Localization in situ of specific mRNA using thymine-thymine dimerized DNA probes. Sensitive and reliable non-radioactive in situ hybridization

Abstract

In situ hybridization has been recognized as a powerful technique for localizing specific nucleic acids such as mRNA and viral DNA in individual cells. For in situ hybridization, the use of a non-radioactive probe is considered superior to that of a radioactive one from viewpoints of resolution, probe stability and personal safety. Although various non-radioactive labels are currently available, some interfere with the formation of hybrids and some increase steric hindrance and prevent penetration of the labeled nucleic acid probe into cells and tissues. Recently, we have developed a method using thymine-thymine (T-T) dimerized DNA as a non-radioactive probe. This is simple to make, since it dose not require separation of labeled DNA from unreacted labeling compounds, and T-T dimer will not alter the chemical and physical nature of the probe. In this paper, we describe the tissue processing procedures that are suited for the T-T dimerized DNA probes, and the recent new findings on methodological aspects, particularly the use of synthetic oligodeoxynucleotide probes.