Biotin-Labeled Oligonucleotides: Enzymatic Synthesis and Use as Hybridization Probes

Abstract

An oligonucleotide probe (23-mer) containing a single biotinylated deoxyuridine residue at the 3′ terminus was prepared by a primer extention reaction using Escherichia coli DNA polymerase I (Klenow fragment). For efficient synthesis of the probe, it was necessary to add about 16-fold molar excess of the template oligonucleotide (pentadecanucleotide) to the primer oligonucleotide (nonadecanucleotide) in the reaction mixture and to continue the reaction for 2.5 hr at 4°C. The probe was purified by polyacrylamide gel electrophoresis under denaturing conditions. The probe could be specifically and tightly bound with Avidin D (Vector Laboratories) in 1 M NaCl. It could be hybridized to a plasmid DNA containing a perfectly matched complementary sequence, but not to a DNA containing five consecutive noncomplementary bases. The hybridized biotinylated probe could be detected by Avidin D and biotinylated alkaline phosphatase, even when 1.8 ng of the plasmid DNA (0.5 fmoles) was used. A general approach to the enzymatic synthesis of oligonucleotides containing a single biotinylated deoxyuridine at the 3′ end is described.