The biotin-streptavidin interaction can be reversibly broken using water at elevated temperaturesThe biotin-streptavidin system is the strongest noncovalent biological interaction known, having a dissociation constant, K d , in the order of 4610 214 M. The strength and specificity of the interaction has led it to be one of the most widely used affinity pairs in molecular, immunological, and cellular assays. However, it has previously been impossible to re-use any streptavidin solid support, since the conditions needed to break the interaction with biotin has led to the denaturation of the streptavidin. Here, we show that a short incubation in nonionic aqueous solutions at temperatures above 707C can efficiently break the interaction without denaturing the streptavidin tetramer. Both biotin and the streptavidin remain active after dissociation and both molecules can therefore be re-used. The efficiency of the regeneration allowed solid supports with streptavidin to be used many times, here exemplified with the multiple re-use of streptavidin beads used for sample preparation prior to automated DNA sequencing. The results suggest that streptavidin regeneration can be introduced as an improvement in existing methods and assays based on the streptavidin system as well as emerging solid phase applications in fields, such as microfluidics and nanotechnology. IntroductionThe strong interaction between avidin and biotin was discovered as early as 1941 [1]. Avidin is a protein commonly purified from chicken egg white while biotin is a vitamin found in all cells. Streptavidin, a bacterial homologous protein to avidin, isolated from the actinobacterium Streptomyces avidinii, is more frequently used than avidin and is commercially available also in a number of engineered forms. The structure of the biotin-streptavidin complex has been described by several groups [2,3], showing a b-barrel structure of streptavidin binding biotin into its interior. The binding between avidin/streptavidin and biotin has long been regarded as the strongest, noncovalent, biological interaction known, having a dissociation constant, K d , in the order of 4610 214 M [4]. The bond forms very rapidly and is stable in wide ranges of pH and temperature [1,5].The strong interaction has led to a large number of research and diagnostic applications using avidin-biotin or streptavidin-biotin technology. The strength and reliability of the interaction underlie its importance in biotechnology, but the interaction is also a model for high-affinity receptor ligand binding. In most assays, streptavidin is coupled to a solid phase, such as a magnetic bead, a microtiter plate, or a biosensor chip, while biotin is coupled to the moiety of interest, often a nucleic acid, protein, or antibody. However, harsh conditions, such as formamide treatment combined with high temperatures, have been required to separate biotin from streptavidin, resulting in not only denatured streptavidin molecules [5] but also in limitations of downstream applications due to deterioration ...

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Nucleic acid hybridization is an essential component in many of today's standard molecular biology techniques. In a recent study, we investigated whether nucleic acid capture could be improved by taking advantage of stacking hybridization, which refers to the stabilizing effect that exists between oligonucleotides when they hybridize in a contiguous tandem fashion. Here, we describe a specific approach for purification of sequencing products using cooperative probes that hybridize to single-strand targets where one of the probes has been coupled to a magnetic bead. This approach has been developed for standard sequencing primers and has been applied to shotgun plasmid libraries. The cooperative probes have been designed to anneal within the common vector sequence and to avoid co-purification of nonextended sequencing primers and misprimed sequencing products. The reuse of magnetic beads, together with salt independent elution, makes the approach suitable for high-capacity capillary electrophoresis instruments.