High-throughput fluorescent DNA sequence analysis: Methods and automation

Abstract

A strategy for high-throughput DNA sequence analysis using the fluorescent dye-primer chemistry is described. This strategy combines rapid preparation of template DNA using a modification of the polymerase chain reaction (PCR), automation of the DNA sequencing reactions using a robotic laboratory workstation, and subsequent analysis of the fluorescent-labeled reaction products on a commercial automated fluorescent sequencer. Asymmetric PCR provides a reliable means of simultaneously producing sufficient and uniform quantities of several template DNAs directly from bacterial colonies or bacteriophage plaques. The automation scheme for subsequent processing and analysis of DNA samples allows a DNA sequencing facility to operate the fluorescent sequencer twice daily at its full capacity of 24 samples. Furthermore, the methods and instruments described eliminate much of the labor required by technicians to obtain a high level of data output. As described here, a DNA sequencing laboratory equipped with a single automated fluorescent sequencer can perform analysis of up to 48 fluorescent-labeled reactions every day, with an average output of over 10,000 bp per sequence run.