Sequential Injection Analysis System for the Sandwich Hybridization-Based Detection of Nucleic Acids

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• 作者：Katie A. Edwards and Antje J. Baeumner
• 刊名：Analytical Chemistry
• 出版年：2006
• 出版时间：March 15, 2006
• 年：2006
• 卷：78
• 期：6
• 页码：1958 - 1966
• 全文大小：273K
• 年卷期：v.78,no.6(March 15, 2006)
• ISSN：1520-6882

文摘

A sequential injection analysis lab-on-valve (SIA-LOV)system was developed for the specific detection of single-stranded nucleic acid sequences via sandwich hybridization of specific DNA probes to the target sequence. OneDNA probe was tagged with fluorescein; the other wasbiotinylated and immobilized to streptavidin-coated porous beads. The system was optimized with respect tobuffer composition, length of hybridization and washsteps, and volumes and concentrations of componentsused. On-bead oligonucleotide hybridization was studiedusing UV detection at 260 nm, while a final dose responsecurve was quantified using fluorescence detection. Adynamic range of 1-1000 pmol was obtained for asynthetic DNA sequence that was homologous to a segment in the B. anthracis atxA mRNA. A within-dayvariation of 7.2% and a day-to-day variation of 9.9% wasobserved. Each analysis was completed within 20 min.Subsequently, the system was applied to the detection ofatxA mRNA expressed in a surrogate organism andamplified using NASBA. The SIA-LOV will find its application in routine laboratory-based analysis of specific single-stranded DNA/RNA sequences. Future improvements willinclude the integration of dye-encapsulating liposomes forsignal enhancement used in lieu of the single fluorophore-labeled probe in order to lower the limit of detection.