Sequential injection analysis system for the sandwich hybridization-based detection of nucleic acids.
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. One DNA probe was tagged with fluorescein; the other was biotinylated and immobilized to streptavidin-coated porous beads. The system was optimized with respect to buffer composition, length of hybridization and wash steps, and volumes and concentrations of components used. On-bead oligonucleotide hybridization was studied using UV detection at 260 nm, while a final dose response curve was quantified using fluorescence detection. A dynamic range of 1-1000 pmol was obtained for a synthetic DNA sequence that was homologous to a segment in the B. anthracis atxA mRNA. A within-day variation of 7.2% and a day-to-day variation of 9.9% was observed. Each analysis was completed within 20 min. Subsequently, the system was applied to the detection of atxA mRNA expressed in a surrogate organism and amplified using NASBA. The SIA-LOV will find its application in routine laboratory-based analysis of specific single-stranded DNA/RNA sequences. Future improvements will include the integration of dye-encapsulating liposomes for signal enhancement used in lieu of the single fluorophore-labeled probe in order to lower the limit of detection.
Edwards
Katie A
KA
Department of Biological and Environmental Engineering, Cornell University, Ithaca, New York 14853, USA.
Baeumner
Antje J
AJ
D015374:Biosensing Techniques / Q000295:instrumentation* / Q000379:methods*; D017022:Flow Injection Analysis / Q000295:instrumentation* / Q000379:methods*; D009693:Nucleic Acid Hybridization / Q000379:methods*; D009696:Nucleic Acids / Q000032:analysis*; D012680:Sensitivity and Specificity; D013997:Time Factors
D009696:Nucleic Acids