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Proceedings Paper

Enhanced throughput for infrared automated DNA sequencing
Author(s): Lyle Richard Middendorf; Bill O. Gartside; Pat G. Humphrey; Stephen C. Roemer; David R. Sorensen; David L. Steffens; Scott L. Sutter
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Paper Abstract

Several enhancements have been developed and applied to infrared automated DNA sequencing resulting in significantly higher throughput. A 41 cm sequencing gel (31 cm well- to-read distance) combines high resolution of DNA sequencing fragments with optimized run times yielding two runs per day of 500 bases per sample. A 66 cm sequencing gel (56 cm well-to-read distance) produces sequence read lengths of up to 1000 bases for ds and ss templates using either T7 polymerase or cycle-sequencing protocols. Using a multichannel syringe to load 64 lanes allows 16 samples (compatible with 96-well format) to be visualized for each run. The 41 cm gel configuration allows 16,000 bases per day (16 samples X 500 bases/sample X 2 ten hour runs/day) to be sequenced with the advantages of infrared technology. Enhancements to internal labeling techniques using an infrared-labeled dATP molecule (Boehringer Mannheim GmbH, Penzberg, Germany) and Sequenase (U.S. Biochemical) have also been made. The inclusion of glycerol in the sequencing reactions yields greatly improved results for some primer and template combinations. The inclusion of (alpha) -Thio-dNTP's in the labeling reaction increases signal intensity two- to three-fold.

Paper Details

Date Published: 3 April 1995
PDF: 13 pages
Proc. SPIE 2386, Ultrasensitive Instrumentation for DNA Sequencing and Biochemical Diagnostics, (3 April 1995); doi: 10.1117/12.206009
Show Author Affiliations
Lyle Richard Middendorf, LI-COR, Inc. (United States)
Bill O. Gartside, LI-COR, Inc. (United States)
Pat G. Humphrey, LI-COR, Inc. (United States)
Stephen C. Roemer, LI-COR, Inc. (United States)
David R. Sorensen, LI-COR, Inc. (United States)
David L. Steffens, LI-COR, Inc. (United States)
Scott L. Sutter, LI-COR, Inc. (United States)

Published in SPIE Proceedings Vol. 2386:
Ultrasensitive Instrumentation for DNA Sequencing and Biochemical Diagnostics
Gerald E. Cohn; Jeremy M. Lerner; Kevin J. Liddane; Alexander Scheeline; Steven A. Soper, Editor(s)

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