Non-radioactive 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 principle. Oligonucleotides labelled with methylene blue (a photosensitizer) are hybridized on a membrane to immobilized DNA target sequences. After hybridization and stringency washing 2(-)[3-(hydroxyphenyl)methoxymethylene] adamantane is added to the membrane and the membrane is irradiated with a tungsten lamp light source through a cut-off filter. Thermally stable dioxetanes are amplified during irradiation at the positions 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 the method allows detection of 0.3 pg single-stranded M13mp18(+) plasmid DNA in dot blots and 75 pg in Southern blots. Additional immunological reaction steps and washing steps with blocking reagents and buffers are avoided. Furthermore, expensive reagents and equipment for physical detection are not necessary. The method might be particularly useful for fast routine analysis in forensic and medical applications. The synthesis of the olefin, conditions of hybridization and the protocol of detection are described in detail.
Schubert
F
F
Humboldt-Universität zu Berlin, Institut für Chemie, Germany.
Knaf
A
A
Möller
U
U
Cech
D
D
D000218:Adamantane / Q000031:analogs & derivatives*; D001483:Base Sequence; D015139:Blotting, Southern / Q000379:methods; D004247:DNA / Q000737:chemistry; D004923:Erythrosine; D005452:Fluoresceins; D006571:Heterocyclic Compounds; D007202:Indicators and Reagents; D008163:Luminescent Measurements; D009682:Magnetic Resonance Spectroscopy; D008751:Methylene Blue; D008969:Molecular Sequence Data; D015394:Molecular Structure; D009693:Nucleic Acid Hybridization; D015345:Oligonucleotide Probes / Q000032:analysis* / Q000737:chemistry; D010636:Phenols; D017319:Photosensitizing Agents; D010957:Plasmids / Q000032:analysis*; D012395:Rose Bengal; D012680:Sensitivity and Specificity
C097219:2-(3-(hydroxyphenyl)methoxymethylene)adamantane; D005452:Fluoresceins; D006571:Heterocyclic Compounds; D007202:Indicators and Reagents; D015345:Oligonucleotide Probes; D010636:Phenols; D017319:Photosensitizing Agents; D012395:Rose Bengal; D004247:DNA; D000218:Adamantane; D004923:Erythrosine; D008751:Methylene Blue