Biotin is widely used for DNA/RNA detection/isolation due to the extremely high affinity of the biotin-streptavidin interaction (association constant 1015/M). Biotin moieties can be incorporated within an oligo on any place and in any number. We have long and super-long tethering arms covalently attached to biotin for improved binding kinetics, increased binding capacity of large DNA fragments, and for accessibility to enzymatic events occurring at the solid-phase surface.
The size of the DNA oligonucleotide covalently attached to biotin seems to limit the binding capacity to immobilized streptavidin (Sabanayagam et al., Nucleic Acids Res., 2000, 28, e33). We have discovered that in the use of biotinylated primers for sequencing reactions, the ability to capture large DNA Sanger fragment pools with paramagnetic streptavidin beads was directly related to the length of linker arms holding the biotin moiety. The capturing efficiency for these long DNA fragments was maximized when using our “super-long” tethering arms with biotin. We speculate that steric interference of the DNA to the surface-bound streptavidin is minimized by increasing the length of the tethering arm (NNNA 2003, Oligonucleotide Synthesis 2004).
Our long tethers can be used in overcoming unfavorable interactions of bis-biotinylated dsDNA to recombinant streptavidin with a covalently attached oligonucleotide (Niemeyer et al., Bioconj. Chem., 2001, 12, 364). These conjugates can be used as reagents in immuno-PCR or as building blocks in molecular nanotechnology. However, a significant decrease of the size of the dsDNA-linked oligomeric aggregrates was attributed to steric or electrostatic repulsion of the DNA moieties (dsDNA and the covalently bound oligonucleotide). By increasing the tethering length of the dsDNA to the biotin, some of these repulsive forces may be reduced, if not eliminated.
cDNA/screening procedures involving magnetic bead capture
Magnetic bead capture has successfully been used to obtain cDNAs form double-stranded plasmid cDNA libraries (Shepard and Rae, Nucleic Acids Res., 1997, 25, 3183) and fewer rounds of enrichment may be necessary utilizing our “super-long” biotinylated oligonucleotides.
Biotinylated molecular beacon probes
Internally biotinylated oligonucleotides can be used to immobilize a molecular beacon probe to a glass surface (Li et al., Anal. Sci., 2001, 17, 1149). A bridge structure for immobilization has been proposed to reduce steric hindrances. Further reductions in unfavorable sterics and improved fluorescence may be achieved by increasing the tether arm of biotin allowing optimum hybridization of target to probe.
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