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Title
| - Rotating Rod Renewable Microcolumns forAutomated, Solid-Phase DNA Hybridization Studies
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Abstract
| - The development of a new temperature-controlled renewable microcolumn flow cell for solid-phase nucleic acidhybridization in an automated sequential injection systemis described. The flow cell included a stepper motor-driven rotating rod with the working end cut to a 45°angle. In one position, the end of the rod preventedpassage of microbeads while allowing fluid flow; rotationof the rod by 180° releases the beads. This system wasused to rapidly test many hybridization and elutionprotocols to examine the temperature and solution conditions required for sequence-specific nucleic acid hybridization. Target nucleic acids labeled with a near-infraredfluorescent dye were detected immediately postcolumnduring all column perfusion and elution steps using a flow-through fluorescence detector. Temperature control of thecolumn and the presence of Triton X-100 surfactant werecritical for specific hybridization. Perfusion of the columnwith complementary oligonucleotide (200 μL, 10 nM)resulted in hybridization with 8% of the DNA binding siteson the microbeads with a solution residence time of lessthan 1 s and a total sample perfusion time of 40 s. Theuse of the renewable column system for detection of anunlabeled PCR product in a sandwich assay was alsodemonstrated.
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