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

Stephen C. Roemer; Craig M. Johnson; Vince R. Boveia; Philip R. Buzby; James J. DiMeo; Dan Draney; Narasimhachari Narayanan; D. Michael Olive

doi:10.1117/12.424593

16 April 2001 Novel fluorescent nonnucleoside triphosphates as terminators of enzyme-directed DNA synthesis

Stephen C. Roemer, Craig M. Johnson, Vince R. Boveia, Philip R. Buzby, James J. DiMeo, Dan Draney, Narasimhachari Narayanan, D. Michael Olive

Author Affiliations +

Proceedings Volume 4264, Genomics and Proteomics Technologies; (2001) https://doi.org/10.1117/12.424593
Event: BiOS 2001 The International Symposium on Biomedical Optics, 2001, San Jose, CA, United States

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.

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Stephen C. Roemer, Craig M. Johnson, Vince R. Boveia, Philip R. Buzby, James J. DiMeo, Dan Draney, Narasimhachari Narayanan, and D. Michael Olive "Novel fluorescent nonnucleoside triphosphates as terminators of enzyme-directed DNA synthesis", Proc. SPIE 4264, Genomics and Proteomics Technologies, (16 April 2001); https://doi.org/10.1117/12.424593

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KEYWORDS

Polymers

Analog electronics

Life sciences

Luminescence

Absorbance

Near infrared

Chromium

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