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Ultraviolet imaging: A simple method for detecting nucleic acids in preparative gels

1982; Elsevier BV; Volume: 124; Issue: 1 Linguagem: Inglês

10.1016/0003-2697(82)90224-x

1096-0309

Patrick Clarke, Hun-Chi Lin, Gary Wilcox,

Mass Spectrometry Techniques and Applications

A method and apparatus for the detection and quantification of large fragments of unlabelled deoxyribonucleic acid (DNA) in agarose gels is presented. The technique is based on ultra-violet (UV) absorption by nucleotides. Chemical vapour deposition (CVD) diamond has a high indirect band gap of 5.45 eV and is therefore sensitive to UV photons of wavelengths <224 nm. Many bio-molecules display high absorption in this region. The diamond is used to locate bands of nucleic acids as they pass by the detector during electrophoresis. We have constructed and tested a CVD diamond based system that uses the intrinsic absorption of DNA at 260 nm to locate, identify and quantify DNA in gels. A deuterium lamp is used to illuminate a sample lane of an electrophoresis gel using quartz lenses. The transmitted light is detected using a CVD diamond detector. As DNA bands pass through the illuminated region of the gel the intensity of UV light transmitted is reduced due to DNA absorption, and the signal generated in the diamond detector by UV photons is reduced accordingly. The system has a detection limit of 20 ng compared with 2–10 ng for the most popular conventional technique, using ethidium bromide staining and the technique is inherently quantitative. Using our detection technique, the DNA sample remains in its native state, and can easily be used for further experimentation. The removal of carcinogenic dyes from the detection procedure greatly reduces associated biological hazards.