In situ hybridization of ?Nick-translated? 3H-ribosomal DNA to chromosomes from salamanders

Abstract

A technique is described for preparation of ³H-labelled DNA by nick-translation employing deoxyribonuclease I and DNA polymerase I. The labelled DNA can be obtained in high yield with specific activities of 10⁶ cpm/μg or more. Ribosomal DNA, isolated from ovaries of young Xenopus laevis, and whole DNA from Plethodon cinereus were labelled in this way. The rDNA was used for in situ hybridization to meiotic chromosomes from P. cinereus, P. vehiculum and P. dunni. Autoradiographs of in situ hybrids were exposed for 5 to 10 days, by which time nucleolus organizer regions on the chromosomes of all 3 species were clearly and specifically labelled. In all eases, labelling was confined to a short region near the middle of the short arm of both halves of a medium length bivalent. It is concluded that nick-translation is a useful and altogether efficient method of labelling nucleic acids for subsequent use in experiments involving in situ hybridizations.