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Title: Fully automated DNA reaction and analysis in a fluidic capillary instrument

Abstract

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. 56 refs.,more » 3 figs.« less

Authors:
; ;  [1]
  1. Univ. of Utah, Salt Lake City, UT (United States)
Publication Date:
OSTI Identifier:
466343
Resource Type:
Journal Article
Journal Name:
Analytical Chemistry (Washington)
Additional Journal Information:
Journal Volume: 69; Journal Issue: 5; Other Information: PBD: 1 Mar 1997
Country of Publication:
United States
Language:
English
Subject:
55 BIOLOGY AND MEDICINE, BASIC STUDIES; 40 CHEMISTRY; 66 PHYSICS; DNA POLYMERASES; CHAIN REACTIONS; DNA SEQUENCING; CAPILLARY FLOW; ELECTROPHORESIS; LIQUID COLUMN CHROMATOGRAPHY; THERMAL CYCLING; GENETICS; FLUIDIC DEVICES; DESIGN; SIGNAL-TO-NOISE RATIO; RELIABILITY; AUTOMATION

Citation Formats

Swerdlow, H, Jones, B J, and Wittwer, C T. Fully automated DNA reaction and analysis in a fluidic capillary instrument. United States: N. p., 1997. Web. doi:10.1021/ac961104o.
Swerdlow, H, Jones, B J, & Wittwer, C T. Fully automated DNA reaction and analysis in a fluidic capillary instrument. United States. https://doi.org/10.1021/ac961104o
Swerdlow, H, Jones, B J, and Wittwer, C T. 1997. "Fully automated DNA reaction and analysis in a fluidic capillary instrument". United States. https://doi.org/10.1021/ac961104o.
@article{osti_466343,
title = {Fully automated DNA reaction and analysis in a fluidic capillary instrument},
author = {Swerdlow, H and Jones, B J and Wittwer, C T},
abstractNote = {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. 56 refs., 3 figs.},
doi = {10.1021/ac961104o},
url = {https://www.osti.gov/biblio/466343}, journal = {Analytical Chemistry (Washington)},
number = 5,
volume = 69,
place = {United States},
year = {1997},
month = {3}
}