FULLY AUTOMATED DNA REACTION AND ANALYSIS IN A FLUIDIC CAPILLARY INSTRUMENT

A simple, reliable, automated genetic analysis instrument has been des igned and prototyped. The system uses novel fluidic technology, coupli ng thermal cycling, reaction purification, in-line loading, and capill ary electrophoresis in a single instrument. Samples in the loop of an injection valve are amplified inside a rapid air thermal cycler. A liq uid chromatographic separation eliminates contaminants and excess salt . The sample is loaded in an efficient, continuous, flow-through manne r onto a polymer-filled separation capillary. Detection by laser-induc ed fluorescence produces signal-to-noise ratios of 1000:1 or greater. Refilling of the polymer-filled capillary is automatic; during the run , the system is reconditioned for injection of another sample. Since a ll components and connections are fluidic, automation is natural and s imple. The instrument is reliable and fast, performing PCR reaction cy cling, purification and analysis, all in 20 min. Reproducibility (CV) of retention times is 2% (n = 129) and of peak areas 9% (n = 34). Bubb les and particulates are eliminated by the chromatography column. Adap tation of the instrument prototype for separation of DNA-sequencing re actions is described; cycle sequencing and electrophoresis of a single lane are complete in 90 min. Implications and challenges for developm ent of fully automated fluidic instruments for genomic sequencing are discussed.