Hybridization of RNA labelled with ¹²⁵I to High Specific Activity

Abstract

Hybridization techniques have been used frequently in studies requiring the preparation or comparison of specific nucleic acids. The feasibility of annealing reactions for a particular application depends on the ability to separate the duplex nucleic acid from the single stranded forms, the complexity of the parental nucleic acid pool and the specific activity of isotopic labelling. The latter factor, that is, an inadequacy in isotopic labelling, has been a frequent handicap, particularly in hybridization analysis of the eukaryote nucleic acids. Recently, the sensitivity of hybridization experiments has been substantially improved through in vitro enzymatic copying of nucleic acids with RNA polymerase or reverse transcriptase in the presence of labelled nucleotide^1–4. In the work described here, the recently introduced procedure for derivatization with ¹²⁵I-labelled iodine catalysed by thallic trichloride⁵ was applied to obtain ribonucleic acids of high specific activity. The labelled product was tested for capacity to anneal to DNA. Standard hybridization procedures entailing elevated temperature incubation in the presence of a filter gave high background values. At low temperature in solutions containing dimethylformamide, annealing reactions proceeded without this complication and yielded gene frequency comparable to established levels.