J. Lesley Woodhead

Detection of DNA sequences using biotinylated probes.

It was found (1) that biotinylated analogs of dUTP, which contain a biotin molecule covalently bound to the C-5 position of the pyrimidine via an allylamine linker arm, can be used as substrates for DNA polymerase. Figure 1 shows the structure of biotin-11-dUTP, a commonly used substrate for biotinylation by the method of nick translation (2). The substituted DNA molecule will have similar hybridization properties to the un-substituted DNA. After hybridization to the test DNA, the biotin probe can be located by a variety of methods, including incubation with streptavidin-peroxidase conjugate, which will be described here. Fig. 1. Structure of biotin-11-UTP.

Luminescent Detection of Specific DNA Sequences

The process whereby part of the energy evolved in a chemical reaction is emitted as light is known as luminescence. When luminol is oxidized by hydrogen peroxide in the presence of horseradish peroxidase (HRP), light is evolved (1). This is shown in Fig. 1. Luciferin not only enhances the reaction, but prolongs the emission over several minutes (2). The light may be measured in a luminometer or visualized as the blackening of photographic film, as will be described. Fig. 1. Oxidation of luminol in the presence of hydrogen peroxide and HRP (the catalyst).

Crosslinking of single-stranded DNA to resins.

Specific fragments of single-stranded DNA may be covalently immobilized to solid supports for various reasons. The support can be used as an affinity column for purifying DNA from a test sample, to detect viral or bacterial DNAs, or for antenatal diagnosis of genetic diseases such as sickle cell anemia using a sandwich hybridization method developed in this laboratory (1).