Koch Lab:Protocols/Microsphere-DNA tethering
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Principle
A microsphere can be tethered to a microscope slide (or other surface) via a single double-stranded DNA molecule (dsDNA). Commonly, the microsphere is about a 500 nm polystyrene or silica microsphere, but of course many other possibilities exist, such as: 2.8 micron polystyrene microspheres with embedded maghemite superparamagnetic nanocrystals; small gold nanoparticles; quantum dots. Once tethers have been formed, they can be stretched with optical tweezers (e.g., to measure the elastic properties of DNA, or unzip the DNA), or the DNA tether length can be monitored via analysis of the Brownian motion of the microsphere (Tethered Particle Motion, TPM...perhaps to detect DNA looping by a protein).
To achieve tethering, you need to attach one end of the DNA to the glass slide, and the other end to the microsphere. Generically, this is what needs to be done:
glass --- connection#1 --- endDNA******DNAend --- connection#2 --- microsphere
A very common method that is used is to label the DNA with digoxigenin (dig) on one end and biotin on the other end. The surface of the glass slide is coated with a polyclonal antibody against dig (anti-dig) and the microspheres are coated with streptavidin (which binds to biotin with high affinity). This makes the tethering scheme:
glass --- anti-dig --- digDNA******DNAbiotin --- streptavidin --- microsphere
There are also many other methods. Because of the availability of a variety of streptavidin coated microspheres, biotin-streptavidin connection seems to be by far the most popular. Dig/anti-dig is also very common, but other methods for tethering that end of the dNA to the class are:
- anti-HA --- HA-RNA polymerase-DNA
- gold --- thiol-DNA
- anti-his --- his-Protein-DNA
- anti-fluorescein --- fluoresceinDNA
Some general thoughts--not rigorously tested
Specific protocols
- A specific protocol, 4411 basepair PCR-labeled DNA, 0.5 micron streptavidin microspheres
