Photo-Biotin Modification of DNA

When photobiotin is irradiated in the presence of DNA the reaction process non-selectively couples a biotin label to every 100—200 base residues. The result is a oligonucleotide probe detectable by the use of avidin or streptavidin conjugates. The uses of photobiotin for DNA or RNA modification are summarized in Chapter 8, Section 3.4. 

The following protocol is based on the method of Forster et al. (1985). Some optimization may be necessary to obtain the best signal and activity for particular probes in hybridization assays. 

In subdued lighting conditions, dissolve photobiotin in water at a concentration of 1 (xg/pl. Protect from light. 

Mix an equal volume of the photobiotin solution with the DNA probe solution. Place the solution in an ice bath and irradiate from above (about 10 cm away) for 15 min using a sunlamp (such as Philips Ultrapnil MLU 300 W, General Elearic sunlamp RSM 275 W, or National Self-BallastedBHRF 240—250 V 250 W W-P lamp). 

Add 50 pi of 0.1 M Tris, pH 9, and increase the total volume of the solution to 100 pi (if it is less than this amount). 

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