A method for biotinylating oligonucleotide probes for use in molecular hybridizations.

A new method is described for biotinylation of oligonucleotide probes for use in molecular hybridization reactions. Biotin-11-dUTP residues were added enzymatically, using terminal deoxynucleotidyl transferase, to the 3' terminus of a synthetic oligonucleotide prepared from the known nucleotide sequence for adenosine deaminase. The biotinylated probe was hybridized to DNA and mRNA selectively immobilized on nitrocellulose and detected by sequential incubation of the nitrocellulose membrane with avidin and biotinylated polyalkaline phosphatase, followed by colorimetric development. The biotinylated oligonucleotide probe proved useful for the qualitative detection of complementary DNA and mRNA sequences but was unsatisfactory for quantitative determinations using reflective densitometry.