Determination of base ratios on sub-microgram quantities of RNA

Abstract

1. Introduction The determination of base compositions of ribo- nucleic acids is an important step in sequence analysis. As RNAs are often not available in milligram quan- tities for the determination of base compositions using spectrophotometric procedures [l-6] , more sensitive radioactive labelling methods have been developed. The procedures of Sanger et al. [7] can be used directly for base composition analysis of RNA uniformly labelled with 32P to a high specific activity. But not all RNAs can be so labelled in vivo, and 32P also has an inconveniently short half-life. The Ran- deraths [8,9] .developed a method using sodium boro- [3H]hydride to reduce periodate-oxidised nucleosides to stable tritiated nucleoside tri-alcohols. Tritium has a low energy of radiation so it is difficult to detect by autoradiography, especially when picomole quantities of tritiated nucleoside tri-alcohols are analysed. We previously described a simple, sensitive proce- dure for chemically labelling RNA and its derivatives which uses p-hydrazinobenzene-[35S]sulphonic acid (35S-p-HBSA) as the radioactive labelling reagent [lo] We now wish to describe routine labelling procedure for determining base compositions of RNA and ribo- oligonucleotides. The procedure is simple and very sensitive requiring only 0.5 pg of the polymer for at least a triplicate determination. 2. Materials and methods 2.1.