Fully automated DNA reaction and analysis in a fluidic capillary instrument.
A simple, reliable, automated genetic analysis instrument has been designed and prototyped. The system uses novel fluidic technology, coupling thermal cycling, reaction purification, in-line loading, and capillary electrophoresis in a single instrument. Samples in the loop of an injection valve are amplified inside a rapid air thermal cycler. A liquid chromatographic separation eliminates contaminants and excess salt. The sample is loaded in an efficient, continuous, flow-through manner onto a polymer-filled separation capillary. Detection by laser-induced 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 all components and connections are fluidic, automation is natural and simple. The instrument is reliable and fast, performing PCR reaction cycling, purification and analysis, all in 20 min. Reproducibility (CV) of retention times is 2% (n = 129) and of peak areas 9% (n = 34). Bubbles and particulates are eliminated by the chromatography column. Adaptation of the instrument prototype for separation of DNA-sequencing reactions is described; cycle sequencing and electrophoresis of a single lane are complete in 90 min. Implications and challenges for development of fully automated fluidic instruments for genomic sequencing are discussed.
Swerdlow
H
H
Department of Human Genetics, University of Utah, Salt Lake City 84112, USA.
Jones
B J
BJ
Wittwer
C T
CT
D002851:Chromatography, High Pressure Liquid; D004247:DNA / Q000737:chemistry*; D019075:Electrophoresis, Capillary / Q000379:methods*; D016133:Polymerase Chain Reaction; D013050:Spectrometry, Fluorescence
D004247:DNA