Detection of Specific DNA Sequences With Short Biotin-Labeled Probes

Abstract

We have developed a simple, general synthesis of nonradioactive DNA probes in which biotin is attached to the 5'-terminal phosphate of an oligodeoxyribonucleotide 16 bases long via an ethylenediamine or hexamethylenediamine linker. The products are stable under normal hybridization conditions. They hybridize to target DNA as efficiently as the underivatized oligodeoxyribonucleotide. Color development, using a commercially available kit, is complete within 3 hr using the biotin-detection method. The sensitivity of detection of homologous DNA with a probe to which biotin was attached via a hexamethylenediamine linker is about one-tenth of that achieved overnight by autoradiography with the corresponding 32P-labeled probe.

A Method for Biotinylating Oligonucleotide Probes for Use in Molecular Hybridizations

“…This technique, however, requires the synthesis of both a template and a primer oligonucleotide. Recently, methods have been described for chemical labeling of oligonucleotides (Challot and Kawashima, 1985; Chu and Orgel, 1985). The disadvantage to these methods is the necessity to purify the adduct prior to conversion to the biotinylated derivatives.…”

“…Several methods have been reported for preparation of biotin-labeled oligonucleotides. They have been labeled chemically using a phosphate linker (Chollet and Kawashima, 1985;Chu and Orgel, 1985), enzymatically (Murasugi and Wallace, 1984), or by a combination of chemical and enzymatic reactions (Kempe et al, 1985). Biotinylated oligomers have also been synthesized using biotin-labeled nucleotides (Bryan and Arnold, 1984;Ruth, 1984).…”

The use of gel electrophoresis to study the reactions of activated amino acids with oligonucleotides

“…Amino acids containing nucleoside bases (I and II; Scheme 1) were a gift from Dr. Azzouz Ben Cheikh (Ben Cheikh and Orgel 1990 atives using polynucleotide kinase and 5'-[~2p]-ATP (Maniatis et al 1982). Ethylenediamine was attached to p(dT)10 and p(dT)t5 via a phosphoramidate bond using a published procedure (Chu and Orgel 1985) and purified by electrophoresis on an 8 M urea-20% polyacrylamide gel (32 V/cm). Products were extracted from the gel with 0.5 M Tris -HC1 containing 1 mM EDTA at pH 7.7 and stored in 10 mM EDTA at pH 9.0 and 4°C.…”

Detection of DNA via an Ion Channel Switch Biosensor

“…Detection of the binding event is via either incorporation of a directly detectable label in the capture probe (e.g., fluorophores, acridinium esters) (9,17) or indirect detection via a second binding event (e.g., biotin or digoxigenin) (4,18). In the latter case, streptavidin (avidin) or antidigoxigenin antibodies are conjugated to enzymes which mediate reactions producing colorimetric or chemiluminescent signals upon addition of substrate (3,19) which allows measurement of a signal.…”