Simultaneous Bi-Directional Cycle Sequencing.

Stephen C. Roemer, Kurt A. Brumbaugh, Vince Boveia, Mary Jensen, and John Gardner¹
LI-COR, inc., Biotechnology Division, 4647 Superior St., Lincoln, NE 68504 and ¹CEPRAP, University of California, Davis, CA

Standard sequencing primers are end-labeled with a near-infrared (NIR) fluorescent dye (IRD700 or IRD800). Simultaneous sequencing on both strands of duplex DNA is accomplished when a forward and reverse primer (each tagged with a different fluorescent dye) are used in the same sequencing reaction.

The LI-COR IR² Automated DNA Sequencing System is capable of analyzing the forward and reverse sequences of a bidirectional sequencing reaction in parallel. In the schematic, DNA sequencing products tagged with IRD700 represent plus strand (forward) sequence and are symbolized by red bands. Blue bands symbolize minus strand (reverse) sequence labeled with IRD800. Bands in which both IRD700- and IRD800-labeled sequencing products co-migrate are shown in black.

Dye-labeled DNA fragments are detected during electrophoresis by a scanning fluorescence microscope. The microscope has two diode lasers (l ca. 700 and 800 nm) for exciting the fluorophores, a microscope objective for collecting the emission radiation, and two cooled avalanche photodiodes, each filtered for detecting a different NIR emission.

Two separate gel images, corresponding to reactions primed with IRD700- or IRD800-labeled oligonucleotides, are generated in real time for the same four sequencing lanes (A, T, G, and C). These images are interpreted using the automated base-calling algorithms of LI-COR's Base ImagIR^TM software.