as terminators of enzyme-directed DNA synthesis
Author(s): Stephen C. Roemer; Craig M. Johnson; Vince R. Boveia; Philip R. Buzby; James J. DiMeo; Dan Draney; Narasimhachari Narayanan; D. Michael Olive
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Paper Abstract

Near infrared (NTR)fluorescentacycloterniinators were tested as substrates in the Sanger enzymatic method ofDNA sequencing. The acyclic triphosphates ofadenosine, 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 ofthe ribose functioned similarly to chain-terminating dideoxynucleotides (ddNTPs). These fluorescent nonnucleotide analogs were incorporated by a mutant, thermostable Taq DNA polymerase with the same efficacy and fidelity as traditional ddNTPs. Sequence read length and basecalling accuracy were comparable for both dye-acyclotenninator and dye-primer sequencing methods. In 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.

Paper Details

Date Published: 16 April 2001
PDF: 8 pages
Proc. SPIE 4264, Genomics and Proteomics Technologies, (16 April 2001); doi: 10.1117/12.424593
Show Author Affiliations
Stephen C. Roemer, Orchid BioSciences, Inc. (United States)
Craig M. Johnson, LI-COR, Inc. (United States)
Vince R. Boveia, LI-COR, Inc. (United States)
Philip R. Buzby, Perkin Elmer Life Sciences (United States)
James J. DiMeo, Perkin Elmer Life Sciences (United States)
Dan Draney, LI-COR, Inc. (United States)
Narasimhachari Narayanan, LI-COR, Inc. (United States)
D. Michael Olive, LI-COR, Inc. (United States)

Published in SPIE Proceedings Vol. 4264:
Genomics and Proteomics Technologies
Ramesh Raghavachari; Weihong Tan, Editor(s)

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