Single-lane single-fluor sequencing using dideoxy-labeled, heavy-atom-modified near-IR fluorescent dyes

Daryl C. Williams; James H. Flanagan Jr.; Benjamin L. Legendre Jr.; Robert P. Hammer; Steven A. Soper

doi:10.1117/12.206008

3 April 1995 Single-lane single-fluor sequencing using dideoxy-labeled, heavy-atom-modified near-IR fluorescent dyes

Daryl C. Williams, James H. Flanagan Jr., Benjamin L. Legendre Jr., Robert P. Hammer, Steven A. Soper

Proceedings Volume 2386, Ultrasensitive Instrumentation for DNA Sequencing and Biochemical Diagnostics; (1995) https://doi.org/10.1117/12.206008
Event: Photonics West '95, 1995, San Jose, CA, United States

Abstract

Using a near-IR (NIR) fluorescence detection system and labels synthesized in our laboratories, electropherograms of oligonucleotides separated by capillary gel electrophoresis and detected using NIR fluorescence will be presented. The sequence of nucleotide bases was determined using a single-lane, single-dye technique. The molar concentrations of the ddNTP's used during extension reactions were varied in order to achieve a ratio of 4:2:1:0 (A:C:G:T) which allowed the identification of each terminal base via fluorescence intensity measurements. Sequencing ladders were prepared from the template, M13mp18, using standard Sanger dideoxy chain termination techniques, the modified T7 DNA polymerase, and a NIR-labeled M13 primer. The data indicated reliable sequence determination up to 300 bases with a base-calling accuracy of 90%. In order to eliminate the need for dye-labeled primers and the T7 DNA polymerase enzyme, we have developed a sequencing strategy which utilizes dye-labeled dideoxy nucleotides in a single-lane, single-fluor approach. Base-calling is accomplished by measuring the fluorescence lifetime of intramolecular heavy-atom modified dyes.

Citation Download Citation

Daryl C. Williams, James H. Flanagan Jr., Benjamin L. Legendre Jr., Robert P. Hammer, and Steven A. Soper "Single-lane single-fluor sequencing using dideoxy-labeled, heavy-atom-modified near-IR fluorescent dyes", Proc. SPIE 2386, Ultrasensitive Instrumentation for DNA Sequencing and Biochemical Diagnostics, (3 April 1995); https://doi.org/10.1117/12.206008

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