Use of sodium trichloroacetate and mung bean nuclease to increase sensitivity and precision during transcript mapping

Abstract

An improved method for mapping RNA transcript boundaries by the nuclease protection technique is presented. This method exploits the large (greater than 20 degrees C) difference in the thermal stability of RNA:DNA and DNA:DNA duplexes in concentrated chaotropic salt solutions. At 45 degrees C in 3.0 M sodium trichloroacetate RNA:DNA hybridization is very efficient but DNA:DNA duplexes remain completely denatured. For many applications, this solvent system can eliminate the need to prepare probes that are free of competing or irrelevant DNA molecules. Fifty- to 100-fold more RNA:DNA hybridization is observed when reassociation is performed in 3.0 M sodium trichloroacetate than in solutions containing high concentrations of formamide. A comparison of the use of S1 nuclease or mung bean nuclease suggests that mung bean nuclease can produce more precise and less ambiguous nuclease protection patterns.