Edwards Katie A, Baeumner Antje J
Department of Biological and Environmental Engineering, Cornell University, Ithaca, New York 14853, USA.
Anal Chem. 2006 Mar 15;78(6):1958-66. doi: 10.1021/ac051768a.
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.
开发了一种顺序注射分析阀上实验室（SIA-LOV）系统，用于通过特定DNA探针与靶序列的夹心杂交来特异性检测单链核酸序列。一种DNA探针用荧光素标记；另一种用生物素标记并固定在链霉亲和素包被的多孔珠上。对该系统在缓冲液组成、杂交和洗涤步骤的时长以及所用组分的体积和浓度方面进行了优化。使用260nm的紫外检测研究了珠上寡核苷酸杂交，而使用荧光检测对最终的剂量响应曲线进行了定量。对于与炭疽芽孢杆菌atxA mRNA中的一个片段同源的合成DNA序列，获得了1-1000 pmol的动态范围。观察到日内变化为7.2%，日间变化为9.9%。每次分析在20分钟内完成。随后，该系统应用于检测在替代生物体中表达并使用核酸序列扩增技术（NASBA）扩增的atxA mRNA。SIA-LOV将在基于实验室的特定单链DNA/RNA序列的常规分析中得到应用。未来的改进将包括整合用于信号增强的染料包封脂质体，以替代单荧光团标记探针，从而降低检测限。
