BIOTINYLATED OLIGONUCLEOTIDE HYBRIDIZATION PROBES: PROGRESS TOWARDS A NON-RADIOACTIVE METHOD FOR THE DIAGNOSIS OF HUMAN GENETIC DISEASES.

Abstract

Under appropriate conditions synthetic oligonucleotide hybridization probes display essentially absolute hybridization specificity. All of the non-Watson-Crick base pairs, including G-T, have a destabilizing effect. Thus, it is possible to choose stringent conditions of hybridization such that, while a perfectly matched duplex between an oligonucleotide and complementary DNA will form, duplexes mismatched at one or more positions will not. The hybridization of oligonucleotides to regions of DNA containing the single base changes responsible for many human genetic disease, thus, provides a means of detecting these diseases. In an attempt to develop a non-radioactive method for the detection of human genetic diseases, we have prepared biotinylated oligonucleotides by an enzymatic method. An oligonucleotide probe (23-mer) containing a single biotinylated deoxyuridine residue at the 3’-terminus was prepared by a primer extention reaction using E. coli DNA polymerase I (Klenow fragment). The biotinylated probe could be hybridized to a complementary sequence and visualized by Avidin D and biotinylated alkaline phosphatase. A general approach to the enzymatic synthesis of oligonucleotides containing a single biotinylated deoxyuridine at the 3’ end is described.