Immobilization of Long Biotinylated DNA Fragments
The Dynabeads® kilobaseBINDER™ Kit is designed to immobilize long (>2 kb) double-stranded DNA molecules. Traditional immobilization of PCR products to Streptavidin-coupled Dynabeads® has shown that the amount of DNA immobilized is dependent on fragment size. The binding efficiency is significantly reduced, due to steric hindrance, when fragment size exceeds 2 kb (Figure 1). The kilobaseBINDER™ kit includes a patented immobilization activator in the Binding Solution, which significantly increases binding efficiency of long, biotinylated DNA molecules.
- Increased binding capacity for long (>2 kb)
- Biotinylated dsDNA fragments
- Rapid, efficient and easy purification of nuclei from cytoplasm
- Chromatin isolation within minutes
Figure 1. B&W buffer (red) is the standard Binding and Washing Buffer recommended for use with Streptavidin-coupled Dynabeads®. The specially designed Binding Solution (green) significantly increases the binding capacity.
Figure 2. DNA-coated Dynabeads® (red) assemble a mitotic spindle (green) in Xenopus egg extracts, indistinguishable from those in normal dividing cells (1). Binding capacity for 8kb biotinylated DNA fragments was routinely 20 µg (3.8 pmol) per mg Dynabeads®. Courtesy of R. Heald, E Karsenti and A. Hyman, EMBL, Heidelberg, Germany.
Various innovative applications in cell biology have utilized this approach, including
- Chromatin beads mimicking chromosomes for studying self-organization of microtubules into bipolar spindles (ref. 1 and Figure 2)
- Synthetic nuclei for studying RNA export from the nucleus in vitro (2)
- Reconstitution of nuclear movement along microtubules and examination of the molecular basis of this movement in vitro (3)
- In vitro analysis of glucocorticoid receptor binding sites on reconstituted chromatin DNA fragments using restriction enzyme assay and DNase footprinting (4)
- Recruitment analysis of Xenopus and Drosophila DNA binding proteins (5, 6)
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- Ikeda K et al (2002) Mol. Cell. Biol. 22(19):6759-6766