NONRADIOACTIVE DETECTION OF OLIGONUCLEOTIDE PROBES BY PHOTOCHEMICAL AMPLIFICATION OF DIOXETANES

We describe a new method of non-radioactive labelling and detection of oligonucleotide probes. The approach is based on a simple chemical pr inciple. Oligonucleotides labelled with methylene blue (a photosensiti zer) are hybridized on a membrane to immobilized DNA target sequences. After hybridization and stringency washing 2-[3-(hydroxyphenyl)methox ymethylene] adamantane is added to the membrane and the membrane is ir radiated with a tungsten lamp light source through a cut-off filter. T hermally stable dioxetanes are amplified during irradiation at the pos itions of the labelled probe. These amplified dioxetanes are detected using chemically triggered chemiluminescent decay Signals are recorded on commercial X-ray film. Detection is possible immediately after the last washing step and a hard copy of the blot is obtained within 1 h. Dependent on the level of the target sequences, the sensitivity of th e method allows detection of 0.3 pg single-stranded M13mp18(+) plasmid DNA in dot blots and 75 pg in Southern blots. Additional immunologica l reaction steps and washing steps with blocking reagents and buffers are avoided. Furthermore, expensive reagents and equipment for physica l detection are not necessary. The method might be particularly useful for fast routine analysis in forensic and medical applications. The s ynthesis of the olefin, conditions of hybridization and the protocol o f detection are described in detail.