Kiyama H, Emson P C, Tohyama M
MRC Group, Institute of Animal Physiology and Genetics Research, Babraham, Cambridge, U.K.
Neurosci Res. 1990 Sep;9(1):1-21. doi: 10.1016/0168-0102(90)90041-c.
Recent developments in DNA and oligonucleotide chemistry have made it possible to modify nucleotides and link quite complex molecules to the modified nucleotides. These advancements in DNA chemistry provide a number of possibilities for labelling oligonucleotide probes for DNA or RNA detection by non-radioactive methods. Over the years a number of non-radioactive detection systems for mRNA or chromosomal DNA have been developed. As reporter molecules, biotin, acetylaminofluorene, dinitrophenol, digoxigenin, sulfonized nucleotides, and mercury have been used and may be detected with a variety of high-affinity detectors, e.g. avidin (in the case of biotin) or antibodies specific to digoxigenin. These various 'indirect methods' of detection have used a number of chemical amplification procedures in attempts to improve their sensitivity. However, the sensitivity of these methods is often less than that of conventional radioactive methods. A sensitive non-radioactive technique would have a number of advantages over the complex and specialized radioactive in situ hybridization methods. In our laboratory we have recently found that simple enzyme-labelled probes provide excellent sensitivity (equivalent to that found with radioactive methods) and substantially improved cellular resolution. In this article, we describe the principle of the method and illustrate some applications of this novel non-radioactive in situ method.
